Research ArticleTrichostatin A-Mediated Epigenetic Transformationof Adult Bone Marrow-Derived Mesenchymal Stem CellsBiases the In Vitro Developmental Capability Quality andPluripotency Extent of Porcine Cloned Embryos
Marcin Samiec1 Jolanta Opiela1 Daniel LipiNski23 and Joanna Romanek1
1Department of Biotechnology of Animal Reproduction National Research Institute of Animal ProductionKrakowska 1 Street 32-083 Balice n Krakow Poland2Department of Biochemistry and Biotechnology Poznan University of Life Sciences Dojazd 11 Street 60-632 Poznan Poland3Institute of Human Genetics Polish Academy of Sciences Strzeszynska 32 Street 60-479 Poznan Poland
Correspondence should be addressed to Marcin Samiec marcinsamiecizookrakowpl
Received 1 October 2014 Accepted 21 February 2015
Academic Editor Kui Li
Copyright copy 2015 Marcin Samiec et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
The current research was conducted to explore the in vitro developmental outcome and cytologicalmolecular quality of porcinenuclear-transferred (NT) embryos reconstituted with adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) that wereepigenetically transformed by treatment with nonspecific inhibitor of histone deacetylases known as trichostatin A (TSA) Thecytological quality of cloned blastocysts was assessed by estimation of the total cells number (TCN) and apoptotic index Theirmolecular quality was evaluated by real-time PCR-mediated quantification of gene transcripts for pluripotency- and multipotentstemness-related markers (Oct4 Nanog and Nestin) The morula and blastocyst formation rates of NT embryos derived fromABM-MSCs undergoing TSA treatment were significantly higher than in the TSA-unexposed group Moreover the NT blastocystsgenerated using TSA-treated ABM-MSCs exhibited significantly higher TCN and increased pluripotency extent measured withrelative abundance of Oct4 and Nanog mRNAs as compared to the TSA-untreated group Altogether the improvements inmorulablastocyst yields and quality of cloned pig embryos seem to arise from enhanced abilities for promotion of correct epigeneticreprogramming of TSA-exposed ABM-MSC nuclei in a cytoplasm of reconstructed oocytes To our knowledge we are the firstto report the successful production of mammalian high-quality NT blastocysts using TSA-dependent epigenomic modulation ofABM-MSCs
1 Introduction
The efficiency of somatic cell cloning in pigs which is meas-ured with the rate of embryos developing up to blastocyststage or the rate of born offspring in relation to the num-ber of reconstructed oocytes remains disappointingly lowMoreover despite tremendous improvement of somatic cellnuclear transfer (SCNT) technique in pigs high early- mid-and late-gestation mortality rates of nuclear-transferredembryosfetuses as well as numerous malformations of resul-tant cloned offspring still often appear in this species Incom-plete and aberrant reprogramming of epigenetic memory
of somatic cell nuclei in preimplanted nuclear-transferred(NT) embryos is one of the most important factors that limitcloning effectiveness in this species [1ndash4]
The process of epigenomically dependent reprogram-ming is related to the stable erasure (vanishing) of donor cellnuclear DNA-epigenetic status and turning back (molecularnulling) the somatogenic ldquotranscriptional and translationalclockrdquo This contributes to recapitulation of a particular pro-gram of the embryonic genome expression which is inducedby the reestablishment of the embryo cell genome-associatedmethylation and embryo cell chromatin-associated acetyla-tion patterns [5ndash7] SCNT-linked problems are hypothesized
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 814686 13 pageshttpdxdoiorg1011552015814686
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to result from aberrant gene dedifferentiation of somatic cellnuclei at the levels of epigenomic genomic and molecularmemory The mechanism underlying the dedifferentiationprocess of donor nuclei is the cessation of their owngene expression and reversal of the differentiated (special-ized) somatic nucleus to a totipotentpluripotent embryonic(undifferentiatedunspecialized) state within the host oop-lasm and cytoplasm of cleavage descendant blastomeres ofNT embryos [8ndash10] In turn impaired restoration (reestab-lishment) of the totipotencypluripotency of embryonic celllines in the first phase of epigenetic reprogramming (iegene dedifferentiation) during preimplantation developmentby the blastocyst stage may trigger disadvantageous alter-ations in the second phase of donor nuclear reprogram-ming These are connected with improper redifferentiationof somatic cell-inherited genes throughout postimplantationfetalplacental development Additional work is needed todetermine whether failures in the early-stage reprogrammingare magnified downstream in development [11ndash13]
In vitro cultured fibroblast cells which had been derivedfrom the dermointegumentary tissue of fetuses and adultspecimens are the commonly used source of nuclear donorcells in the pig cloning procedure [14ndash18] The degree ofmolecular and epigenetic differentiation of these cells thatis related both to the advanced methylation profile of DNAcytosine residues and to the lysine deacetylation profile ofhistones forming nucleosomal core of nuclear chromatinoften seems to make the converting of the abovementionedcovalentmodifications back to a totipotent state of embryonic(zygotic) cells impossibleThis leadsmainly to decrease in theabilities of differentiated fibroblast cells for supporting the invitro development of cloned embryos to the blastocyst stage[1 19] As a rule the percentage of blastocysts originatingfrom the porcine oocytes reconstructed with fetal or adultcutaneous fibroblast cell nuclei oscillates from 10 to 30[20ndash23]
It is hypothesized that the use of undifferentiated mes-enchymal stem cells (MSCs) isolated from adult bone mar-row which are characterized by the high multipotencylevel and genomicepigenomic plasticity allows increasingthe preimplantation developmental potential of mammaliancloned embryos As compared to hematopoietic stem cellsthe MSCs exhibit the lower activity of histone deacetylases(HDACs) and DNA methyltransferases (DNMTs) whichresults in hyperacetylation of histone lysine moieties anddemethylation of DNA cytosine residues [24ndash27] The lasttwo processes affect the inhibition of transcriptional suppres-sion of many regions in the nuclear genome of multipotentMSCs All these properties are responsible for the highsusceptibility of the MSC nuclei to correct and completeepigenetic reprogramming in the cytoplasm of NT embryoblastomeres Therefore the genome of undifferentiated stemcells may be more easily reprogrammed to resemble thegenome of the zygote which may make stem cells moreefficient as nuclear donors in the somatic cell cloning [28ndash30] Generally porcine NT embryos reconstituted with adultbone marrow-derived mesenchymal stem cells displayedconsiderably higher blastocyst formation rates than those
reconstitutedwith adult cutaneous or fetal fibroblast cells [31ndash33]
Transcriptional activity of somatogenic nuclear genomeduring embryo pre- andor postimplantation development aswell as fetogenesis is correlated with the frequencies forspatial remodeling of chromatin architecture and reprogram-ming of cellular epigenetic memory This former andthis latter process include such covalent modifications asdemethylationde novo methylation of DNA cytosine res-idues and acetylationdeacetylation as well as demethylationremethylation of lysine residues of nucleosomal core-derivedH3 and H4 histones [2 30 34 35] The level of progressionfor the processes of epigenetic genome-wide alterationsthat are mediated by DNA methyltransferases (DNMTs 1oand 3a3b) and histone deacetylases (HDACs) can be mod-ulated (ie reversed) via exogenous inhibitors of theseenzymes throughout in vitro culture of nuclear donor cellsandor cloned embryos [19 36ndash39] Moreover the use ofthe artificial modifiers of epigenomically conditioned geneexpression leads to the inhibition of both chromatin con-densation and transcriptional silencing of the genomic DNAof cultured somatic cells that are applied as a source ofdonor nuclei for the reconstruction of enucleated oocytesand subsequent generation of cloned embryos The membersof these epigenetic modifiers are 5-aza-21015840-deoxycytidine (5-aza-dC) [37 39ndash41] S-adenosylhomocysteine (SAH) [42]trichostatin A (TSA) [6 19 36 38 43] valproic acidsodiumvalproate (VPASV) [44 45] 6-(13-dioxo-1H3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide called scrip-taid [46ndash49] sodium butyrate (NaBu) [50 51] m-carboxy-cinnamic acid bishydroxamide (CBHA) [30 52] and oxam-flatin [29 53] The onset of chromatin decondensationand gene transcriptional activity is evoked by nonspecificnonselective blocking of the activity of either DNMTs by5-aza-dC and SAH [41 42] or HDACs by the TSA VPAscriptaid NaBu CBHA and oxamflatin [30 36 44 4851 53] Such exogenous epigenomic modulation (epigenetictransformation) of nuclear donor cells or cloned embryosmay facilitate and accelerate the reprogrammability for geneexpression of donor cell nuclei that have been transplantedinto cytoplasmic microenvironment of recipient oocytesSubsequently these cell nuclei undergo the proper dedif-ferentiating and faithful reestablishing of the epigeneticallydependent status of their transcriptional activity during thepreimplantation development of cloned embryos [43 54 55]
To increase the developmental capacity of porcine clonedembryos by enhancement of donor cell nuclear repro-grammability a nonselective inhibitor of HDACs designatedas TSA was used for epigenetic transformation of adult bonemarrow-derived mesenchymal stem cells (ABM-MSCs) thatprovided a source of nuclear donor cells In the currentinvestigation we focused on determining the impact of TSA-dependent epigenomic modulation of cultured MSCs on thesomatic cell cloning efficiency measured with in vitro devel-opmental capability of NT pig embryos and their cytologicalquality at the blastocyst stage that was evaluated on thebasis of both total blastomere number and apoptotic indexestimated by TUNEL analysis Furthermore we examinedwhether exposure of MSCs to TSA affects the pluripotency
BioMed Research International 3
status of cloned pig blastocysts For this reason the quanti-tative expression profiles for gene transcripts encoding suchproteins as pluripotency-related markers (Oct4 and Nanog)and multipotent stemness-associated marker (Nestin) wereevaluated in nuclear-transferred embryos reconstructed withepigenetically modified MSCs To the best of our knowledgethis report is the first in which the effect of TSA-mediatedepigenetic transformation of nuclear donor ABM-MSCs onthe extracorporeal developmental competences of porcinecloned embryos their cytological quality and quantitativepluripotency profile at the DNA transcription level wascomprehensively explored
2 Materials and Methods
21 Animals Four outbred Polish LargeWhite (PLW) pigs ofeither sex weighing approximately 20 kg each were main-tained under conventional conditions in the pigsty of theDepartment of Biotechnology of Animal Reproduction fromNational Research Institute of Animal Production in Bal-ice Poland The veterinary care was provided All animalprocedures were approved by the Local Animal Care EthicsCommittee Number II in Krakow
22 Chemicals and Supplies The reagents used in the presentexperiments were purchased from Sigma-Aldrich (PoznanPoland) unless otherwise indicated
23 Recovery and In Vitro Culture of Porcine MesenchymalStem Cells Mesenchymal stem cells (MSCs) were isolatedfrom the pig bone marrow as described by Opiela et al[56 57] Briefly the bone marrow was aspirated undergeneral anesthesia Bone marrow samples were placed inphosphate-buffered saline (PBS Biomed Lublin Poland)(1 1) and 6mL of this solution was layered over 3mLof Ficoll-Paque (Stem-cells Technologies USA) Followingcentrifugation the mononuclear cell fraction was collectedand rinsed twice with PBS The pelleted cells were trans-ferred to 75 cm2 tissue culture flasks (T75) filled with 17mLof MSC growthexpansion medium (Dulbeccorsquos ModifiedEaglersquos Medium DMEM) that was formulated to contain lowconcentration (1mgmL) ofD-glucose and enrichedwith 10fetal bovine serum (FBS) and 1 antibiotics1 Glutamax(Invitrogen USA) The culture medium was replenishedon the next day and the adherent cells were allowed toform colonies until reaching confluence The cell colonieswere harvested by trypsinEDTA treatment and passagedAfterwards the cells were seeded de novo at a concentrationof 025 to 05 times 106 into one T75 flask To confirm the mes-enchymal stemness origin of isolated cells flow cytometry-based detection of positive and negative expression of specificsurface cluster of differentiation (CD) antigens and ex vivodifferentiation of established MSC lines into adipocytes andosteocytes were accomplished as described by Opiela et al[56 58]
24 Simultaneous Cell Cycle Synchronization and EpigenomicModulation (Epigenetic Transformation) of Porcine MSCs
prior to SCNT Before trypsin-mediated detachment and usefor somatic cell cloning the cryopreserved permanent MSClines (between passages 1 and 2) that had been establishedfrom the primary cultures originating from bone marrowbiopsies of the prepubertal boars or gilts were thawed andcultured in vitro in DMEM medium enriched with relevantsupplementations The mitotic cycle of MSCs was synchro-nized at G1G0 stages through 24 to 48 h contact inhibition oftheir proliferative activity after reaching the total confluencestate under culture conditions in the medium enriched with10FBSDuring artificial synchronization of cell cycleMSCswere epigenetically transformed by 24 h exposure to 50 nMTSA
25 Preparation of Porcine Nuclear Recipient Oocytes forSomatic Cell Cloning Retrieval selection for extracorporealmeiotic maturation and in vitro culture of pig oocytes forthe purposes of SCNT were accomplished according to themethods applied by Samiec and Skrzyszowska [16] Samiecet al [22] and Skrzyszowska et al [20] Briefly slaughter-house ovaries were collected from both prepubertal femalepigs (gilts) and postpubertal female pigs (gilts and sows)Cumulus-oocyte complexes (COCs) were recovered by aspi-ration of follicular fluid from 2 to 6mm antral ovarian folli-cles The COCs with evenly granulated ooplasm and severaluniform layers of compact cumulus cells were selected for invitro maturation The maturation medium was comprised ofHEPES- and NaHCO
3-buffered Tissue Culture Medium 199
(TCM199) thatwas supplementedwith 10porcine follicularfluid (pFF) 10 FBS 5 ngmL recombinant human basicfibroblast growth factor (rh-bFGF) 10 ngmL recombinanthuman epidermal growth factor (rhEGF) 06mM L-cysteine1mMdibutyryl cyclic adenosinemonophosphate (db-cAMPbucladesine) 01 IUmL human menopausal gonadotropin(hMG) and 5mIUmL porcine follicle-stimulating hormone(pFSH) Approximately 50 to 60 COCs were cultured inthe db-cAMP- and hMG + pFSH-supplemented medium for20 h at 39∘C in a 100 water-saturated atmosphere of 5CO2and 95 air The oocytes were then cultured for 22 to
24 h in fresh maturation medium that did not contain db-cAMP hMG or pFSH After maturation expanded cumuluscells and corona cells were completely removed by vigorouspipetting of the COCs in the presence of 01 hyaluronidasefor 1 to 2min The metaphase II-stage oocytes which hadbeen selected on the basis of accepted morphological criteriainvolving evenly granulated dark ooplasm and the presenceof distinctly extruded first polar bodies provided a source ofhost cytoplasm for the cell nuclei of TSA-exposed or TSA-unexposed bone marrow-derived MSCs in the somatic cellcloning procedure
26 Production of Porcine Nuclear-Transferred Embryos UsingMSCs Modulated or Not Modulated Epigenomically by TSATreatment The approaches to somatic cell cloning and invitro culture of nuclear-transferred pig embryos that wereused in the current experiments were thoroughly presentedin our previous studies [15 16 18 59 60] Conciselyprior to the SCNT procedure cumulus-denuded in vitro
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matured giltsow oocytes were incubated in the matura-tion medium supplemented with 04 120583gmL demecolcine(DMCC) for 50 to 60min The DMCC-treated oocyteswere subsequently transferred into a micromanipulationchamber filled with TC 199 medium containing 4mgmLbovine serum albumin (fraction V BSA-V) and 75 120583gmLcytochalasin B (CB) Metaphase chromosomes which hadbeen allocated into the chemically induced protrusion ofthe plasma membrane were removed microsurgically Thechemically assisted enucleation was accomplished by gentlyaspirating the ooplasmic cone which contained the con-densed chromosome cluster with the aid of a bevelledmicropipette The reconstruction of enucleated oocytes wasachieved by their electrofusion with TSA-treated or TSA-untreated MSCs Single nuclear donor cells were insertedinto the perivitelline space of previously enucleated oocytes(ie ooplastscytoplasts) The resulting somatic cell-ooplastcouplets were placed into a fusionactivation chamber filledwith electroporation medium (dielectric solution) SCNT-derived oocytes were artificially stimulated using the protocolof simultaneous fusion and electrical activation (SF-EA) Inthe SF-EA protocol electric pulses that induced a fusion ofMSC-cytoplast couplets were simultaneously the stimuli ini-tiating the activation of reconstructed oocytesThe complexesof ooplasts and MSCs were subjected to plasma membraneelectroporation by application of two successive DC pulsesof 12 kVcm for 60120583s The electropermeabilization of cellplasma membranes was performed in an isotonic dielectricsolution (03M D-mannitol) with concentration of CaCl
2
increased up to 10mM Following SF-EA porcine clonedembryos were exposed to 5120583gmL CB for 2 h and subse-quently cultured in vitro in North Carolina State University-23 (NCSU-23) medium supplemented with 4mgmL BSA-V1 Eaglersquos Minimum Essential Medium-nonessential aminoacid solution (MEM-NEAA) and 2 MEM-essential aminoacid solution (MEM-EAA) for 72ndash96 h Afterwards cleavedembryos were cultured in NCSU-23BSA-V + MEM-NEAA+ MEM-EAA medium supplemented with 10 FBS for anadditional 72 h up to morula and blastocyst stages
27 Assessment of Cloned Blastocyst Quality by TUNELAssay The blastocysts developed from NT oocytes recon-structed with TSA-treated and -untreated MSCs wereanalyzed using a Deadend Fluorometric TUNEL System(Promega Warsaw Poland) according to the protocol ofterminal deoxynucleotidyl transferase- (TdT-) mediateddUTP (21015840-deoxyuridine-51015840-triphosphate) nick-end labelling(TUNEL) that was dependent on the fluorescein isothio-cyanate (FITC) conjugated with dUTP This protocol wasthoroughly described by Opiela et al [61 62] Briefly afterfixationwith 1paraformaldehyde (PFA) diluted inPBS solu-tion the embryos were rinsed extensively (3 times) Subse-quently they were exposed to 02 Triton X-100 solution for5min followed by 1 h incubation in the reactionmixture con-sisting of equilibration buffer a cocktail of nucleotides andTdT enzyme at a maximum humidity Finally the embryoswere placed into a 2x concentrated solution of salinesodiumchloride-sodiumtrisodium citrate (SSC) buffer for 15min
After all the incubations carried out cloned blastocystswere washed three times in PBSpolyvinylpyrrolidone (PVP)solution for at least 5min and lastly transferred into a dropof VECTASHIELD mounting medium This medium wassupplemented with 410158406-diamidino-2-phenylindole (DAPI)counterstain and has been found to be unsurpassed inpreventing photobleaching of DAPI fluorochrome in orderto retain antifading ability during long-term storage of dyedembryos The evaluation of embryos that had been subjectedto TUNEL analysis was performed under a fluorescencemicroscope (Nicon Eclipse E600 Tokyo Japan) The assess-ment of blastocyst cytological quality enabled visualizingthe total number of blastomere nuclei in each embryo andthe number of cell nuclei exhibiting internucleosomallyfragmented DNA into which a FITC-conjugated dUTP wasincorporated The fluorescence filters adjusted to excitationwavelength higher than 460 nm and the filters adapted toemission wavelengths ranging from 480 nm to 540 nm wereapplied to detect the blue fluorescence emitted by cell nucleiof all the DAPI-tagged blastocyst blastomeres and the FITC-derived green fluorescence emitted by cell nuclei of late-apoptotic blastomeres
28 Determination of Apoptotic Index (DCI) in Cloned Blasto-cysts All the blastocysts generated were subjected to TUNELanalysisThenumber of all DAPI-dyed blastomere nuclei (iethe total number of inner cell massICM and trophoblastcell nuclei) and the number of all TUNEL-positiveFITC-stained (ie apoptotic) cell nuclei were estimated for eachembryo To assess the apoptotic index (dead cell index DCI)in the single blastocyst the sum of all apoptotic cell nucleidetected in the analyzed blastocyst was divided by the sumof all ICMtrophoblast cell nuclei identified in this blastocystand subsequently multiplied by 100 [62]
29 Analysis of Porcine Cloned Embryos for Oct4 Nanog andNestin mRNA Expression Profile Using Quantitative ReverseTranscriptase Real-Time PCR (qRT-PCR) The qRT-PCR wasapplied to estimate the expression levels of mRNA tran-scripts for pluripotency-related target genes (Oct4 Nanog)and multipotent stemness-associated target gene (Nestin) intwo groups of SCNT-derived blastocysts encompassing theembryos developed from oocytes reconstructed with eitherTSA-treated or -untreated MSC nuclei Briefly 40 120583L ofSideStep Lysis amp Stabilization Buffer (Agilent Technologies)was added to 10 120583L of blastocysts sample (10 blastocysts)in PBS The SideStep Buffer was used to lyse the cells ofblastocysts and ensure RNA stabilization The lysed embryosamples were vortexed at room temperature for 1min Theone-step Brilliant II SYBR Green QRT-PCR Master MixKit (Agilent Technologies) was used to perform relativequantification of gene expression Each PCR probe (totalvolume of 25 120583L) was comprised of 1 120583L of embryo cell lysateand 24120583L of reaction mixture which consisted of 125 120583L 2xSYBRGreen QRT-PCRmaster mix (containing an optimizedRT-PCR buffer MgCl
2 nucleotides (GAUC) SureStart Taq
DNA polymerase daggerSYBR Green and stabilizers) and 04 120583Lof each 200 nM forward and reverse primer (Table 1) 1 120583L
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Table 1 Primers used in quantitative reverse transcriptase real-time PCR (qRT-PCR)
of RTRNase block enzyme mixture and 97 120583L RNase-freewater Thermal cycling conditions were as follows 30min at50∘C (for the first-strand synthesis) 10min at 95∘C 40 cyclesof 30 s at 95∘C for denaturing 60 s at 57∘C for annealing and30 s at 72∘C for extension
The glyceraldehyde-3-phosphate dehydrogenase(GAPDH) housekeeping gene was used as an endogenousstandard The results for expression levelsprofiles of indi-vidual target genes (Oct4 Nanog and Nestin) were normal-ized according to the relative concentration of the endog-enous standard Each reaction was run in triplicate and theobtained results were averaged By sample we mean mRNAderived from a pool of 10 blastocysts We made six PCR runsfrom one mRNA sample 3 for three analyzed genes (Oct4Nanog and Nestin) and 3 for the GAPDH control gene EachcDNA synthesis and PCR amplification were achieved inone tube and one buffer All data are presented as the meanvalues of the relative abundance (RA) of Oct4 Nanog andNestin mRNA transcripts Expression of these transcriptsisolated from blastocysts that originated from TSA-treatedand -untreated groups was compared to that for controlblastocysts which served as a calibrator Experiments werecarried out on a Mastercycler Realplex (Eppendorf UK Lim-ited Cambridge)
210 Statistical Analysis The 1205942 test was applied to esti-mate the differences in the in vitro developmental capa-bilities of porcine NT embryos originating from TSA-treated and -untreated MSCs The statistical analysis of vari-ance (ANOVA) and Tukeyrsquos Honestly Significant Difference(HSD) post hoc test for multiple ranges were used for com-parison of the levels of Oct4 Nanog and Nestin transcriptsbetween blastocysts derived from oocytes reconstituted withthe cell nuclei of TSA-exposed and -unexposed MSCs TheANOVA and subsequent Tukeyrsquos HSD post hoc test werealso applied to compare the mean values calculated fortotal number of DAPI-labelled cell nuclei total number ofTUNEL-positiveFITC-labelled (ie apoptotic) cell nucleiand apoptotic index (DCI) between blastocysts developedfrom NT oocytes generated using epigenetically modulatedand nonmodulated MSCs The differences with a probability(P) less than 005 were considered to be significant
3 Results
31 The In Vitro Developmental Potential of Cloned EmbryosDerived from the Cell Nuclei of MSCs Undergoing or NotUndergoing TSA-Based Epigenomic Modulation Althoughthe cleavage activity did not vary between cultured nuclear-transferred embryos reconstituted fromMSCs subjected andnot subjected to TSA-dependent epigenetic transformation(119875 ge 005) the morula and blastocyst formation rates werecharacterized by statistically significant differences A signif-icant variability in the in vitro developmental competencesto reach the morula stage (119875 lt 005) and a very highlysignificant variability in the developmental competences toreach the blastocyst stage (119875 lt 0001 Figure 1) were observedbetween these two groups of cloned embryos respectivelyThe detailed data that are focused on the assessment ofextracorporeal developmental capability of NT embryosreconstituted with epigenetically nonmodulated or modu-lated MSCs are presented in Table 2 As compared to thepresent investigation in our previous studies in which the invitro developmental potential of cloned pig embryos derivedfrom the cell nuclei of fetal fibroblast cells (being currentlyused source of nuclear donor cells) and generated applyingthe same experimental protocols of SF-EA for artificialstimulation of nuclear-transferred oocytes was explored thepercentages of the obtained morulae and blastocysts rangedfrom 582 to 613 and from 296 to 332 respectively[16 18 20] It is worth noting that all these percentagesturned out to be considerably lower than themorula and blas-tocyst formation rates achieved for NT embryos producedusing epigenomically nontransformed or transformed MSCs(Table 2)
32 Dependence of the Cloned Blastocyst Quality Evaluated byTUNEL Assay on the TSA-Based Epigenomic Modulation ofMSCs The quality of nuclear-transferred embryos derivedfrom TSA-exposed MSCs was considerably higher than thatof NT embryos derived from TSA-unexposed MSCs Veryhighly significant differences in the mean number of DAPI-stained cell nuclei per blastocyst were shown between thesetwo groups of embryos (119875 lt 0005 Table 3 Figure 2) Inturn no significant differences in the mean number of
6 BioMed Research International
005mm
(a)
005mm
(b)
005mm
(c)
005mm
(d)
Figure 1 Porcine cloned blastocysts developed fromnuclear-transferred oocytes reconstitutedwith adult bonemarrow-derivedmesenchymalstem cells undergoing trichostatinA- (TSA-) dependent epigenomicmodulation (photographs (a) and (b)) or not undergoingTSA-dependentepigenomic modulation (photographs (c) and (d)) Images were taken at magnification times200
Table 2 Effect of the TSA-dependent epigenomic modulation of MSCs on the in vitro developmental outcome of cloned pig embryos
TSA-mediated epigenetictransformation of MSCs
Number of oocytesembryos Development toEnucleated Electrofused () Cleaved () Morulae () Blastocysts ()
minus 293 275293 (939) 262275 (953) 216275 (785)b 125275 (455)B
TSA trichostatin A MSC mesenchymal stem cellValues with different small superscript letters (a and b) within the same column denote statistically significant differences between experimental groups (119875 lt005 1205942 test) Values with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0001 1205942 test)Number of replicates ge6
Table 3 Effect of the TSA-mediated epigenomic modulation of MSCs on the cytological quality of cloned pig embryos assessed by TUNELassay
TSA-mediated epigenetictransformation of MSCs
Number ofanalyzedblastocysts
Mean number ofDAPI-tagged cell nuclei per
blastocyst plusmn SD
Mean number ofTUNEL-positive (apoptotic) cell
nuclei per blastocyst plusmn SD
Mean apoptotic index(DCI) per blastocyst plusmn
SD ()+ 31 4797 plusmn 2743A 161 plusmn 338 495 plusmn 1398minus 26 2777 plusmn 1582B 158 plusmn 269 713 plusmn 1244TSA trichostatin A MSC mesenchymal stem cell TUNEL terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling DAPI 410158406-diamidino-2-phenylindole SD standard deviation DCI dead cell indexValues with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0005 ANOVA followed byTukeyrsquos HSD post hoc test) Number of replicates = 6
TUNEL-positive (ie late-apoptotic) cell nuclei per blasto-cyst were identified between NT embryos reconstituted withMSCs undergoing and not undergoing TSA-mediated epige-netic transformation (119875 ge 005 Table 3 Figure 2) Althoughno significant differences in the DCI per blastocyst were
found between both experimental groups (119875 ge 005) theincidence of late apoptosis-related internucleosomal DNAfragmentation trended slightly upwards for NT embryosgenerated using TSA-untreated MSCs as compared to thoseproduced using TSA-treated MSCs (Table 3 Figure 2)
BioMed Research International 7
005mm
(a)
005mm
(b)
005mm
(c)
005mm
(d)
005mm
(e)
005mm
(f)
Figure 2 Evaluation of cytological quality of porcine cloned blastocysts on the basis of simultaneous determination of total nuclear numberand detection of apoptotic cell nuclei by terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling (TUNEL) analysis Photographs (a) to (d) depict embryos originating from nuclear-transferred (NT) oocytes reconstitutedwith adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) subjected to epigenetic transformation via trichostatin A (TSA)treatment Photographs (e) and (f) depict embryos originating from NT oocytes reconstituted with ABM-MSCs not subjected to epigenetictransformation via TSA treatment In each blastocyst the cell nuclei of all the blastomeres (both inner cell mass (ICM) and trophectoderm(TE) cells) had been tagged with 410158406-diamidino-2-phenylindole (DAPI) counterstain and subsequently fluoresced in blue The cell nucleiof late-apoptotic blastomeres (ICM andor TE cells) exhibiting internucleosomal DNA fragmentation had been dyed with fluoresceinisothiocyanate (FITC) and then fluoresced in bright green In each photograph the DAPI-derived blue and FITC-derived green fluorescentsignals merge into one another Photographs (a) to (f) represent the blastocysts displaying different incidence of blastomere apoptosisand thereby varied advancement of internucleosomal DNA fragmentation ((a) (c) (d) and (e) the lack of apoptotic intranuclear DNAfragmentation (b) and (e) few apoptotic cell nuclei (f) increased extent of apoptotic intranuclear DNA fragmentation) Images were takenat magnification times200
8 BioMed Research International
0
1
2
3
4
5
6
B
A
Oct4 Nanog Nestin
MSCTSAMSC
Figure 3 Relative abundance (mean plusmn SD) of Oct4 Nanog andNestin mRNAs in blastocysts developed from nuclear-transferredpig embryos descended from MSCs undergoing or not undergoingTSA-dependent epigenomic transformation The highly significantdifferences were designated with the A and B letters (119875 lt 001ANOVA followed by Tukeyrsquos HSD post hoc test)
33 The Pluripotency Status of Porcine Nuclear-TransferredEmbryos Originating from Epigenomically Modulated or Non-modulated MSCs The total number of analyzed clonedblastocysts was 60 To assess relative abundance (RA) of threeanalyzed mRNA transcripts for pluripotency-related targetgenes (Oct4 Nanog) multipotent stemness-associated targetgene (Nestin) and control GAPDH gene transcript we used3 times 10 blastocysts from both TSA-treated and -untreatedgroups
Highly significant differences were indicated for theOct4gene transcript quantitative profile between blastocysts orig-inating from TSA-treated and -untreated groups (119875 lt 001Figure 3) Although no significant intergroup variability wasshown in the RA of Nanog and Nestin mRNAs (119875 ge 005)their expression levels tended to be higher in blastocystsderived from NT embryos reconstructed with TSA-exposedMSCs as compared to the TSA-untreated group (Figure 3)
4 Discussion
The abundance of the morulae and blastocysts of highercytological and molecular quality generated in the presentinvestigation confirms that the reprogrammability of adultbone marrow-derived mesenchymal stem cells which hadbeen epigenetically modified via exposure to trichostatinA underwent considerable improvement in a cytoplasm ofporcine nuclear-transferred oocytes and resultant in vitrocultured cloned embryos It is beyond any doubt thatthe impact of TSA-based epigenomic transformation ofbone marrow-retrieved MSCs representing multipotent andundifferentiated stem cells on their competence for SCNThas not yet been explored not only in pigs but also inother mammalian species So far TSA-mediated epigeneticmodulation has been applied only for differentiated somatic
cells that commonly provide the source of nuclear donorsfor cloning procedure in pigs The results of the study byDiao et al [55] proved that porcineNT embryos reconstitutedwith TSA-treated fetal fibroblast cells were characterized bytwofold higher developmental potential to reach the blasto-cyst stage (30) than the NT embryos reconstituted withTSA-untreated fibroblast cells (15) Similar tendency hasbeen also shown in our current study inwhich trichostatinA-mediated epigenetic transformation of bone marrow-derivedMSCs gave rise to significant enhancement of capability ofcloned embryos to complete their development to blastocyststage (65) as compared to that observed for embryosderived from TSA-unexposed MSC nuclei (46) Nonethe-less NT pig embryos originating from epigenomically trans-formed fetal fibroblast cells displayed considerably lowerblastocyst formation rate [55] than the rate indicated in ourpresent study for NT embryos reconstructed with epige-nomically nontransformed MSCs Moreover treatment ofnuclear donor MSCs with trichostatin A resulted in one anda half times to severalfold increase of blastocyst percentage(65) as compared to the blastocyst yields obtained by otherinvestigators using undifferentiated or in vitro differentiatedMSCs that were not modulated epigenetically [26ndash28 31 32]In the study by Kumar et al [28] the abilities of epige-nomically nontransformed bone marrow-descended MSCnuclei to direct the in vitro development of porcine clonedembryos to blastocyst stage decreased above three times(approximately 20) as compared to our present work Inturn nuclear-transferred embryos that had originated fromporcine undifferentiated bone marrow-retrieved MSCs andtheir derivatives along the osteogenic lineage were able tocomplete their in vitro development to blastocyst stage atthe rates ranging from 33 to 45 [31] In contrast Jin etal [32] and Li et al [27] reported that blastocyst yields ofNT embryos derived from porcine epigenetically unmodu-lated MSCs were maintained at the relatively low levels ofapproximately 18 and 16 respectively Nevertheless in thestudy by Lee et al [26] the in vitro developmental outcometo the blastocyst stage of NT embryos originating fromundifferentiated MSCs achieved the rate of approximately48 but even this blastocyst formation rate was considerablylower than that noticed in our current investigation
The results of the current study have also clearly demon-strated that the TSA-dependent epigenomic transformationof nuclear donor MSCs contributes to improvement of notonly in vitro developmental competences but also qualityand transcription level-related pluripotency extent of porcinecloned embryos The utilization of undifferentiated mes-enchymal stem cells and the modification of the molecularmechanisms of transcriptional reprogramming of the donornuclear genome via HDAC inhibitor-mediated epigeneticmodulation of MSCs before their use for SCNT led to correctand complete adaptation of MSC-inherited genomic DNA tothe cytoplasmic environment of enucleated oocytes and invitro cultured NT pig embryos Epigenetic reprogrammingof donor cell nuclei suggests that a new program for theirtranscriptional activity is loaded and reloaded immediatelyfollowing reconstruction of enucleated oocytes The successof SCNT may depend upon both genomic DNA-associated
BioMed Research International 9
reprogramming of gene expression for dedifferentiation ofthe donor somatic cell nuclei during early preimplantationdevelopment of cloned embryos and reprogramming of geneexpression for onset of somatogenic nuclear redifferentiationduring blastocyst formation [7 10 63 64] It has been ascer-tained that somatic cell nuclei should undergo the wide DNAcytosine residue demethylation changes throughout the earlydevelopment of NT embryos to erase and then reset theirown overall epigenetic as well as parental genomic imprintingmemory which has been established by remethylation ofthe nuclear genome within the framework of the specificpathway of somatic and germ cell lineage commitment anddifferentiation [2 4 13 30 65ndash67]
It appears that the enhanced cytological quality of clonedpig blastocysts that originated from enucleated oocytesreceiving epigenetically modulated MSC nuclei could beassociated with more faithful and faultless pattern for repro-gramming of transcriptional activity of genomic DNA inher-ited from undifferentiated and multipotent stem cells Thesecells exhibit increased genomic and epigenomic plasticity inrearrangements of their gene expression in the blastomeres ofpreimplanted NT embryos The fluorocytochemical analysisof overall mean nuclear counts revealed almost twofoldhigher total cells number in porcine blastocysts developedfrom NT oocytes reconstituted with TSA-exposed MSCs (48blastomeres) than that identified in blastocysts derived fromNT oocytes reconstituted with TSA-unexposed MSCs (28blastomeres) The quality of cloned blastocysts generated inour present study using epigenomically transformed MSCswas comparable to that noticed in the study by Lee et al[26] for porcine NT blastocysts derived from epigenomicallynontransformed MSCs These blastocysts were also charac-terized by a total cells number equal to 48 In contrast to theabovementioned findings in the investigations carried outby Li et al [27] Kumar et al [28] and Jin et al [32] theoverall number of ICM and trophectoderm cells in clonedpig blastocysts originating from embryos reconstituted withTSA-untreated MSCs ranged from 28 to 35 and decreasedapproximately one and a half times as compared to the totalcells number estimated for NT blastocysts obtained by usin the TSA-exposed MSC group Nevertheless the meanapoptotic index (ie DCI = 495) that was calculated by usfor blastocysts developed from cloned embryos reconstitutedwith epigenetically modified MSC nuclei was similar to theratios of TUNEL-positive cells (46 to 47) observed by Jinet al [32] andKumar et al [28] among porcineNT blastocystsderived from epigenetically nonmodulated counterparts Inturn cloned blastocysts produced in our study using TSA-untreated MSCs displayed the apoptotic index (713) thatwas comparable or slightly lower to indexes identified forcloned embryos created with the aid of fetal fibroblast cellsnot undergoing exposure to trichostatin A [28 32]The latterindexes (DCIs) oscillated between 73 and 78 Generally inour current work the proportion of TUNEL-positive cellsin relation to a total cells number tended to insignificantlydecrease among NT blastocysts generated using undifferen-tiated mesenchymal stem cells subjected to TSA treatmentcompared to those generated using nuclear donor cells not
exposed to TSA (495 versus 713) Analogous slight down-ward tendency in the ratios of TUNEL-positive cells wasindicated byDiao et al [55] comparing blastocysts developedfrom cloned embryos reconstructed with cell nuclei of fetalfibroblasts treated with TSA and the counterparts producedusing TSA-unexposed fetal fibroblast cell nuclei (nearly 3versus 45)
Improved reprogrammability of transcriptional activityfor nuclear genome of epigenetically modulated MSCs inthe cells of preimplanted NT embryos turned out to bepositively correlated with enhanced molecular quality ofporcine cloned blastocysts assessed on the basis of theirpluripotency extent whichwasmeasuredwith the expressionprofiles identified for Oct4 and Nanog genes A 38-kDaprotein Oct4 (ie octamer-binding transcription factor 4)that is a member of the family of POU- (Pit-Oct-Unc-)domain and homeodomain transcription factors acts as avital regulator of pluripotency extent playing an importantrole in not only controlling preimplantation embryonicdevelopment but also maintenance of ICM cell fate inblastocysts and pluripotency status of embryonic stem cells(ESCs) [68ndash70] A 35-kDa protein designated as Nanog fromCelticIrish mythical Tır na nOg (Tir Na Nog The Landof the Ever-Young) is another homeobox-containing tran-scription factor that represents the group of pivotal proteinsmodulating pluripotency degree [70 71] The homeoproteinNanog can act synergistically with Oct4 protein in retainingthe pluripotent status of blastocyst-descended ICM andepiblast cells as well as in sustaining the undifferentiatedstatus and ability for self-renewal of ESCs [71 72] Theaberrant (ie downregulated) expression levels and patternsof Oct4 and Nanog transcripts in both bovine NT blastocystsproduced using epigenetically nonmodulated calf dermalfibroblast cells [73] and porcine NT blastocysts producedusing either epigenetically nonmodulated adult MSCs orfetal fibroblast cells [28] have been found to be a majorcause of not only their declined cytological and molecularquality but also decreased in vitro developmental potentialof cloned embryos However in our current study it seemsthat TSA-dependent epigenomic transformation of nucleardonor MSCs biases correctingly the expression profile ofOct4 andNanog mRNAs in cloned pig blastocysts triggeringboth significant enhancement of relative abundance (RA)of Oct4 transcripts and slight (nonsignificant) increase inNanog transcript RA as compared to the TSA-untreatedMSCgroup On the one hand this influence could be exerted bydirect diminishment in the deacetylation level within thenucleosomal core-derived histone lysine moieties that wasevoked by trichostatin A-mediated nonspecific inhibition ofHDACs On the other hand it could be probably elicitedvia indirect nonselective suppression of cytosine residuemethylation processes within the Oct4 and Nanog gene pro-moters andor enhancers leading to upregulated expressionof these crucial pluripotency-related genes Similar resultshave been shown in the study by Wang et al [73] in whichthe sequential treatment of bovine differentiated nucleardonor fibroblast cells and resultant cloned embryos by bothnonspecific inhibitors of DNMTs and HDACs (ie 5-aza-dC
10 BioMed Research International
and TSA resp) contributed to increase of Oct4 transcriptRA in the blastocysts obtained simultaneously sustainingthe expression of Nanog mRNAs on the unchanged levelas compared to the 5-aza-dC- and TSA-unexposed groupIn this case 5-aza-21015840-deoxycytidine- and trichostatin A-mediated epigenetic modulation of both nuclear donor cellsand cloned embryos gave rise presumably to direct down-regulation in the methylation of cytosine residues withinthe Oct4 gene promoter andor enhancer that resultedfrom 5-aza-dC-dependent nonspecific inhibition of DNMTsFurthermore this two-factor epigenetic modulation couldaffect the enhanced incidence of acetylation level within thenucleosomal core-derived histone lysine moieties that wastriggered by trichostatin A-mediated nonselective inhibitionof HDACs consequently leading to indirect upregulatedexpression of the Oct4 gene Taking into consideration allthe abovementioned findings considerable transcriptionalupregulation in the expression of Oct4 gene (confirmedamong porcine NT blastocysts in our present study andamong bovineNT blastocysts in the study byWang et al [73])can be found to be pivotal indicator of increased pluripotencyextent of cloned embryos This process appears to be alsoassociated with improved cytological quality of NT blasto-cysts that was measured with their total cell counts In thecurrent investigation we have shown that the overall numberof ICM and trophoblast cells in blastocysts was significantlyhigher following the reconstruction of NT pig embryos withepigenomically transformed mesenchymal stem cells
5 Conclusions and Future Goals
Summing up artificial epigenomic modulation of in vitrocultured MSCs using the nonspecific HDAC inhibitor desig-nated as TSA seems to facilitate much more the reprogram-ming process for epigenetically determined transcriptionalactivity of somatic cell-inherited nuclear genome in theNT pig embryos The use of ectopic HDAC inhibitors forepigenetic transformation of mesenchymal stem cells whosecell nuclei were transferred into enucleated oocytes is thecompletely new approach in the studies involving somaticcell cloning of pigs and other mammalian species Enhancedreprogrammability of nuclear genome descended from TSA-exposed MSCs in the blastomeres of porcine preimplantedcloned embryos resulted in the improvements of not onlytheir capacity to complete in vitro development to themorula and blastocyst stages but also cytological and molec-ular quality of the blastocysts produced Therefore furtherinvestigations are also necessary to determine whether thenovel strategy of TSA-dependent epigenetic modification ofnuclear donor MSCs which has been recently utilized in ourlaboratory for the future goals of generating cloned pigletsenables retaining the in vivo developmental competences ofthe high-quality NT-derived blastocysts to reach full term
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Marcin Samiec and Jolanta Opiela contributed equally to thepreparation of this paper
Acknowledgments
The project was funded by the Polish National ScienceCentre resources allocated on the basis of Decision no DEC-201103DNZ905537 The authors would like to thank DrJarosław Wieczorek DVM for bone marrow retrieval
References
[1] A J Bonk H-T Cheong R Li et al ldquoCorrelation of develop-mental differences of nuclear transfer embryos cells to themethylation profiles of nuclear transfer donor cells in swinerdquoEpigenetics vol 2 no 3 pp 179ndash186 2007
[2] A J Bonk R Li L Lai et al ldquoAberrant DNA methylation inporcine in vitro- parthenogenetic- and somatic cell nucleartransfer-produced blastocystsrdquo Molecular Reproduction andDevelopment vol 75 no 2 pp 250ndash264 2008
[3] M Samiec ldquoDevelopment of pig cloning studies past presentand futurerdquo Journal of Animal and Feed Sciences vol 13 no 2pp 211ndash238 2004
[4] K MWhitworth and R S Prather ldquoSomatic cell nuclear trans-fer efficiency how can it be improved through nuclear remod-eling and reprogrammingrdquoMolecular Reproduction and Devel-opment vol 77 no 12 pp 1001ndash1015 2010
[5] K-I Yamanaka S Sugimura T Wakai M Kawahara and ESato ldquoAcetylation level of histone H3 in early embryonic stagesaffects subsequent development of miniature pig somatic cellnuclear transfer embryosrdquo The Journal of Reproduction andDevelopment vol 55 no 6 pp 638ndash644 2009
[6] R P Cervera N Martı-Gutierrez E Escorihuela R Morenoand M Stojkovic ldquoTrichostatin A affects histone acetylationand gene expression in porcine somatic cell nucleus transferembryosrdquoTheriogenology vol 72 no 8 pp 1097ndash1110 2009
[7] J Zhao J Whyte and R S Prather ldquoEffect of epigeneticregulation during swine embryogenesis and on cloning bynuclear transferrdquo Cell and Tissue Research vol 341 no 1 pp13ndash21 2010
[8] WReik ldquoStability and flexibility of epigenetic gene regulation inmammalian developmentrdquo Nature vol 447 no 7143 pp 425ndash432 2007
[9] R S Prather J W Ross S C Isom and J A Green ldquoTranscrip-tional post-transcriptional and epigenetic control of porcineoocyte maturation and embryogenesisrdquo Society of Reproductionand Fertility supplement vol 66 pp 165ndash176 2009
[10] N Rodriguez-Osorio R Urrego J B Cibelli K Eilertsen andE Memili ldquoReprogramming mammalian somatic cellsrdquo Theri-ogenology vol 78 no 9 pp 1869ndash1886 2012
[11] K J Eilertsen R A Power L L Harkins and P Misica ldquoTar-geting cellular memory to reprogram the epigenome restorepotential and improve somatic cell nuclear transferrdquo AnimalReproduction Science vol 98 no 1-2 pp 129ndash146 2007
[12] M Samiec and M Skrzyszowska ldquoMolecular conditions of thecell nucleus remodellingreprogramming process and nucleartransferred embryo development in the intraooplasmic kary-oplast injection technique a reviewrdquo Czech Journal of AnimalScience vol 50 no 5 pp 185ndash195 2005
BioMed Research International 11
[13] P Narbonne K Miyamoto and J B Gurdon ldquoReprogrammingand development in nuclear transfer embryos and in interspe-cific systemsrdquo Current Opinion in Genetics amp Development vol22 no 5 pp 450ndash458 2012
[14] J-I Bang J-G Yoo M-R Park et al ldquoThe effects of artificialactivation timing on the development of SCNT-derivedembryos and newborn pigletsrdquoReproductive Biology vol 13 no2 pp 127ndash132 2013
[15] M Samiec and M Skrzyszowska ldquoHigh developmental capa-bility of porcine cloned embryos following trichostatin A-dependent epigenomic transformation during in vitro matura-tion of oocytes pre-exposed to R-roscovitinerdquo Animal SciencePapers and Reports vol 30 no 4 pp 383ndash393 2012
[16] M Samiec and M Skrzyszowska ldquoRoscovitine is a novel agentthat can be used for the activation of porcine oocytes recon-structed with adult cutaneous or fetal fibroblast cell nucleirdquoTheriogenology vol 78 no 8 pp 1855ndash1867 2012
[17] Z Li J Shi D Liu et al ldquoEffects of donor fibroblast cell typeand transferred cloned embryo number on the efficiency of pigcloningrdquo Cellular Reprogramming vol 15 no 1 pp 35ndash42 2013
[18] M Samiec and M Skrzyszowska ldquoBiological transcomple-mentary activation as a novel and effective strategy appliedto the generation of porcine somatic cell cloned embryosrdquoReproductive Biology vol 14 no 2 pp 128ndash139 2014
[19] J Li O Svarcova K Villemoes et al ldquoHigh in vitro developmentafter somatic cell nuclear transfer and trichostatin A treatmentof reconstructed porcine embryosrdquoTheriogenology vol 70 no5 pp 800ndash808 2008
[20] M Skrzyszowska M Samiec R Słomski D Lipinski and EMały ldquoDevelopment of porcine transgenic nuclear-transferredembryos derived from fibroblast cells transfected by the noveltechnique of nucleofection or standard lipofectionrdquo Theri-ogenology vol 70 no 2 pp 248ndash259 2008
[21] Y-W Pang L An P Wang et al ldquoTreatment of porcine donorcells and reconstructed embryoswith the antioxidantmelatoninenhances cloning efficiencyrdquo Journal of Pineal Research vol 54no 4 pp 389ndash397 2013
[22] M Samiec M Skrzyszowska and J Opiela ldquoCreation of clonedpig embryos using contact-inhibited or serum-starved fibrob-last cells analysed intra vitam for apoptosis occurrencerdquo Annalsof Animal Science vol 13 no 2 pp 275ndash293 2013
[23] H Wei Y Qing W Pan et al ldquoComparison of the efficiency ofBannaminiature inbred pig somatic cell nuclear transfer amongdifferent donor cellsrdquo PLoSONE vol 8 no 2 Article ID e577282013
[24] K Inoue N Ogonuki H Miki et al ldquoInefficient reprogram-ming of the hematopoietic stem cell genome following nucleartransferrdquo Journal of Cell Science vol 119 no 10 pp 1985ndash19912006
[25] K Inoue S Noda N Ogonuki et al ldquoDifferential developmen-tal ability of embryos cloned from tissue-specific stem cellsrdquoStem Cells vol 25 no 5 pp 1279ndash1285 2007
[26] S L Lee E J Kang G H Maeng et al ldquoDevelopmental abilityof miniature pig embryos cloned withmesenchymal stem cellsrdquoThe Journal of Reproduction and Development vol 56 no 2 pp256ndash262 2010
[27] Z Li X He L Chen et al ldquoBone marrow mesenchymal stemcells are an attractive donor cell type for production of clonedpigs as well as genetically modified cloned pigs by somatic cellnuclear transferrdquo Cellular Reprogramming vol 15 no 5 pp459ndash470 2013
[28] B M Kumar H-F Jin J-G Kim et al ldquoDifferential geneexpression patterns in porcine nuclear transfer embryos recon-structed with fetal fibroblasts and mesenchymal stem cellsrdquoDevelopmental Dynamics vol 236 no 2 pp 435ndash446 2007
[29] J Su Y Wang Y Li et al ldquoOxamflatin significantly improvesnuclear reprogramming blastocyst quality and in vitro devel-opment of bovine SCNT embryosrdquo PLoS ONE vol 6 no 8Article ID e23805 2011
[30] Y Song THai YWang et al ldquoEpigenetic reprogramming geneexpression and in vitro development of porcine SCNT embryosare significantly improved by a histone deacetylase inhibitor-m-carboxycinnamic acid bishydroxamide (CBHA)rdquo Protein ampCell vol 5 no 5 pp 382ndash393 2014
[31] S Colleoni G Donofrio I Lagutina R Duchi C Galli andG Lazzari ldquoEstablishment differentiation electroporationviral transduction and nuclear transfer of bovine and porcinemesenchymal stem cellsrdquo Cloning and Stem Cells vol 7 no 3pp 154ndash166 2005
[32] H F Jin B M Kumar J G Kim et al ldquoEnhanced developmentof porcine embryos cloned from bone marrow mesenchymalstem cellsrdquoThe International Journal of Developmental Biologyvol 51 no 1 pp 85ndash90 2007
[33] J Opiela and M Samiec ldquoCharacterization of mesenchymalstem cells and their application in experimental embryologyrdquoPolish Journal of Veterinary Sciences vol 16 no 3 pp 593ndash5992013
[34] X Yang S L Smith X C Tian H A Lewin J-P Renard and TWakayama ldquoNuclear reprogramming of cloned embryos and itsimplications for therapeutic cloningrdquo Nature Genetics vol 39no 3 pp 295ndash302 2007
[35] XWu Y Li G-P Li et al ldquoTrichostatin A improved epigeneticmodifications of transfected cells but did not improve subse-quent cloned embryo developmentrdquo Animal Biotechnology vol19 no 4 pp 211ndash224 2008
[36] F Bo L Di F Qing-Chang et al ldquoEffect of trichostatin A ontransfected donor cells and subsequent development of porcinecloned embryosrdquo Zygote vol 19 no 3 pp 237ndash243 2011
[37] X Ding Y Wang D Zhang Z Guo and Y Zhang ldquoIncreasedpre-implantation development of cloned bovine embryostreated with 5-aza-21015840-deoxycytidine and trichostatin ArdquoTheri-ogenology vol 70 no 4 pp 622ndash630 2008
[38] H S Lee X F Yu J I Bang et al ldquoEnhanced histone acetylationin somatic cells induced by a histone deacetylase inhibitorimproved inter-generic cloned leopard cat blastocystsrdquo Theri-ogenology vol 74 no 8 pp 1439ndash1449 2010
[39] S-F Ning Q-Y Li M-M Liang et al ldquoMethylation charac-teristics and developmental potential of Guangxi Bamaminipig(Sus scrofa domestica) cloned embryos from donor cells treatedwith trichostatin A and 5-aza-21015840-deoxycytidinerdquo Zygote vol 21no 2 pp 178ndash186 2013
[40] B P Enright L-Y Sung C-C Chang X Yang and X C TianldquoMethylation and acetylation characteristics of cloned bovineembryos from donor cells treated with 5-aza-21015840-deoxycytidinerdquoBiology of Reproduction vol 72 no 4 pp 944ndash948 2005
[41] Y J Huan J Zhu B T Xie et al ldquoTreating cloned embryos butnot donor cells with 5-aza-21015840-deoxycytidine enhances thedevelopmental competence of porcine cloned embryosrdquo TheJournal of Reproduction andDevelopment vol 59 no 5 pp 442ndash449 2013
[42] B-G JeonG Coppola S D Perrault G-J RhoDH Betts andWAKing ldquoS-adenosylhomocysteine treatment of adult female
12 BioMed Research International
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
to result from aberrant gene dedifferentiation of somatic cellnuclei at the levels of epigenomic genomic and molecularmemory The mechanism underlying the dedifferentiationprocess of donor nuclei is the cessation of their owngene expression and reversal of the differentiated (special-ized) somatic nucleus to a totipotentpluripotent embryonic(undifferentiatedunspecialized) state within the host oop-lasm and cytoplasm of cleavage descendant blastomeres ofNT embryos [8ndash10] In turn impaired restoration (reestab-lishment) of the totipotencypluripotency of embryonic celllines in the first phase of epigenetic reprogramming (iegene dedifferentiation) during preimplantation developmentby the blastocyst stage may trigger disadvantageous alter-ations in the second phase of donor nuclear reprogram-ming These are connected with improper redifferentiationof somatic cell-inherited genes throughout postimplantationfetalplacental development Additional work is needed todetermine whether failures in the early-stage reprogrammingare magnified downstream in development [11ndash13]
In vitro cultured fibroblast cells which had been derivedfrom the dermointegumentary tissue of fetuses and adultspecimens are the commonly used source of nuclear donorcells in the pig cloning procedure [14ndash18] The degree ofmolecular and epigenetic differentiation of these cells thatis related both to the advanced methylation profile of DNAcytosine residues and to the lysine deacetylation profile ofhistones forming nucleosomal core of nuclear chromatinoften seems to make the converting of the abovementionedcovalentmodifications back to a totipotent state of embryonic(zygotic) cells impossibleThis leadsmainly to decrease in theabilities of differentiated fibroblast cells for supporting the invitro development of cloned embryos to the blastocyst stage[1 19] As a rule the percentage of blastocysts originatingfrom the porcine oocytes reconstructed with fetal or adultcutaneous fibroblast cell nuclei oscillates from 10 to 30[20ndash23]
It is hypothesized that the use of undifferentiated mes-enchymal stem cells (MSCs) isolated from adult bone mar-row which are characterized by the high multipotencylevel and genomicepigenomic plasticity allows increasingthe preimplantation developmental potential of mammaliancloned embryos As compared to hematopoietic stem cellsthe MSCs exhibit the lower activity of histone deacetylases(HDACs) and DNA methyltransferases (DNMTs) whichresults in hyperacetylation of histone lysine moieties anddemethylation of DNA cytosine residues [24ndash27] The lasttwo processes affect the inhibition of transcriptional suppres-sion of many regions in the nuclear genome of multipotentMSCs All these properties are responsible for the highsusceptibility of the MSC nuclei to correct and completeepigenetic reprogramming in the cytoplasm of NT embryoblastomeres Therefore the genome of undifferentiated stemcells may be more easily reprogrammed to resemble thegenome of the zygote which may make stem cells moreefficient as nuclear donors in the somatic cell cloning [28ndash30] Generally porcine NT embryos reconstituted with adultbone marrow-derived mesenchymal stem cells displayedconsiderably higher blastocyst formation rates than those
reconstitutedwith adult cutaneous or fetal fibroblast cells [31ndash33]
Transcriptional activity of somatogenic nuclear genomeduring embryo pre- andor postimplantation development aswell as fetogenesis is correlated with the frequencies forspatial remodeling of chromatin architecture and reprogram-ming of cellular epigenetic memory This former andthis latter process include such covalent modifications asdemethylationde novo methylation of DNA cytosine res-idues and acetylationdeacetylation as well as demethylationremethylation of lysine residues of nucleosomal core-derivedH3 and H4 histones [2 30 34 35] The level of progressionfor the processes of epigenetic genome-wide alterationsthat are mediated by DNA methyltransferases (DNMTs 1oand 3a3b) and histone deacetylases (HDACs) can be mod-ulated (ie reversed) via exogenous inhibitors of theseenzymes throughout in vitro culture of nuclear donor cellsandor cloned embryos [19 36ndash39] Moreover the use ofthe artificial modifiers of epigenomically conditioned geneexpression leads to the inhibition of both chromatin con-densation and transcriptional silencing of the genomic DNAof cultured somatic cells that are applied as a source ofdonor nuclei for the reconstruction of enucleated oocytesand subsequent generation of cloned embryos The membersof these epigenetic modifiers are 5-aza-21015840-deoxycytidine (5-aza-dC) [37 39ndash41] S-adenosylhomocysteine (SAH) [42]trichostatin A (TSA) [6 19 36 38 43] valproic acidsodiumvalproate (VPASV) [44 45] 6-(13-dioxo-1H3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide called scrip-taid [46ndash49] sodium butyrate (NaBu) [50 51] m-carboxy-cinnamic acid bishydroxamide (CBHA) [30 52] and oxam-flatin [29 53] The onset of chromatin decondensationand gene transcriptional activity is evoked by nonspecificnonselective blocking of the activity of either DNMTs by5-aza-dC and SAH [41 42] or HDACs by the TSA VPAscriptaid NaBu CBHA and oxamflatin [30 36 44 4851 53] Such exogenous epigenomic modulation (epigenetictransformation) of nuclear donor cells or cloned embryosmay facilitate and accelerate the reprogrammability for geneexpression of donor cell nuclei that have been transplantedinto cytoplasmic microenvironment of recipient oocytesSubsequently these cell nuclei undergo the proper dedif-ferentiating and faithful reestablishing of the epigeneticallydependent status of their transcriptional activity during thepreimplantation development of cloned embryos [43 54 55]
To increase the developmental capacity of porcine clonedembryos by enhancement of donor cell nuclear repro-grammability a nonselective inhibitor of HDACs designatedas TSA was used for epigenetic transformation of adult bonemarrow-derived mesenchymal stem cells (ABM-MSCs) thatprovided a source of nuclear donor cells In the currentinvestigation we focused on determining the impact of TSA-dependent epigenomic modulation of cultured MSCs on thesomatic cell cloning efficiency measured with in vitro devel-opmental capability of NT pig embryos and their cytologicalquality at the blastocyst stage that was evaluated on thebasis of both total blastomere number and apoptotic indexestimated by TUNEL analysis Furthermore we examinedwhether exposure of MSCs to TSA affects the pluripotency
BioMed Research International 3
status of cloned pig blastocysts For this reason the quanti-tative expression profiles for gene transcripts encoding suchproteins as pluripotency-related markers (Oct4 and Nanog)and multipotent stemness-associated marker (Nestin) wereevaluated in nuclear-transferred embryos reconstructed withepigenetically modified MSCs To the best of our knowledgethis report is the first in which the effect of TSA-mediatedepigenetic transformation of nuclear donor ABM-MSCs onthe extracorporeal developmental competences of porcinecloned embryos their cytological quality and quantitativepluripotency profile at the DNA transcription level wascomprehensively explored
2 Materials and Methods
21 Animals Four outbred Polish LargeWhite (PLW) pigs ofeither sex weighing approximately 20 kg each were main-tained under conventional conditions in the pigsty of theDepartment of Biotechnology of Animal Reproduction fromNational Research Institute of Animal Production in Bal-ice Poland The veterinary care was provided All animalprocedures were approved by the Local Animal Care EthicsCommittee Number II in Krakow
22 Chemicals and Supplies The reagents used in the presentexperiments were purchased from Sigma-Aldrich (PoznanPoland) unless otherwise indicated
23 Recovery and In Vitro Culture of Porcine MesenchymalStem Cells Mesenchymal stem cells (MSCs) were isolatedfrom the pig bone marrow as described by Opiela et al[56 57] Briefly the bone marrow was aspirated undergeneral anesthesia Bone marrow samples were placed inphosphate-buffered saline (PBS Biomed Lublin Poland)(1 1) and 6mL of this solution was layered over 3mLof Ficoll-Paque (Stem-cells Technologies USA) Followingcentrifugation the mononuclear cell fraction was collectedand rinsed twice with PBS The pelleted cells were trans-ferred to 75 cm2 tissue culture flasks (T75) filled with 17mLof MSC growthexpansion medium (Dulbeccorsquos ModifiedEaglersquos Medium DMEM) that was formulated to contain lowconcentration (1mgmL) ofD-glucose and enrichedwith 10fetal bovine serum (FBS) and 1 antibiotics1 Glutamax(Invitrogen USA) The culture medium was replenishedon the next day and the adherent cells were allowed toform colonies until reaching confluence The cell colonieswere harvested by trypsinEDTA treatment and passagedAfterwards the cells were seeded de novo at a concentrationof 025 to 05 times 106 into one T75 flask To confirm the mes-enchymal stemness origin of isolated cells flow cytometry-based detection of positive and negative expression of specificsurface cluster of differentiation (CD) antigens and ex vivodifferentiation of established MSC lines into adipocytes andosteocytes were accomplished as described by Opiela et al[56 58]
24 Simultaneous Cell Cycle Synchronization and EpigenomicModulation (Epigenetic Transformation) of Porcine MSCs
prior to SCNT Before trypsin-mediated detachment and usefor somatic cell cloning the cryopreserved permanent MSClines (between passages 1 and 2) that had been establishedfrom the primary cultures originating from bone marrowbiopsies of the prepubertal boars or gilts were thawed andcultured in vitro in DMEM medium enriched with relevantsupplementations The mitotic cycle of MSCs was synchro-nized at G1G0 stages through 24 to 48 h contact inhibition oftheir proliferative activity after reaching the total confluencestate under culture conditions in the medium enriched with10FBSDuring artificial synchronization of cell cycleMSCswere epigenetically transformed by 24 h exposure to 50 nMTSA
25 Preparation of Porcine Nuclear Recipient Oocytes forSomatic Cell Cloning Retrieval selection for extracorporealmeiotic maturation and in vitro culture of pig oocytes forthe purposes of SCNT were accomplished according to themethods applied by Samiec and Skrzyszowska [16] Samiecet al [22] and Skrzyszowska et al [20] Briefly slaughter-house ovaries were collected from both prepubertal femalepigs (gilts) and postpubertal female pigs (gilts and sows)Cumulus-oocyte complexes (COCs) were recovered by aspi-ration of follicular fluid from 2 to 6mm antral ovarian folli-cles The COCs with evenly granulated ooplasm and severaluniform layers of compact cumulus cells were selected for invitro maturation The maturation medium was comprised ofHEPES- and NaHCO
3-buffered Tissue Culture Medium 199
(TCM199) thatwas supplementedwith 10porcine follicularfluid (pFF) 10 FBS 5 ngmL recombinant human basicfibroblast growth factor (rh-bFGF) 10 ngmL recombinanthuman epidermal growth factor (rhEGF) 06mM L-cysteine1mMdibutyryl cyclic adenosinemonophosphate (db-cAMPbucladesine) 01 IUmL human menopausal gonadotropin(hMG) and 5mIUmL porcine follicle-stimulating hormone(pFSH) Approximately 50 to 60 COCs were cultured inthe db-cAMP- and hMG + pFSH-supplemented medium for20 h at 39∘C in a 100 water-saturated atmosphere of 5CO2and 95 air The oocytes were then cultured for 22 to
24 h in fresh maturation medium that did not contain db-cAMP hMG or pFSH After maturation expanded cumuluscells and corona cells were completely removed by vigorouspipetting of the COCs in the presence of 01 hyaluronidasefor 1 to 2min The metaphase II-stage oocytes which hadbeen selected on the basis of accepted morphological criteriainvolving evenly granulated dark ooplasm and the presenceof distinctly extruded first polar bodies provided a source ofhost cytoplasm for the cell nuclei of TSA-exposed or TSA-unexposed bone marrow-derived MSCs in the somatic cellcloning procedure
26 Production of Porcine Nuclear-Transferred Embryos UsingMSCs Modulated or Not Modulated Epigenomically by TSATreatment The approaches to somatic cell cloning and invitro culture of nuclear-transferred pig embryos that wereused in the current experiments were thoroughly presentedin our previous studies [15 16 18 59 60] Conciselyprior to the SCNT procedure cumulus-denuded in vitro
4 BioMed Research International
matured giltsow oocytes were incubated in the matura-tion medium supplemented with 04 120583gmL demecolcine(DMCC) for 50 to 60min The DMCC-treated oocyteswere subsequently transferred into a micromanipulationchamber filled with TC 199 medium containing 4mgmLbovine serum albumin (fraction V BSA-V) and 75 120583gmLcytochalasin B (CB) Metaphase chromosomes which hadbeen allocated into the chemically induced protrusion ofthe plasma membrane were removed microsurgically Thechemically assisted enucleation was accomplished by gentlyaspirating the ooplasmic cone which contained the con-densed chromosome cluster with the aid of a bevelledmicropipette The reconstruction of enucleated oocytes wasachieved by their electrofusion with TSA-treated or TSA-untreated MSCs Single nuclear donor cells were insertedinto the perivitelline space of previously enucleated oocytes(ie ooplastscytoplasts) The resulting somatic cell-ooplastcouplets were placed into a fusionactivation chamber filledwith electroporation medium (dielectric solution) SCNT-derived oocytes were artificially stimulated using the protocolof simultaneous fusion and electrical activation (SF-EA) Inthe SF-EA protocol electric pulses that induced a fusion ofMSC-cytoplast couplets were simultaneously the stimuli ini-tiating the activation of reconstructed oocytesThe complexesof ooplasts and MSCs were subjected to plasma membraneelectroporation by application of two successive DC pulsesof 12 kVcm for 60120583s The electropermeabilization of cellplasma membranes was performed in an isotonic dielectricsolution (03M D-mannitol) with concentration of CaCl
2
increased up to 10mM Following SF-EA porcine clonedembryos were exposed to 5120583gmL CB for 2 h and subse-quently cultured in vitro in North Carolina State University-23 (NCSU-23) medium supplemented with 4mgmL BSA-V1 Eaglersquos Minimum Essential Medium-nonessential aminoacid solution (MEM-NEAA) and 2 MEM-essential aminoacid solution (MEM-EAA) for 72ndash96 h Afterwards cleavedembryos were cultured in NCSU-23BSA-V + MEM-NEAA+ MEM-EAA medium supplemented with 10 FBS for anadditional 72 h up to morula and blastocyst stages
27 Assessment of Cloned Blastocyst Quality by TUNELAssay The blastocysts developed from NT oocytes recon-structed with TSA-treated and -untreated MSCs wereanalyzed using a Deadend Fluorometric TUNEL System(Promega Warsaw Poland) according to the protocol ofterminal deoxynucleotidyl transferase- (TdT-) mediateddUTP (21015840-deoxyuridine-51015840-triphosphate) nick-end labelling(TUNEL) that was dependent on the fluorescein isothio-cyanate (FITC) conjugated with dUTP This protocol wasthoroughly described by Opiela et al [61 62] Briefly afterfixationwith 1paraformaldehyde (PFA) diluted inPBS solu-tion the embryos were rinsed extensively (3 times) Subse-quently they were exposed to 02 Triton X-100 solution for5min followed by 1 h incubation in the reactionmixture con-sisting of equilibration buffer a cocktail of nucleotides andTdT enzyme at a maximum humidity Finally the embryoswere placed into a 2x concentrated solution of salinesodiumchloride-sodiumtrisodium citrate (SSC) buffer for 15min
After all the incubations carried out cloned blastocystswere washed three times in PBSpolyvinylpyrrolidone (PVP)solution for at least 5min and lastly transferred into a dropof VECTASHIELD mounting medium This medium wassupplemented with 410158406-diamidino-2-phenylindole (DAPI)counterstain and has been found to be unsurpassed inpreventing photobleaching of DAPI fluorochrome in orderto retain antifading ability during long-term storage of dyedembryos The evaluation of embryos that had been subjectedto TUNEL analysis was performed under a fluorescencemicroscope (Nicon Eclipse E600 Tokyo Japan) The assess-ment of blastocyst cytological quality enabled visualizingthe total number of blastomere nuclei in each embryo andthe number of cell nuclei exhibiting internucleosomallyfragmented DNA into which a FITC-conjugated dUTP wasincorporated The fluorescence filters adjusted to excitationwavelength higher than 460 nm and the filters adapted toemission wavelengths ranging from 480 nm to 540 nm wereapplied to detect the blue fluorescence emitted by cell nucleiof all the DAPI-tagged blastocyst blastomeres and the FITC-derived green fluorescence emitted by cell nuclei of late-apoptotic blastomeres
28 Determination of Apoptotic Index (DCI) in Cloned Blasto-cysts All the blastocysts generated were subjected to TUNELanalysisThenumber of all DAPI-dyed blastomere nuclei (iethe total number of inner cell massICM and trophoblastcell nuclei) and the number of all TUNEL-positiveFITC-stained (ie apoptotic) cell nuclei were estimated for eachembryo To assess the apoptotic index (dead cell index DCI)in the single blastocyst the sum of all apoptotic cell nucleidetected in the analyzed blastocyst was divided by the sumof all ICMtrophoblast cell nuclei identified in this blastocystand subsequently multiplied by 100 [62]
29 Analysis of Porcine Cloned Embryos for Oct4 Nanog andNestin mRNA Expression Profile Using Quantitative ReverseTranscriptase Real-Time PCR (qRT-PCR) The qRT-PCR wasapplied to estimate the expression levels of mRNA tran-scripts for pluripotency-related target genes (Oct4 Nanog)and multipotent stemness-associated target gene (Nestin) intwo groups of SCNT-derived blastocysts encompassing theembryos developed from oocytes reconstructed with eitherTSA-treated or -untreated MSC nuclei Briefly 40 120583L ofSideStep Lysis amp Stabilization Buffer (Agilent Technologies)was added to 10 120583L of blastocysts sample (10 blastocysts)in PBS The SideStep Buffer was used to lyse the cells ofblastocysts and ensure RNA stabilization The lysed embryosamples were vortexed at room temperature for 1min Theone-step Brilliant II SYBR Green QRT-PCR Master MixKit (Agilent Technologies) was used to perform relativequantification of gene expression Each PCR probe (totalvolume of 25 120583L) was comprised of 1 120583L of embryo cell lysateand 24120583L of reaction mixture which consisted of 125 120583L 2xSYBRGreen QRT-PCRmaster mix (containing an optimizedRT-PCR buffer MgCl
2 nucleotides (GAUC) SureStart Taq
DNA polymerase daggerSYBR Green and stabilizers) and 04 120583Lof each 200 nM forward and reverse primer (Table 1) 1 120583L
BioMed Research International 5
Table 1 Primers used in quantitative reverse transcriptase real-time PCR (qRT-PCR)
of RTRNase block enzyme mixture and 97 120583L RNase-freewater Thermal cycling conditions were as follows 30min at50∘C (for the first-strand synthesis) 10min at 95∘C 40 cyclesof 30 s at 95∘C for denaturing 60 s at 57∘C for annealing and30 s at 72∘C for extension
The glyceraldehyde-3-phosphate dehydrogenase(GAPDH) housekeeping gene was used as an endogenousstandard The results for expression levelsprofiles of indi-vidual target genes (Oct4 Nanog and Nestin) were normal-ized according to the relative concentration of the endog-enous standard Each reaction was run in triplicate and theobtained results were averaged By sample we mean mRNAderived from a pool of 10 blastocysts We made six PCR runsfrom one mRNA sample 3 for three analyzed genes (Oct4Nanog and Nestin) and 3 for the GAPDH control gene EachcDNA synthesis and PCR amplification were achieved inone tube and one buffer All data are presented as the meanvalues of the relative abundance (RA) of Oct4 Nanog andNestin mRNA transcripts Expression of these transcriptsisolated from blastocysts that originated from TSA-treatedand -untreated groups was compared to that for controlblastocysts which served as a calibrator Experiments werecarried out on a Mastercycler Realplex (Eppendorf UK Lim-ited Cambridge)
210 Statistical Analysis The 1205942 test was applied to esti-mate the differences in the in vitro developmental capa-bilities of porcine NT embryos originating from TSA-treated and -untreated MSCs The statistical analysis of vari-ance (ANOVA) and Tukeyrsquos Honestly Significant Difference(HSD) post hoc test for multiple ranges were used for com-parison of the levels of Oct4 Nanog and Nestin transcriptsbetween blastocysts derived from oocytes reconstituted withthe cell nuclei of TSA-exposed and -unexposed MSCs TheANOVA and subsequent Tukeyrsquos HSD post hoc test werealso applied to compare the mean values calculated fortotal number of DAPI-labelled cell nuclei total number ofTUNEL-positiveFITC-labelled (ie apoptotic) cell nucleiand apoptotic index (DCI) between blastocysts developedfrom NT oocytes generated using epigenetically modulatedand nonmodulated MSCs The differences with a probability(P) less than 005 were considered to be significant
3 Results
31 The In Vitro Developmental Potential of Cloned EmbryosDerived from the Cell Nuclei of MSCs Undergoing or NotUndergoing TSA-Based Epigenomic Modulation Althoughthe cleavage activity did not vary between cultured nuclear-transferred embryos reconstituted fromMSCs subjected andnot subjected to TSA-dependent epigenetic transformation(119875 ge 005) the morula and blastocyst formation rates werecharacterized by statistically significant differences A signif-icant variability in the in vitro developmental competencesto reach the morula stage (119875 lt 005) and a very highlysignificant variability in the developmental competences toreach the blastocyst stage (119875 lt 0001 Figure 1) were observedbetween these two groups of cloned embryos respectivelyThe detailed data that are focused on the assessment ofextracorporeal developmental capability of NT embryosreconstituted with epigenetically nonmodulated or modu-lated MSCs are presented in Table 2 As compared to thepresent investigation in our previous studies in which the invitro developmental potential of cloned pig embryos derivedfrom the cell nuclei of fetal fibroblast cells (being currentlyused source of nuclear donor cells) and generated applyingthe same experimental protocols of SF-EA for artificialstimulation of nuclear-transferred oocytes was explored thepercentages of the obtained morulae and blastocysts rangedfrom 582 to 613 and from 296 to 332 respectively[16 18 20] It is worth noting that all these percentagesturned out to be considerably lower than themorula and blas-tocyst formation rates achieved for NT embryos producedusing epigenomically nontransformed or transformed MSCs(Table 2)
32 Dependence of the Cloned Blastocyst Quality Evaluated byTUNEL Assay on the TSA-Based Epigenomic Modulation ofMSCs The quality of nuclear-transferred embryos derivedfrom TSA-exposed MSCs was considerably higher than thatof NT embryos derived from TSA-unexposed MSCs Veryhighly significant differences in the mean number of DAPI-stained cell nuclei per blastocyst were shown between thesetwo groups of embryos (119875 lt 0005 Table 3 Figure 2) Inturn no significant differences in the mean number of
6 BioMed Research International
005mm
(a)
005mm
(b)
005mm
(c)
005mm
(d)
Figure 1 Porcine cloned blastocysts developed fromnuclear-transferred oocytes reconstitutedwith adult bonemarrow-derivedmesenchymalstem cells undergoing trichostatinA- (TSA-) dependent epigenomicmodulation (photographs (a) and (b)) or not undergoingTSA-dependentepigenomic modulation (photographs (c) and (d)) Images were taken at magnification times200
Table 2 Effect of the TSA-dependent epigenomic modulation of MSCs on the in vitro developmental outcome of cloned pig embryos
TSA-mediated epigenetictransformation of MSCs
Number of oocytesembryos Development toEnucleated Electrofused () Cleaved () Morulae () Blastocysts ()
minus 293 275293 (939) 262275 (953) 216275 (785)b 125275 (455)B
TSA trichostatin A MSC mesenchymal stem cellValues with different small superscript letters (a and b) within the same column denote statistically significant differences between experimental groups (119875 lt005 1205942 test) Values with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0001 1205942 test)Number of replicates ge6
Table 3 Effect of the TSA-mediated epigenomic modulation of MSCs on the cytological quality of cloned pig embryos assessed by TUNELassay
TSA-mediated epigenetictransformation of MSCs
Number ofanalyzedblastocysts
Mean number ofDAPI-tagged cell nuclei per
blastocyst plusmn SD
Mean number ofTUNEL-positive (apoptotic) cell
nuclei per blastocyst plusmn SD
Mean apoptotic index(DCI) per blastocyst plusmn
SD ()+ 31 4797 plusmn 2743A 161 plusmn 338 495 plusmn 1398minus 26 2777 plusmn 1582B 158 plusmn 269 713 plusmn 1244TSA trichostatin A MSC mesenchymal stem cell TUNEL terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling DAPI 410158406-diamidino-2-phenylindole SD standard deviation DCI dead cell indexValues with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0005 ANOVA followed byTukeyrsquos HSD post hoc test) Number of replicates = 6
TUNEL-positive (ie late-apoptotic) cell nuclei per blasto-cyst were identified between NT embryos reconstituted withMSCs undergoing and not undergoing TSA-mediated epige-netic transformation (119875 ge 005 Table 3 Figure 2) Althoughno significant differences in the DCI per blastocyst were
found between both experimental groups (119875 ge 005) theincidence of late apoptosis-related internucleosomal DNAfragmentation trended slightly upwards for NT embryosgenerated using TSA-untreated MSCs as compared to thoseproduced using TSA-treated MSCs (Table 3 Figure 2)
BioMed Research International 7
005mm
(a)
005mm
(b)
005mm
(c)
005mm
(d)
005mm
(e)
005mm
(f)
Figure 2 Evaluation of cytological quality of porcine cloned blastocysts on the basis of simultaneous determination of total nuclear numberand detection of apoptotic cell nuclei by terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling (TUNEL) analysis Photographs (a) to (d) depict embryos originating from nuclear-transferred (NT) oocytes reconstitutedwith adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) subjected to epigenetic transformation via trichostatin A (TSA)treatment Photographs (e) and (f) depict embryos originating from NT oocytes reconstituted with ABM-MSCs not subjected to epigenetictransformation via TSA treatment In each blastocyst the cell nuclei of all the blastomeres (both inner cell mass (ICM) and trophectoderm(TE) cells) had been tagged with 410158406-diamidino-2-phenylindole (DAPI) counterstain and subsequently fluoresced in blue The cell nucleiof late-apoptotic blastomeres (ICM andor TE cells) exhibiting internucleosomal DNA fragmentation had been dyed with fluoresceinisothiocyanate (FITC) and then fluoresced in bright green In each photograph the DAPI-derived blue and FITC-derived green fluorescentsignals merge into one another Photographs (a) to (f) represent the blastocysts displaying different incidence of blastomere apoptosisand thereby varied advancement of internucleosomal DNA fragmentation ((a) (c) (d) and (e) the lack of apoptotic intranuclear DNAfragmentation (b) and (e) few apoptotic cell nuclei (f) increased extent of apoptotic intranuclear DNA fragmentation) Images were takenat magnification times200
8 BioMed Research International
0
1
2
3
4
5
6
B
A
Oct4 Nanog Nestin
MSCTSAMSC
Figure 3 Relative abundance (mean plusmn SD) of Oct4 Nanog andNestin mRNAs in blastocysts developed from nuclear-transferredpig embryos descended from MSCs undergoing or not undergoingTSA-dependent epigenomic transformation The highly significantdifferences were designated with the A and B letters (119875 lt 001ANOVA followed by Tukeyrsquos HSD post hoc test)
33 The Pluripotency Status of Porcine Nuclear-TransferredEmbryos Originating from Epigenomically Modulated or Non-modulated MSCs The total number of analyzed clonedblastocysts was 60 To assess relative abundance (RA) of threeanalyzed mRNA transcripts for pluripotency-related targetgenes (Oct4 Nanog) multipotent stemness-associated targetgene (Nestin) and control GAPDH gene transcript we used3 times 10 blastocysts from both TSA-treated and -untreatedgroups
Highly significant differences were indicated for theOct4gene transcript quantitative profile between blastocysts orig-inating from TSA-treated and -untreated groups (119875 lt 001Figure 3) Although no significant intergroup variability wasshown in the RA of Nanog and Nestin mRNAs (119875 ge 005)their expression levels tended to be higher in blastocystsderived from NT embryos reconstructed with TSA-exposedMSCs as compared to the TSA-untreated group (Figure 3)
4 Discussion
The abundance of the morulae and blastocysts of highercytological and molecular quality generated in the presentinvestigation confirms that the reprogrammability of adultbone marrow-derived mesenchymal stem cells which hadbeen epigenetically modified via exposure to trichostatinA underwent considerable improvement in a cytoplasm ofporcine nuclear-transferred oocytes and resultant in vitrocultured cloned embryos It is beyond any doubt thatthe impact of TSA-based epigenomic transformation ofbone marrow-retrieved MSCs representing multipotent andundifferentiated stem cells on their competence for SCNThas not yet been explored not only in pigs but also inother mammalian species So far TSA-mediated epigeneticmodulation has been applied only for differentiated somatic
cells that commonly provide the source of nuclear donorsfor cloning procedure in pigs The results of the study byDiao et al [55] proved that porcineNT embryos reconstitutedwith TSA-treated fetal fibroblast cells were characterized bytwofold higher developmental potential to reach the blasto-cyst stage (30) than the NT embryos reconstituted withTSA-untreated fibroblast cells (15) Similar tendency hasbeen also shown in our current study inwhich trichostatinA-mediated epigenetic transformation of bone marrow-derivedMSCs gave rise to significant enhancement of capability ofcloned embryos to complete their development to blastocyststage (65) as compared to that observed for embryosderived from TSA-unexposed MSC nuclei (46) Nonethe-less NT pig embryos originating from epigenomically trans-formed fetal fibroblast cells displayed considerably lowerblastocyst formation rate [55] than the rate indicated in ourpresent study for NT embryos reconstructed with epige-nomically nontransformed MSCs Moreover treatment ofnuclear donor MSCs with trichostatin A resulted in one anda half times to severalfold increase of blastocyst percentage(65) as compared to the blastocyst yields obtained by otherinvestigators using undifferentiated or in vitro differentiatedMSCs that were not modulated epigenetically [26ndash28 31 32]In the study by Kumar et al [28] the abilities of epige-nomically nontransformed bone marrow-descended MSCnuclei to direct the in vitro development of porcine clonedembryos to blastocyst stage decreased above three times(approximately 20) as compared to our present work Inturn nuclear-transferred embryos that had originated fromporcine undifferentiated bone marrow-retrieved MSCs andtheir derivatives along the osteogenic lineage were able tocomplete their in vitro development to blastocyst stage atthe rates ranging from 33 to 45 [31] In contrast Jin etal [32] and Li et al [27] reported that blastocyst yields ofNT embryos derived from porcine epigenetically unmodu-lated MSCs were maintained at the relatively low levels ofapproximately 18 and 16 respectively Nevertheless in thestudy by Lee et al [26] the in vitro developmental outcometo the blastocyst stage of NT embryos originating fromundifferentiated MSCs achieved the rate of approximately48 but even this blastocyst formation rate was considerablylower than that noticed in our current investigation
The results of the current study have also clearly demon-strated that the TSA-dependent epigenomic transformationof nuclear donor MSCs contributes to improvement of notonly in vitro developmental competences but also qualityand transcription level-related pluripotency extent of porcinecloned embryos The utilization of undifferentiated mes-enchymal stem cells and the modification of the molecularmechanisms of transcriptional reprogramming of the donornuclear genome via HDAC inhibitor-mediated epigeneticmodulation of MSCs before their use for SCNT led to correctand complete adaptation of MSC-inherited genomic DNA tothe cytoplasmic environment of enucleated oocytes and invitro cultured NT pig embryos Epigenetic reprogrammingof donor cell nuclei suggests that a new program for theirtranscriptional activity is loaded and reloaded immediatelyfollowing reconstruction of enucleated oocytes The successof SCNT may depend upon both genomic DNA-associated
BioMed Research International 9
reprogramming of gene expression for dedifferentiation ofthe donor somatic cell nuclei during early preimplantationdevelopment of cloned embryos and reprogramming of geneexpression for onset of somatogenic nuclear redifferentiationduring blastocyst formation [7 10 63 64] It has been ascer-tained that somatic cell nuclei should undergo the wide DNAcytosine residue demethylation changes throughout the earlydevelopment of NT embryos to erase and then reset theirown overall epigenetic as well as parental genomic imprintingmemory which has been established by remethylation ofthe nuclear genome within the framework of the specificpathway of somatic and germ cell lineage commitment anddifferentiation [2 4 13 30 65ndash67]
It appears that the enhanced cytological quality of clonedpig blastocysts that originated from enucleated oocytesreceiving epigenetically modulated MSC nuclei could beassociated with more faithful and faultless pattern for repro-gramming of transcriptional activity of genomic DNA inher-ited from undifferentiated and multipotent stem cells Thesecells exhibit increased genomic and epigenomic plasticity inrearrangements of their gene expression in the blastomeres ofpreimplanted NT embryos The fluorocytochemical analysisof overall mean nuclear counts revealed almost twofoldhigher total cells number in porcine blastocysts developedfrom NT oocytes reconstituted with TSA-exposed MSCs (48blastomeres) than that identified in blastocysts derived fromNT oocytes reconstituted with TSA-unexposed MSCs (28blastomeres) The quality of cloned blastocysts generated inour present study using epigenomically transformed MSCswas comparable to that noticed in the study by Lee et al[26] for porcine NT blastocysts derived from epigenomicallynontransformed MSCs These blastocysts were also charac-terized by a total cells number equal to 48 In contrast to theabovementioned findings in the investigations carried outby Li et al [27] Kumar et al [28] and Jin et al [32] theoverall number of ICM and trophectoderm cells in clonedpig blastocysts originating from embryos reconstituted withTSA-untreated MSCs ranged from 28 to 35 and decreasedapproximately one and a half times as compared to the totalcells number estimated for NT blastocysts obtained by usin the TSA-exposed MSC group Nevertheless the meanapoptotic index (ie DCI = 495) that was calculated by usfor blastocysts developed from cloned embryos reconstitutedwith epigenetically modified MSC nuclei was similar to theratios of TUNEL-positive cells (46 to 47) observed by Jinet al [32] andKumar et al [28] among porcineNT blastocystsderived from epigenetically nonmodulated counterparts Inturn cloned blastocysts produced in our study using TSA-untreated MSCs displayed the apoptotic index (713) thatwas comparable or slightly lower to indexes identified forcloned embryos created with the aid of fetal fibroblast cellsnot undergoing exposure to trichostatin A [28 32]The latterindexes (DCIs) oscillated between 73 and 78 Generally inour current work the proportion of TUNEL-positive cellsin relation to a total cells number tended to insignificantlydecrease among NT blastocysts generated using undifferen-tiated mesenchymal stem cells subjected to TSA treatmentcompared to those generated using nuclear donor cells not
exposed to TSA (495 versus 713) Analogous slight down-ward tendency in the ratios of TUNEL-positive cells wasindicated byDiao et al [55] comparing blastocysts developedfrom cloned embryos reconstructed with cell nuclei of fetalfibroblasts treated with TSA and the counterparts producedusing TSA-unexposed fetal fibroblast cell nuclei (nearly 3versus 45)
Improved reprogrammability of transcriptional activityfor nuclear genome of epigenetically modulated MSCs inthe cells of preimplanted NT embryos turned out to bepositively correlated with enhanced molecular quality ofporcine cloned blastocysts assessed on the basis of theirpluripotency extent whichwasmeasuredwith the expressionprofiles identified for Oct4 and Nanog genes A 38-kDaprotein Oct4 (ie octamer-binding transcription factor 4)that is a member of the family of POU- (Pit-Oct-Unc-)domain and homeodomain transcription factors acts as avital regulator of pluripotency extent playing an importantrole in not only controlling preimplantation embryonicdevelopment but also maintenance of ICM cell fate inblastocysts and pluripotency status of embryonic stem cells(ESCs) [68ndash70] A 35-kDa protein designated as Nanog fromCelticIrish mythical Tır na nOg (Tir Na Nog The Landof the Ever-Young) is another homeobox-containing tran-scription factor that represents the group of pivotal proteinsmodulating pluripotency degree [70 71] The homeoproteinNanog can act synergistically with Oct4 protein in retainingthe pluripotent status of blastocyst-descended ICM andepiblast cells as well as in sustaining the undifferentiatedstatus and ability for self-renewal of ESCs [71 72] Theaberrant (ie downregulated) expression levels and patternsof Oct4 and Nanog transcripts in both bovine NT blastocystsproduced using epigenetically nonmodulated calf dermalfibroblast cells [73] and porcine NT blastocysts producedusing either epigenetically nonmodulated adult MSCs orfetal fibroblast cells [28] have been found to be a majorcause of not only their declined cytological and molecularquality but also decreased in vitro developmental potentialof cloned embryos However in our current study it seemsthat TSA-dependent epigenomic transformation of nucleardonor MSCs biases correctingly the expression profile ofOct4 andNanog mRNAs in cloned pig blastocysts triggeringboth significant enhancement of relative abundance (RA)of Oct4 transcripts and slight (nonsignificant) increase inNanog transcript RA as compared to the TSA-untreatedMSCgroup On the one hand this influence could be exerted bydirect diminishment in the deacetylation level within thenucleosomal core-derived histone lysine moieties that wasevoked by trichostatin A-mediated nonspecific inhibition ofHDACs On the other hand it could be probably elicitedvia indirect nonselective suppression of cytosine residuemethylation processes within the Oct4 and Nanog gene pro-moters andor enhancers leading to upregulated expressionof these crucial pluripotency-related genes Similar resultshave been shown in the study by Wang et al [73] in whichthe sequential treatment of bovine differentiated nucleardonor fibroblast cells and resultant cloned embryos by bothnonspecific inhibitors of DNMTs and HDACs (ie 5-aza-dC
10 BioMed Research International
and TSA resp) contributed to increase of Oct4 transcriptRA in the blastocysts obtained simultaneously sustainingthe expression of Nanog mRNAs on the unchanged levelas compared to the 5-aza-dC- and TSA-unexposed groupIn this case 5-aza-21015840-deoxycytidine- and trichostatin A-mediated epigenetic modulation of both nuclear donor cellsand cloned embryos gave rise presumably to direct down-regulation in the methylation of cytosine residues withinthe Oct4 gene promoter andor enhancer that resultedfrom 5-aza-dC-dependent nonspecific inhibition of DNMTsFurthermore this two-factor epigenetic modulation couldaffect the enhanced incidence of acetylation level within thenucleosomal core-derived histone lysine moieties that wastriggered by trichostatin A-mediated nonselective inhibitionof HDACs consequently leading to indirect upregulatedexpression of the Oct4 gene Taking into consideration allthe abovementioned findings considerable transcriptionalupregulation in the expression of Oct4 gene (confirmedamong porcine NT blastocysts in our present study andamong bovineNT blastocysts in the study byWang et al [73])can be found to be pivotal indicator of increased pluripotencyextent of cloned embryos This process appears to be alsoassociated with improved cytological quality of NT blasto-cysts that was measured with their total cell counts In thecurrent investigation we have shown that the overall numberof ICM and trophoblast cells in blastocysts was significantlyhigher following the reconstruction of NT pig embryos withepigenomically transformed mesenchymal stem cells
5 Conclusions and Future Goals
Summing up artificial epigenomic modulation of in vitrocultured MSCs using the nonspecific HDAC inhibitor desig-nated as TSA seems to facilitate much more the reprogram-ming process for epigenetically determined transcriptionalactivity of somatic cell-inherited nuclear genome in theNT pig embryos The use of ectopic HDAC inhibitors forepigenetic transformation of mesenchymal stem cells whosecell nuclei were transferred into enucleated oocytes is thecompletely new approach in the studies involving somaticcell cloning of pigs and other mammalian species Enhancedreprogrammability of nuclear genome descended from TSA-exposed MSCs in the blastomeres of porcine preimplantedcloned embryos resulted in the improvements of not onlytheir capacity to complete in vitro development to themorula and blastocyst stages but also cytological and molec-ular quality of the blastocysts produced Therefore furtherinvestigations are also necessary to determine whether thenovel strategy of TSA-dependent epigenetic modification ofnuclear donor MSCs which has been recently utilized in ourlaboratory for the future goals of generating cloned pigletsenables retaining the in vivo developmental competences ofthe high-quality NT-derived blastocysts to reach full term
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Marcin Samiec and Jolanta Opiela contributed equally to thepreparation of this paper
Acknowledgments
The project was funded by the Polish National ScienceCentre resources allocated on the basis of Decision no DEC-201103DNZ905537 The authors would like to thank DrJarosław Wieczorek DVM for bone marrow retrieval
References
[1] A J Bonk H-T Cheong R Li et al ldquoCorrelation of develop-mental differences of nuclear transfer embryos cells to themethylation profiles of nuclear transfer donor cells in swinerdquoEpigenetics vol 2 no 3 pp 179ndash186 2007
[2] A J Bonk R Li L Lai et al ldquoAberrant DNA methylation inporcine in vitro- parthenogenetic- and somatic cell nucleartransfer-produced blastocystsrdquo Molecular Reproduction andDevelopment vol 75 no 2 pp 250ndash264 2008
[3] M Samiec ldquoDevelopment of pig cloning studies past presentand futurerdquo Journal of Animal and Feed Sciences vol 13 no 2pp 211ndash238 2004
[4] K MWhitworth and R S Prather ldquoSomatic cell nuclear trans-fer efficiency how can it be improved through nuclear remod-eling and reprogrammingrdquoMolecular Reproduction and Devel-opment vol 77 no 12 pp 1001ndash1015 2010
[5] K-I Yamanaka S Sugimura T Wakai M Kawahara and ESato ldquoAcetylation level of histone H3 in early embryonic stagesaffects subsequent development of miniature pig somatic cellnuclear transfer embryosrdquo The Journal of Reproduction andDevelopment vol 55 no 6 pp 638ndash644 2009
[6] R P Cervera N Martı-Gutierrez E Escorihuela R Morenoand M Stojkovic ldquoTrichostatin A affects histone acetylationand gene expression in porcine somatic cell nucleus transferembryosrdquoTheriogenology vol 72 no 8 pp 1097ndash1110 2009
[7] J Zhao J Whyte and R S Prather ldquoEffect of epigeneticregulation during swine embryogenesis and on cloning bynuclear transferrdquo Cell and Tissue Research vol 341 no 1 pp13ndash21 2010
[8] WReik ldquoStability and flexibility of epigenetic gene regulation inmammalian developmentrdquo Nature vol 447 no 7143 pp 425ndash432 2007
[9] R S Prather J W Ross S C Isom and J A Green ldquoTranscrip-tional post-transcriptional and epigenetic control of porcineoocyte maturation and embryogenesisrdquo Society of Reproductionand Fertility supplement vol 66 pp 165ndash176 2009
[10] N Rodriguez-Osorio R Urrego J B Cibelli K Eilertsen andE Memili ldquoReprogramming mammalian somatic cellsrdquo Theri-ogenology vol 78 no 9 pp 1869ndash1886 2012
[11] K J Eilertsen R A Power L L Harkins and P Misica ldquoTar-geting cellular memory to reprogram the epigenome restorepotential and improve somatic cell nuclear transferrdquo AnimalReproduction Science vol 98 no 1-2 pp 129ndash146 2007
[12] M Samiec and M Skrzyszowska ldquoMolecular conditions of thecell nucleus remodellingreprogramming process and nucleartransferred embryo development in the intraooplasmic kary-oplast injection technique a reviewrdquo Czech Journal of AnimalScience vol 50 no 5 pp 185ndash195 2005
BioMed Research International 11
[13] P Narbonne K Miyamoto and J B Gurdon ldquoReprogrammingand development in nuclear transfer embryos and in interspe-cific systemsrdquo Current Opinion in Genetics amp Development vol22 no 5 pp 450ndash458 2012
[14] J-I Bang J-G Yoo M-R Park et al ldquoThe effects of artificialactivation timing on the development of SCNT-derivedembryos and newborn pigletsrdquoReproductive Biology vol 13 no2 pp 127ndash132 2013
[15] M Samiec and M Skrzyszowska ldquoHigh developmental capa-bility of porcine cloned embryos following trichostatin A-dependent epigenomic transformation during in vitro matura-tion of oocytes pre-exposed to R-roscovitinerdquo Animal SciencePapers and Reports vol 30 no 4 pp 383ndash393 2012
[16] M Samiec and M Skrzyszowska ldquoRoscovitine is a novel agentthat can be used for the activation of porcine oocytes recon-structed with adult cutaneous or fetal fibroblast cell nucleirdquoTheriogenology vol 78 no 8 pp 1855ndash1867 2012
[17] Z Li J Shi D Liu et al ldquoEffects of donor fibroblast cell typeand transferred cloned embryo number on the efficiency of pigcloningrdquo Cellular Reprogramming vol 15 no 1 pp 35ndash42 2013
[18] M Samiec and M Skrzyszowska ldquoBiological transcomple-mentary activation as a novel and effective strategy appliedto the generation of porcine somatic cell cloned embryosrdquoReproductive Biology vol 14 no 2 pp 128ndash139 2014
[19] J Li O Svarcova K Villemoes et al ldquoHigh in vitro developmentafter somatic cell nuclear transfer and trichostatin A treatmentof reconstructed porcine embryosrdquoTheriogenology vol 70 no5 pp 800ndash808 2008
[20] M Skrzyszowska M Samiec R Słomski D Lipinski and EMały ldquoDevelopment of porcine transgenic nuclear-transferredembryos derived from fibroblast cells transfected by the noveltechnique of nucleofection or standard lipofectionrdquo Theri-ogenology vol 70 no 2 pp 248ndash259 2008
[21] Y-W Pang L An P Wang et al ldquoTreatment of porcine donorcells and reconstructed embryoswith the antioxidantmelatoninenhances cloning efficiencyrdquo Journal of Pineal Research vol 54no 4 pp 389ndash397 2013
[22] M Samiec M Skrzyszowska and J Opiela ldquoCreation of clonedpig embryos using contact-inhibited or serum-starved fibrob-last cells analysed intra vitam for apoptosis occurrencerdquo Annalsof Animal Science vol 13 no 2 pp 275ndash293 2013
[23] H Wei Y Qing W Pan et al ldquoComparison of the efficiency ofBannaminiature inbred pig somatic cell nuclear transfer amongdifferent donor cellsrdquo PLoSONE vol 8 no 2 Article ID e577282013
[24] K Inoue N Ogonuki H Miki et al ldquoInefficient reprogram-ming of the hematopoietic stem cell genome following nucleartransferrdquo Journal of Cell Science vol 119 no 10 pp 1985ndash19912006
[25] K Inoue S Noda N Ogonuki et al ldquoDifferential developmen-tal ability of embryos cloned from tissue-specific stem cellsrdquoStem Cells vol 25 no 5 pp 1279ndash1285 2007
[26] S L Lee E J Kang G H Maeng et al ldquoDevelopmental abilityof miniature pig embryos cloned withmesenchymal stem cellsrdquoThe Journal of Reproduction and Development vol 56 no 2 pp256ndash262 2010
[27] Z Li X He L Chen et al ldquoBone marrow mesenchymal stemcells are an attractive donor cell type for production of clonedpigs as well as genetically modified cloned pigs by somatic cellnuclear transferrdquo Cellular Reprogramming vol 15 no 5 pp459ndash470 2013
[28] B M Kumar H-F Jin J-G Kim et al ldquoDifferential geneexpression patterns in porcine nuclear transfer embryos recon-structed with fetal fibroblasts and mesenchymal stem cellsrdquoDevelopmental Dynamics vol 236 no 2 pp 435ndash446 2007
[29] J Su Y Wang Y Li et al ldquoOxamflatin significantly improvesnuclear reprogramming blastocyst quality and in vitro devel-opment of bovine SCNT embryosrdquo PLoS ONE vol 6 no 8Article ID e23805 2011
[30] Y Song THai YWang et al ldquoEpigenetic reprogramming geneexpression and in vitro development of porcine SCNT embryosare significantly improved by a histone deacetylase inhibitor-m-carboxycinnamic acid bishydroxamide (CBHA)rdquo Protein ampCell vol 5 no 5 pp 382ndash393 2014
[31] S Colleoni G Donofrio I Lagutina R Duchi C Galli andG Lazzari ldquoEstablishment differentiation electroporationviral transduction and nuclear transfer of bovine and porcinemesenchymal stem cellsrdquo Cloning and Stem Cells vol 7 no 3pp 154ndash166 2005
[32] H F Jin B M Kumar J G Kim et al ldquoEnhanced developmentof porcine embryos cloned from bone marrow mesenchymalstem cellsrdquoThe International Journal of Developmental Biologyvol 51 no 1 pp 85ndash90 2007
[33] J Opiela and M Samiec ldquoCharacterization of mesenchymalstem cells and their application in experimental embryologyrdquoPolish Journal of Veterinary Sciences vol 16 no 3 pp 593ndash5992013
[34] X Yang S L Smith X C Tian H A Lewin J-P Renard and TWakayama ldquoNuclear reprogramming of cloned embryos and itsimplications for therapeutic cloningrdquo Nature Genetics vol 39no 3 pp 295ndash302 2007
[35] XWu Y Li G-P Li et al ldquoTrichostatin A improved epigeneticmodifications of transfected cells but did not improve subse-quent cloned embryo developmentrdquo Animal Biotechnology vol19 no 4 pp 211ndash224 2008
[36] F Bo L Di F Qing-Chang et al ldquoEffect of trichostatin A ontransfected donor cells and subsequent development of porcinecloned embryosrdquo Zygote vol 19 no 3 pp 237ndash243 2011
[37] X Ding Y Wang D Zhang Z Guo and Y Zhang ldquoIncreasedpre-implantation development of cloned bovine embryostreated with 5-aza-21015840-deoxycytidine and trichostatin ArdquoTheri-ogenology vol 70 no 4 pp 622ndash630 2008
[38] H S Lee X F Yu J I Bang et al ldquoEnhanced histone acetylationin somatic cells induced by a histone deacetylase inhibitorimproved inter-generic cloned leopard cat blastocystsrdquo Theri-ogenology vol 74 no 8 pp 1439ndash1449 2010
[39] S-F Ning Q-Y Li M-M Liang et al ldquoMethylation charac-teristics and developmental potential of Guangxi Bamaminipig(Sus scrofa domestica) cloned embryos from donor cells treatedwith trichostatin A and 5-aza-21015840-deoxycytidinerdquo Zygote vol 21no 2 pp 178ndash186 2013
[40] B P Enright L-Y Sung C-C Chang X Yang and X C TianldquoMethylation and acetylation characteristics of cloned bovineembryos from donor cells treated with 5-aza-21015840-deoxycytidinerdquoBiology of Reproduction vol 72 no 4 pp 944ndash948 2005
[41] Y J Huan J Zhu B T Xie et al ldquoTreating cloned embryos butnot donor cells with 5-aza-21015840-deoxycytidine enhances thedevelopmental competence of porcine cloned embryosrdquo TheJournal of Reproduction andDevelopment vol 59 no 5 pp 442ndash449 2013
[42] B-G JeonG Coppola S D Perrault G-J RhoDH Betts andWAKing ldquoS-adenosylhomocysteine treatment of adult female
12 BioMed Research International
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
status of cloned pig blastocysts For this reason the quanti-tative expression profiles for gene transcripts encoding suchproteins as pluripotency-related markers (Oct4 and Nanog)and multipotent stemness-associated marker (Nestin) wereevaluated in nuclear-transferred embryos reconstructed withepigenetically modified MSCs To the best of our knowledgethis report is the first in which the effect of TSA-mediatedepigenetic transformation of nuclear donor ABM-MSCs onthe extracorporeal developmental competences of porcinecloned embryos their cytological quality and quantitativepluripotency profile at the DNA transcription level wascomprehensively explored
2 Materials and Methods
21 Animals Four outbred Polish LargeWhite (PLW) pigs ofeither sex weighing approximately 20 kg each were main-tained under conventional conditions in the pigsty of theDepartment of Biotechnology of Animal Reproduction fromNational Research Institute of Animal Production in Bal-ice Poland The veterinary care was provided All animalprocedures were approved by the Local Animal Care EthicsCommittee Number II in Krakow
22 Chemicals and Supplies The reagents used in the presentexperiments were purchased from Sigma-Aldrich (PoznanPoland) unless otherwise indicated
23 Recovery and In Vitro Culture of Porcine MesenchymalStem Cells Mesenchymal stem cells (MSCs) were isolatedfrom the pig bone marrow as described by Opiela et al[56 57] Briefly the bone marrow was aspirated undergeneral anesthesia Bone marrow samples were placed inphosphate-buffered saline (PBS Biomed Lublin Poland)(1 1) and 6mL of this solution was layered over 3mLof Ficoll-Paque (Stem-cells Technologies USA) Followingcentrifugation the mononuclear cell fraction was collectedand rinsed twice with PBS The pelleted cells were trans-ferred to 75 cm2 tissue culture flasks (T75) filled with 17mLof MSC growthexpansion medium (Dulbeccorsquos ModifiedEaglersquos Medium DMEM) that was formulated to contain lowconcentration (1mgmL) ofD-glucose and enrichedwith 10fetal bovine serum (FBS) and 1 antibiotics1 Glutamax(Invitrogen USA) The culture medium was replenishedon the next day and the adherent cells were allowed toform colonies until reaching confluence The cell colonieswere harvested by trypsinEDTA treatment and passagedAfterwards the cells were seeded de novo at a concentrationof 025 to 05 times 106 into one T75 flask To confirm the mes-enchymal stemness origin of isolated cells flow cytometry-based detection of positive and negative expression of specificsurface cluster of differentiation (CD) antigens and ex vivodifferentiation of established MSC lines into adipocytes andosteocytes were accomplished as described by Opiela et al[56 58]
24 Simultaneous Cell Cycle Synchronization and EpigenomicModulation (Epigenetic Transformation) of Porcine MSCs
prior to SCNT Before trypsin-mediated detachment and usefor somatic cell cloning the cryopreserved permanent MSClines (between passages 1 and 2) that had been establishedfrom the primary cultures originating from bone marrowbiopsies of the prepubertal boars or gilts were thawed andcultured in vitro in DMEM medium enriched with relevantsupplementations The mitotic cycle of MSCs was synchro-nized at G1G0 stages through 24 to 48 h contact inhibition oftheir proliferative activity after reaching the total confluencestate under culture conditions in the medium enriched with10FBSDuring artificial synchronization of cell cycleMSCswere epigenetically transformed by 24 h exposure to 50 nMTSA
25 Preparation of Porcine Nuclear Recipient Oocytes forSomatic Cell Cloning Retrieval selection for extracorporealmeiotic maturation and in vitro culture of pig oocytes forthe purposes of SCNT were accomplished according to themethods applied by Samiec and Skrzyszowska [16] Samiecet al [22] and Skrzyszowska et al [20] Briefly slaughter-house ovaries were collected from both prepubertal femalepigs (gilts) and postpubertal female pigs (gilts and sows)Cumulus-oocyte complexes (COCs) were recovered by aspi-ration of follicular fluid from 2 to 6mm antral ovarian folli-cles The COCs with evenly granulated ooplasm and severaluniform layers of compact cumulus cells were selected for invitro maturation The maturation medium was comprised ofHEPES- and NaHCO
3-buffered Tissue Culture Medium 199
(TCM199) thatwas supplementedwith 10porcine follicularfluid (pFF) 10 FBS 5 ngmL recombinant human basicfibroblast growth factor (rh-bFGF) 10 ngmL recombinanthuman epidermal growth factor (rhEGF) 06mM L-cysteine1mMdibutyryl cyclic adenosinemonophosphate (db-cAMPbucladesine) 01 IUmL human menopausal gonadotropin(hMG) and 5mIUmL porcine follicle-stimulating hormone(pFSH) Approximately 50 to 60 COCs were cultured inthe db-cAMP- and hMG + pFSH-supplemented medium for20 h at 39∘C in a 100 water-saturated atmosphere of 5CO2and 95 air The oocytes were then cultured for 22 to
24 h in fresh maturation medium that did not contain db-cAMP hMG or pFSH After maturation expanded cumuluscells and corona cells were completely removed by vigorouspipetting of the COCs in the presence of 01 hyaluronidasefor 1 to 2min The metaphase II-stage oocytes which hadbeen selected on the basis of accepted morphological criteriainvolving evenly granulated dark ooplasm and the presenceof distinctly extruded first polar bodies provided a source ofhost cytoplasm for the cell nuclei of TSA-exposed or TSA-unexposed bone marrow-derived MSCs in the somatic cellcloning procedure
26 Production of Porcine Nuclear-Transferred Embryos UsingMSCs Modulated or Not Modulated Epigenomically by TSATreatment The approaches to somatic cell cloning and invitro culture of nuclear-transferred pig embryos that wereused in the current experiments were thoroughly presentedin our previous studies [15 16 18 59 60] Conciselyprior to the SCNT procedure cumulus-denuded in vitro
4 BioMed Research International
matured giltsow oocytes were incubated in the matura-tion medium supplemented with 04 120583gmL demecolcine(DMCC) for 50 to 60min The DMCC-treated oocyteswere subsequently transferred into a micromanipulationchamber filled with TC 199 medium containing 4mgmLbovine serum albumin (fraction V BSA-V) and 75 120583gmLcytochalasin B (CB) Metaphase chromosomes which hadbeen allocated into the chemically induced protrusion ofthe plasma membrane were removed microsurgically Thechemically assisted enucleation was accomplished by gentlyaspirating the ooplasmic cone which contained the con-densed chromosome cluster with the aid of a bevelledmicropipette The reconstruction of enucleated oocytes wasachieved by their electrofusion with TSA-treated or TSA-untreated MSCs Single nuclear donor cells were insertedinto the perivitelline space of previously enucleated oocytes(ie ooplastscytoplasts) The resulting somatic cell-ooplastcouplets were placed into a fusionactivation chamber filledwith electroporation medium (dielectric solution) SCNT-derived oocytes were artificially stimulated using the protocolof simultaneous fusion and electrical activation (SF-EA) Inthe SF-EA protocol electric pulses that induced a fusion ofMSC-cytoplast couplets were simultaneously the stimuli ini-tiating the activation of reconstructed oocytesThe complexesof ooplasts and MSCs were subjected to plasma membraneelectroporation by application of two successive DC pulsesof 12 kVcm for 60120583s The electropermeabilization of cellplasma membranes was performed in an isotonic dielectricsolution (03M D-mannitol) with concentration of CaCl
2
increased up to 10mM Following SF-EA porcine clonedembryos were exposed to 5120583gmL CB for 2 h and subse-quently cultured in vitro in North Carolina State University-23 (NCSU-23) medium supplemented with 4mgmL BSA-V1 Eaglersquos Minimum Essential Medium-nonessential aminoacid solution (MEM-NEAA) and 2 MEM-essential aminoacid solution (MEM-EAA) for 72ndash96 h Afterwards cleavedembryos were cultured in NCSU-23BSA-V + MEM-NEAA+ MEM-EAA medium supplemented with 10 FBS for anadditional 72 h up to morula and blastocyst stages
27 Assessment of Cloned Blastocyst Quality by TUNELAssay The blastocysts developed from NT oocytes recon-structed with TSA-treated and -untreated MSCs wereanalyzed using a Deadend Fluorometric TUNEL System(Promega Warsaw Poland) according to the protocol ofterminal deoxynucleotidyl transferase- (TdT-) mediateddUTP (21015840-deoxyuridine-51015840-triphosphate) nick-end labelling(TUNEL) that was dependent on the fluorescein isothio-cyanate (FITC) conjugated with dUTP This protocol wasthoroughly described by Opiela et al [61 62] Briefly afterfixationwith 1paraformaldehyde (PFA) diluted inPBS solu-tion the embryos were rinsed extensively (3 times) Subse-quently they were exposed to 02 Triton X-100 solution for5min followed by 1 h incubation in the reactionmixture con-sisting of equilibration buffer a cocktail of nucleotides andTdT enzyme at a maximum humidity Finally the embryoswere placed into a 2x concentrated solution of salinesodiumchloride-sodiumtrisodium citrate (SSC) buffer for 15min
After all the incubations carried out cloned blastocystswere washed three times in PBSpolyvinylpyrrolidone (PVP)solution for at least 5min and lastly transferred into a dropof VECTASHIELD mounting medium This medium wassupplemented with 410158406-diamidino-2-phenylindole (DAPI)counterstain and has been found to be unsurpassed inpreventing photobleaching of DAPI fluorochrome in orderto retain antifading ability during long-term storage of dyedembryos The evaluation of embryos that had been subjectedto TUNEL analysis was performed under a fluorescencemicroscope (Nicon Eclipse E600 Tokyo Japan) The assess-ment of blastocyst cytological quality enabled visualizingthe total number of blastomere nuclei in each embryo andthe number of cell nuclei exhibiting internucleosomallyfragmented DNA into which a FITC-conjugated dUTP wasincorporated The fluorescence filters adjusted to excitationwavelength higher than 460 nm and the filters adapted toemission wavelengths ranging from 480 nm to 540 nm wereapplied to detect the blue fluorescence emitted by cell nucleiof all the DAPI-tagged blastocyst blastomeres and the FITC-derived green fluorescence emitted by cell nuclei of late-apoptotic blastomeres
28 Determination of Apoptotic Index (DCI) in Cloned Blasto-cysts All the blastocysts generated were subjected to TUNELanalysisThenumber of all DAPI-dyed blastomere nuclei (iethe total number of inner cell massICM and trophoblastcell nuclei) and the number of all TUNEL-positiveFITC-stained (ie apoptotic) cell nuclei were estimated for eachembryo To assess the apoptotic index (dead cell index DCI)in the single blastocyst the sum of all apoptotic cell nucleidetected in the analyzed blastocyst was divided by the sumof all ICMtrophoblast cell nuclei identified in this blastocystand subsequently multiplied by 100 [62]
29 Analysis of Porcine Cloned Embryos for Oct4 Nanog andNestin mRNA Expression Profile Using Quantitative ReverseTranscriptase Real-Time PCR (qRT-PCR) The qRT-PCR wasapplied to estimate the expression levels of mRNA tran-scripts for pluripotency-related target genes (Oct4 Nanog)and multipotent stemness-associated target gene (Nestin) intwo groups of SCNT-derived blastocysts encompassing theembryos developed from oocytes reconstructed with eitherTSA-treated or -untreated MSC nuclei Briefly 40 120583L ofSideStep Lysis amp Stabilization Buffer (Agilent Technologies)was added to 10 120583L of blastocysts sample (10 blastocysts)in PBS The SideStep Buffer was used to lyse the cells ofblastocysts and ensure RNA stabilization The lysed embryosamples were vortexed at room temperature for 1min Theone-step Brilliant II SYBR Green QRT-PCR Master MixKit (Agilent Technologies) was used to perform relativequantification of gene expression Each PCR probe (totalvolume of 25 120583L) was comprised of 1 120583L of embryo cell lysateand 24120583L of reaction mixture which consisted of 125 120583L 2xSYBRGreen QRT-PCRmaster mix (containing an optimizedRT-PCR buffer MgCl
2 nucleotides (GAUC) SureStart Taq
DNA polymerase daggerSYBR Green and stabilizers) and 04 120583Lof each 200 nM forward and reverse primer (Table 1) 1 120583L
BioMed Research International 5
Table 1 Primers used in quantitative reverse transcriptase real-time PCR (qRT-PCR)
of RTRNase block enzyme mixture and 97 120583L RNase-freewater Thermal cycling conditions were as follows 30min at50∘C (for the first-strand synthesis) 10min at 95∘C 40 cyclesof 30 s at 95∘C for denaturing 60 s at 57∘C for annealing and30 s at 72∘C for extension
The glyceraldehyde-3-phosphate dehydrogenase(GAPDH) housekeeping gene was used as an endogenousstandard The results for expression levelsprofiles of indi-vidual target genes (Oct4 Nanog and Nestin) were normal-ized according to the relative concentration of the endog-enous standard Each reaction was run in triplicate and theobtained results were averaged By sample we mean mRNAderived from a pool of 10 blastocysts We made six PCR runsfrom one mRNA sample 3 for three analyzed genes (Oct4Nanog and Nestin) and 3 for the GAPDH control gene EachcDNA synthesis and PCR amplification were achieved inone tube and one buffer All data are presented as the meanvalues of the relative abundance (RA) of Oct4 Nanog andNestin mRNA transcripts Expression of these transcriptsisolated from blastocysts that originated from TSA-treatedand -untreated groups was compared to that for controlblastocysts which served as a calibrator Experiments werecarried out on a Mastercycler Realplex (Eppendorf UK Lim-ited Cambridge)
210 Statistical Analysis The 1205942 test was applied to esti-mate the differences in the in vitro developmental capa-bilities of porcine NT embryos originating from TSA-treated and -untreated MSCs The statistical analysis of vari-ance (ANOVA) and Tukeyrsquos Honestly Significant Difference(HSD) post hoc test for multiple ranges were used for com-parison of the levels of Oct4 Nanog and Nestin transcriptsbetween blastocysts derived from oocytes reconstituted withthe cell nuclei of TSA-exposed and -unexposed MSCs TheANOVA and subsequent Tukeyrsquos HSD post hoc test werealso applied to compare the mean values calculated fortotal number of DAPI-labelled cell nuclei total number ofTUNEL-positiveFITC-labelled (ie apoptotic) cell nucleiand apoptotic index (DCI) between blastocysts developedfrom NT oocytes generated using epigenetically modulatedand nonmodulated MSCs The differences with a probability(P) less than 005 were considered to be significant
3 Results
31 The In Vitro Developmental Potential of Cloned EmbryosDerived from the Cell Nuclei of MSCs Undergoing or NotUndergoing TSA-Based Epigenomic Modulation Althoughthe cleavage activity did not vary between cultured nuclear-transferred embryos reconstituted fromMSCs subjected andnot subjected to TSA-dependent epigenetic transformation(119875 ge 005) the morula and blastocyst formation rates werecharacterized by statistically significant differences A signif-icant variability in the in vitro developmental competencesto reach the morula stage (119875 lt 005) and a very highlysignificant variability in the developmental competences toreach the blastocyst stage (119875 lt 0001 Figure 1) were observedbetween these two groups of cloned embryos respectivelyThe detailed data that are focused on the assessment ofextracorporeal developmental capability of NT embryosreconstituted with epigenetically nonmodulated or modu-lated MSCs are presented in Table 2 As compared to thepresent investigation in our previous studies in which the invitro developmental potential of cloned pig embryos derivedfrom the cell nuclei of fetal fibroblast cells (being currentlyused source of nuclear donor cells) and generated applyingthe same experimental protocols of SF-EA for artificialstimulation of nuclear-transferred oocytes was explored thepercentages of the obtained morulae and blastocysts rangedfrom 582 to 613 and from 296 to 332 respectively[16 18 20] It is worth noting that all these percentagesturned out to be considerably lower than themorula and blas-tocyst formation rates achieved for NT embryos producedusing epigenomically nontransformed or transformed MSCs(Table 2)
32 Dependence of the Cloned Blastocyst Quality Evaluated byTUNEL Assay on the TSA-Based Epigenomic Modulation ofMSCs The quality of nuclear-transferred embryos derivedfrom TSA-exposed MSCs was considerably higher than thatof NT embryos derived from TSA-unexposed MSCs Veryhighly significant differences in the mean number of DAPI-stained cell nuclei per blastocyst were shown between thesetwo groups of embryos (119875 lt 0005 Table 3 Figure 2) Inturn no significant differences in the mean number of
6 BioMed Research International
005mm
(a)
005mm
(b)
005mm
(c)
005mm
(d)
Figure 1 Porcine cloned blastocysts developed fromnuclear-transferred oocytes reconstitutedwith adult bonemarrow-derivedmesenchymalstem cells undergoing trichostatinA- (TSA-) dependent epigenomicmodulation (photographs (a) and (b)) or not undergoingTSA-dependentepigenomic modulation (photographs (c) and (d)) Images were taken at magnification times200
Table 2 Effect of the TSA-dependent epigenomic modulation of MSCs on the in vitro developmental outcome of cloned pig embryos
TSA-mediated epigenetictransformation of MSCs
Number of oocytesembryos Development toEnucleated Electrofused () Cleaved () Morulae () Blastocysts ()
minus 293 275293 (939) 262275 (953) 216275 (785)b 125275 (455)B
TSA trichostatin A MSC mesenchymal stem cellValues with different small superscript letters (a and b) within the same column denote statistically significant differences between experimental groups (119875 lt005 1205942 test) Values with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0001 1205942 test)Number of replicates ge6
Table 3 Effect of the TSA-mediated epigenomic modulation of MSCs on the cytological quality of cloned pig embryos assessed by TUNELassay
TSA-mediated epigenetictransformation of MSCs
Number ofanalyzedblastocysts
Mean number ofDAPI-tagged cell nuclei per
blastocyst plusmn SD
Mean number ofTUNEL-positive (apoptotic) cell
nuclei per blastocyst plusmn SD
Mean apoptotic index(DCI) per blastocyst plusmn
SD ()+ 31 4797 plusmn 2743A 161 plusmn 338 495 plusmn 1398minus 26 2777 plusmn 1582B 158 plusmn 269 713 plusmn 1244TSA trichostatin A MSC mesenchymal stem cell TUNEL terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling DAPI 410158406-diamidino-2-phenylindole SD standard deviation DCI dead cell indexValues with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0005 ANOVA followed byTukeyrsquos HSD post hoc test) Number of replicates = 6
TUNEL-positive (ie late-apoptotic) cell nuclei per blasto-cyst were identified between NT embryos reconstituted withMSCs undergoing and not undergoing TSA-mediated epige-netic transformation (119875 ge 005 Table 3 Figure 2) Althoughno significant differences in the DCI per blastocyst were
found between both experimental groups (119875 ge 005) theincidence of late apoptosis-related internucleosomal DNAfragmentation trended slightly upwards for NT embryosgenerated using TSA-untreated MSCs as compared to thoseproduced using TSA-treated MSCs (Table 3 Figure 2)
BioMed Research International 7
005mm
(a)
005mm
(b)
005mm
(c)
005mm
(d)
005mm
(e)
005mm
(f)
Figure 2 Evaluation of cytological quality of porcine cloned blastocysts on the basis of simultaneous determination of total nuclear numberand detection of apoptotic cell nuclei by terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling (TUNEL) analysis Photographs (a) to (d) depict embryos originating from nuclear-transferred (NT) oocytes reconstitutedwith adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) subjected to epigenetic transformation via trichostatin A (TSA)treatment Photographs (e) and (f) depict embryos originating from NT oocytes reconstituted with ABM-MSCs not subjected to epigenetictransformation via TSA treatment In each blastocyst the cell nuclei of all the blastomeres (both inner cell mass (ICM) and trophectoderm(TE) cells) had been tagged with 410158406-diamidino-2-phenylindole (DAPI) counterstain and subsequently fluoresced in blue The cell nucleiof late-apoptotic blastomeres (ICM andor TE cells) exhibiting internucleosomal DNA fragmentation had been dyed with fluoresceinisothiocyanate (FITC) and then fluoresced in bright green In each photograph the DAPI-derived blue and FITC-derived green fluorescentsignals merge into one another Photographs (a) to (f) represent the blastocysts displaying different incidence of blastomere apoptosisand thereby varied advancement of internucleosomal DNA fragmentation ((a) (c) (d) and (e) the lack of apoptotic intranuclear DNAfragmentation (b) and (e) few apoptotic cell nuclei (f) increased extent of apoptotic intranuclear DNA fragmentation) Images were takenat magnification times200
8 BioMed Research International
0
1
2
3
4
5
6
B
A
Oct4 Nanog Nestin
MSCTSAMSC
Figure 3 Relative abundance (mean plusmn SD) of Oct4 Nanog andNestin mRNAs in blastocysts developed from nuclear-transferredpig embryos descended from MSCs undergoing or not undergoingTSA-dependent epigenomic transformation The highly significantdifferences were designated with the A and B letters (119875 lt 001ANOVA followed by Tukeyrsquos HSD post hoc test)
33 The Pluripotency Status of Porcine Nuclear-TransferredEmbryos Originating from Epigenomically Modulated or Non-modulated MSCs The total number of analyzed clonedblastocysts was 60 To assess relative abundance (RA) of threeanalyzed mRNA transcripts for pluripotency-related targetgenes (Oct4 Nanog) multipotent stemness-associated targetgene (Nestin) and control GAPDH gene transcript we used3 times 10 blastocysts from both TSA-treated and -untreatedgroups
Highly significant differences were indicated for theOct4gene transcript quantitative profile between blastocysts orig-inating from TSA-treated and -untreated groups (119875 lt 001Figure 3) Although no significant intergroup variability wasshown in the RA of Nanog and Nestin mRNAs (119875 ge 005)their expression levels tended to be higher in blastocystsderived from NT embryos reconstructed with TSA-exposedMSCs as compared to the TSA-untreated group (Figure 3)
4 Discussion
The abundance of the morulae and blastocysts of highercytological and molecular quality generated in the presentinvestigation confirms that the reprogrammability of adultbone marrow-derived mesenchymal stem cells which hadbeen epigenetically modified via exposure to trichostatinA underwent considerable improvement in a cytoplasm ofporcine nuclear-transferred oocytes and resultant in vitrocultured cloned embryos It is beyond any doubt thatthe impact of TSA-based epigenomic transformation ofbone marrow-retrieved MSCs representing multipotent andundifferentiated stem cells on their competence for SCNThas not yet been explored not only in pigs but also inother mammalian species So far TSA-mediated epigeneticmodulation has been applied only for differentiated somatic
cells that commonly provide the source of nuclear donorsfor cloning procedure in pigs The results of the study byDiao et al [55] proved that porcineNT embryos reconstitutedwith TSA-treated fetal fibroblast cells were characterized bytwofold higher developmental potential to reach the blasto-cyst stage (30) than the NT embryos reconstituted withTSA-untreated fibroblast cells (15) Similar tendency hasbeen also shown in our current study inwhich trichostatinA-mediated epigenetic transformation of bone marrow-derivedMSCs gave rise to significant enhancement of capability ofcloned embryos to complete their development to blastocyststage (65) as compared to that observed for embryosderived from TSA-unexposed MSC nuclei (46) Nonethe-less NT pig embryos originating from epigenomically trans-formed fetal fibroblast cells displayed considerably lowerblastocyst formation rate [55] than the rate indicated in ourpresent study for NT embryos reconstructed with epige-nomically nontransformed MSCs Moreover treatment ofnuclear donor MSCs with trichostatin A resulted in one anda half times to severalfold increase of blastocyst percentage(65) as compared to the blastocyst yields obtained by otherinvestigators using undifferentiated or in vitro differentiatedMSCs that were not modulated epigenetically [26ndash28 31 32]In the study by Kumar et al [28] the abilities of epige-nomically nontransformed bone marrow-descended MSCnuclei to direct the in vitro development of porcine clonedembryos to blastocyst stage decreased above three times(approximately 20) as compared to our present work Inturn nuclear-transferred embryos that had originated fromporcine undifferentiated bone marrow-retrieved MSCs andtheir derivatives along the osteogenic lineage were able tocomplete their in vitro development to blastocyst stage atthe rates ranging from 33 to 45 [31] In contrast Jin etal [32] and Li et al [27] reported that blastocyst yields ofNT embryos derived from porcine epigenetically unmodu-lated MSCs were maintained at the relatively low levels ofapproximately 18 and 16 respectively Nevertheless in thestudy by Lee et al [26] the in vitro developmental outcometo the blastocyst stage of NT embryos originating fromundifferentiated MSCs achieved the rate of approximately48 but even this blastocyst formation rate was considerablylower than that noticed in our current investigation
The results of the current study have also clearly demon-strated that the TSA-dependent epigenomic transformationof nuclear donor MSCs contributes to improvement of notonly in vitro developmental competences but also qualityand transcription level-related pluripotency extent of porcinecloned embryos The utilization of undifferentiated mes-enchymal stem cells and the modification of the molecularmechanisms of transcriptional reprogramming of the donornuclear genome via HDAC inhibitor-mediated epigeneticmodulation of MSCs before their use for SCNT led to correctand complete adaptation of MSC-inherited genomic DNA tothe cytoplasmic environment of enucleated oocytes and invitro cultured NT pig embryos Epigenetic reprogrammingof donor cell nuclei suggests that a new program for theirtranscriptional activity is loaded and reloaded immediatelyfollowing reconstruction of enucleated oocytes The successof SCNT may depend upon both genomic DNA-associated
BioMed Research International 9
reprogramming of gene expression for dedifferentiation ofthe donor somatic cell nuclei during early preimplantationdevelopment of cloned embryos and reprogramming of geneexpression for onset of somatogenic nuclear redifferentiationduring blastocyst formation [7 10 63 64] It has been ascer-tained that somatic cell nuclei should undergo the wide DNAcytosine residue demethylation changes throughout the earlydevelopment of NT embryos to erase and then reset theirown overall epigenetic as well as parental genomic imprintingmemory which has been established by remethylation ofthe nuclear genome within the framework of the specificpathway of somatic and germ cell lineage commitment anddifferentiation [2 4 13 30 65ndash67]
It appears that the enhanced cytological quality of clonedpig blastocysts that originated from enucleated oocytesreceiving epigenetically modulated MSC nuclei could beassociated with more faithful and faultless pattern for repro-gramming of transcriptional activity of genomic DNA inher-ited from undifferentiated and multipotent stem cells Thesecells exhibit increased genomic and epigenomic plasticity inrearrangements of their gene expression in the blastomeres ofpreimplanted NT embryos The fluorocytochemical analysisof overall mean nuclear counts revealed almost twofoldhigher total cells number in porcine blastocysts developedfrom NT oocytes reconstituted with TSA-exposed MSCs (48blastomeres) than that identified in blastocysts derived fromNT oocytes reconstituted with TSA-unexposed MSCs (28blastomeres) The quality of cloned blastocysts generated inour present study using epigenomically transformed MSCswas comparable to that noticed in the study by Lee et al[26] for porcine NT blastocysts derived from epigenomicallynontransformed MSCs These blastocysts were also charac-terized by a total cells number equal to 48 In contrast to theabovementioned findings in the investigations carried outby Li et al [27] Kumar et al [28] and Jin et al [32] theoverall number of ICM and trophectoderm cells in clonedpig blastocysts originating from embryos reconstituted withTSA-untreated MSCs ranged from 28 to 35 and decreasedapproximately one and a half times as compared to the totalcells number estimated for NT blastocysts obtained by usin the TSA-exposed MSC group Nevertheless the meanapoptotic index (ie DCI = 495) that was calculated by usfor blastocysts developed from cloned embryos reconstitutedwith epigenetically modified MSC nuclei was similar to theratios of TUNEL-positive cells (46 to 47) observed by Jinet al [32] andKumar et al [28] among porcineNT blastocystsderived from epigenetically nonmodulated counterparts Inturn cloned blastocysts produced in our study using TSA-untreated MSCs displayed the apoptotic index (713) thatwas comparable or slightly lower to indexes identified forcloned embryos created with the aid of fetal fibroblast cellsnot undergoing exposure to trichostatin A [28 32]The latterindexes (DCIs) oscillated between 73 and 78 Generally inour current work the proportion of TUNEL-positive cellsin relation to a total cells number tended to insignificantlydecrease among NT blastocysts generated using undifferen-tiated mesenchymal stem cells subjected to TSA treatmentcompared to those generated using nuclear donor cells not
exposed to TSA (495 versus 713) Analogous slight down-ward tendency in the ratios of TUNEL-positive cells wasindicated byDiao et al [55] comparing blastocysts developedfrom cloned embryos reconstructed with cell nuclei of fetalfibroblasts treated with TSA and the counterparts producedusing TSA-unexposed fetal fibroblast cell nuclei (nearly 3versus 45)
Improved reprogrammability of transcriptional activityfor nuclear genome of epigenetically modulated MSCs inthe cells of preimplanted NT embryos turned out to bepositively correlated with enhanced molecular quality ofporcine cloned blastocysts assessed on the basis of theirpluripotency extent whichwasmeasuredwith the expressionprofiles identified for Oct4 and Nanog genes A 38-kDaprotein Oct4 (ie octamer-binding transcription factor 4)that is a member of the family of POU- (Pit-Oct-Unc-)domain and homeodomain transcription factors acts as avital regulator of pluripotency extent playing an importantrole in not only controlling preimplantation embryonicdevelopment but also maintenance of ICM cell fate inblastocysts and pluripotency status of embryonic stem cells(ESCs) [68ndash70] A 35-kDa protein designated as Nanog fromCelticIrish mythical Tır na nOg (Tir Na Nog The Landof the Ever-Young) is another homeobox-containing tran-scription factor that represents the group of pivotal proteinsmodulating pluripotency degree [70 71] The homeoproteinNanog can act synergistically with Oct4 protein in retainingthe pluripotent status of blastocyst-descended ICM andepiblast cells as well as in sustaining the undifferentiatedstatus and ability for self-renewal of ESCs [71 72] Theaberrant (ie downregulated) expression levels and patternsof Oct4 and Nanog transcripts in both bovine NT blastocystsproduced using epigenetically nonmodulated calf dermalfibroblast cells [73] and porcine NT blastocysts producedusing either epigenetically nonmodulated adult MSCs orfetal fibroblast cells [28] have been found to be a majorcause of not only their declined cytological and molecularquality but also decreased in vitro developmental potentialof cloned embryos However in our current study it seemsthat TSA-dependent epigenomic transformation of nucleardonor MSCs biases correctingly the expression profile ofOct4 andNanog mRNAs in cloned pig blastocysts triggeringboth significant enhancement of relative abundance (RA)of Oct4 transcripts and slight (nonsignificant) increase inNanog transcript RA as compared to the TSA-untreatedMSCgroup On the one hand this influence could be exerted bydirect diminishment in the deacetylation level within thenucleosomal core-derived histone lysine moieties that wasevoked by trichostatin A-mediated nonspecific inhibition ofHDACs On the other hand it could be probably elicitedvia indirect nonselective suppression of cytosine residuemethylation processes within the Oct4 and Nanog gene pro-moters andor enhancers leading to upregulated expressionof these crucial pluripotency-related genes Similar resultshave been shown in the study by Wang et al [73] in whichthe sequential treatment of bovine differentiated nucleardonor fibroblast cells and resultant cloned embryos by bothnonspecific inhibitors of DNMTs and HDACs (ie 5-aza-dC
10 BioMed Research International
and TSA resp) contributed to increase of Oct4 transcriptRA in the blastocysts obtained simultaneously sustainingthe expression of Nanog mRNAs on the unchanged levelas compared to the 5-aza-dC- and TSA-unexposed groupIn this case 5-aza-21015840-deoxycytidine- and trichostatin A-mediated epigenetic modulation of both nuclear donor cellsand cloned embryos gave rise presumably to direct down-regulation in the methylation of cytosine residues withinthe Oct4 gene promoter andor enhancer that resultedfrom 5-aza-dC-dependent nonspecific inhibition of DNMTsFurthermore this two-factor epigenetic modulation couldaffect the enhanced incidence of acetylation level within thenucleosomal core-derived histone lysine moieties that wastriggered by trichostatin A-mediated nonselective inhibitionof HDACs consequently leading to indirect upregulatedexpression of the Oct4 gene Taking into consideration allthe abovementioned findings considerable transcriptionalupregulation in the expression of Oct4 gene (confirmedamong porcine NT blastocysts in our present study andamong bovineNT blastocysts in the study byWang et al [73])can be found to be pivotal indicator of increased pluripotencyextent of cloned embryos This process appears to be alsoassociated with improved cytological quality of NT blasto-cysts that was measured with their total cell counts In thecurrent investigation we have shown that the overall numberof ICM and trophoblast cells in blastocysts was significantlyhigher following the reconstruction of NT pig embryos withepigenomically transformed mesenchymal stem cells
5 Conclusions and Future Goals
Summing up artificial epigenomic modulation of in vitrocultured MSCs using the nonspecific HDAC inhibitor desig-nated as TSA seems to facilitate much more the reprogram-ming process for epigenetically determined transcriptionalactivity of somatic cell-inherited nuclear genome in theNT pig embryos The use of ectopic HDAC inhibitors forepigenetic transformation of mesenchymal stem cells whosecell nuclei were transferred into enucleated oocytes is thecompletely new approach in the studies involving somaticcell cloning of pigs and other mammalian species Enhancedreprogrammability of nuclear genome descended from TSA-exposed MSCs in the blastomeres of porcine preimplantedcloned embryos resulted in the improvements of not onlytheir capacity to complete in vitro development to themorula and blastocyst stages but also cytological and molec-ular quality of the blastocysts produced Therefore furtherinvestigations are also necessary to determine whether thenovel strategy of TSA-dependent epigenetic modification ofnuclear donor MSCs which has been recently utilized in ourlaboratory for the future goals of generating cloned pigletsenables retaining the in vivo developmental competences ofthe high-quality NT-derived blastocysts to reach full term
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Marcin Samiec and Jolanta Opiela contributed equally to thepreparation of this paper
Acknowledgments
The project was funded by the Polish National ScienceCentre resources allocated on the basis of Decision no DEC-201103DNZ905537 The authors would like to thank DrJarosław Wieczorek DVM for bone marrow retrieval
References
[1] A J Bonk H-T Cheong R Li et al ldquoCorrelation of develop-mental differences of nuclear transfer embryos cells to themethylation profiles of nuclear transfer donor cells in swinerdquoEpigenetics vol 2 no 3 pp 179ndash186 2007
[2] A J Bonk R Li L Lai et al ldquoAberrant DNA methylation inporcine in vitro- parthenogenetic- and somatic cell nucleartransfer-produced blastocystsrdquo Molecular Reproduction andDevelopment vol 75 no 2 pp 250ndash264 2008
[3] M Samiec ldquoDevelopment of pig cloning studies past presentand futurerdquo Journal of Animal and Feed Sciences vol 13 no 2pp 211ndash238 2004
[4] K MWhitworth and R S Prather ldquoSomatic cell nuclear trans-fer efficiency how can it be improved through nuclear remod-eling and reprogrammingrdquoMolecular Reproduction and Devel-opment vol 77 no 12 pp 1001ndash1015 2010
[5] K-I Yamanaka S Sugimura T Wakai M Kawahara and ESato ldquoAcetylation level of histone H3 in early embryonic stagesaffects subsequent development of miniature pig somatic cellnuclear transfer embryosrdquo The Journal of Reproduction andDevelopment vol 55 no 6 pp 638ndash644 2009
[6] R P Cervera N Martı-Gutierrez E Escorihuela R Morenoand M Stojkovic ldquoTrichostatin A affects histone acetylationand gene expression in porcine somatic cell nucleus transferembryosrdquoTheriogenology vol 72 no 8 pp 1097ndash1110 2009
[7] J Zhao J Whyte and R S Prather ldquoEffect of epigeneticregulation during swine embryogenesis and on cloning bynuclear transferrdquo Cell and Tissue Research vol 341 no 1 pp13ndash21 2010
[8] WReik ldquoStability and flexibility of epigenetic gene regulation inmammalian developmentrdquo Nature vol 447 no 7143 pp 425ndash432 2007
[9] R S Prather J W Ross S C Isom and J A Green ldquoTranscrip-tional post-transcriptional and epigenetic control of porcineoocyte maturation and embryogenesisrdquo Society of Reproductionand Fertility supplement vol 66 pp 165ndash176 2009
[10] N Rodriguez-Osorio R Urrego J B Cibelli K Eilertsen andE Memili ldquoReprogramming mammalian somatic cellsrdquo Theri-ogenology vol 78 no 9 pp 1869ndash1886 2012
[11] K J Eilertsen R A Power L L Harkins and P Misica ldquoTar-geting cellular memory to reprogram the epigenome restorepotential and improve somatic cell nuclear transferrdquo AnimalReproduction Science vol 98 no 1-2 pp 129ndash146 2007
[12] M Samiec and M Skrzyszowska ldquoMolecular conditions of thecell nucleus remodellingreprogramming process and nucleartransferred embryo development in the intraooplasmic kary-oplast injection technique a reviewrdquo Czech Journal of AnimalScience vol 50 no 5 pp 185ndash195 2005
BioMed Research International 11
[13] P Narbonne K Miyamoto and J B Gurdon ldquoReprogrammingand development in nuclear transfer embryos and in interspe-cific systemsrdquo Current Opinion in Genetics amp Development vol22 no 5 pp 450ndash458 2012
[14] J-I Bang J-G Yoo M-R Park et al ldquoThe effects of artificialactivation timing on the development of SCNT-derivedembryos and newborn pigletsrdquoReproductive Biology vol 13 no2 pp 127ndash132 2013
[15] M Samiec and M Skrzyszowska ldquoHigh developmental capa-bility of porcine cloned embryos following trichostatin A-dependent epigenomic transformation during in vitro matura-tion of oocytes pre-exposed to R-roscovitinerdquo Animal SciencePapers and Reports vol 30 no 4 pp 383ndash393 2012
[16] M Samiec and M Skrzyszowska ldquoRoscovitine is a novel agentthat can be used for the activation of porcine oocytes recon-structed with adult cutaneous or fetal fibroblast cell nucleirdquoTheriogenology vol 78 no 8 pp 1855ndash1867 2012
[17] Z Li J Shi D Liu et al ldquoEffects of donor fibroblast cell typeand transferred cloned embryo number on the efficiency of pigcloningrdquo Cellular Reprogramming vol 15 no 1 pp 35ndash42 2013
[18] M Samiec and M Skrzyszowska ldquoBiological transcomple-mentary activation as a novel and effective strategy appliedto the generation of porcine somatic cell cloned embryosrdquoReproductive Biology vol 14 no 2 pp 128ndash139 2014
[19] J Li O Svarcova K Villemoes et al ldquoHigh in vitro developmentafter somatic cell nuclear transfer and trichostatin A treatmentof reconstructed porcine embryosrdquoTheriogenology vol 70 no5 pp 800ndash808 2008
[20] M Skrzyszowska M Samiec R Słomski D Lipinski and EMały ldquoDevelopment of porcine transgenic nuclear-transferredembryos derived from fibroblast cells transfected by the noveltechnique of nucleofection or standard lipofectionrdquo Theri-ogenology vol 70 no 2 pp 248ndash259 2008
[21] Y-W Pang L An P Wang et al ldquoTreatment of porcine donorcells and reconstructed embryoswith the antioxidantmelatoninenhances cloning efficiencyrdquo Journal of Pineal Research vol 54no 4 pp 389ndash397 2013
[22] M Samiec M Skrzyszowska and J Opiela ldquoCreation of clonedpig embryos using contact-inhibited or serum-starved fibrob-last cells analysed intra vitam for apoptosis occurrencerdquo Annalsof Animal Science vol 13 no 2 pp 275ndash293 2013
[23] H Wei Y Qing W Pan et al ldquoComparison of the efficiency ofBannaminiature inbred pig somatic cell nuclear transfer amongdifferent donor cellsrdquo PLoSONE vol 8 no 2 Article ID e577282013
[24] K Inoue N Ogonuki H Miki et al ldquoInefficient reprogram-ming of the hematopoietic stem cell genome following nucleartransferrdquo Journal of Cell Science vol 119 no 10 pp 1985ndash19912006
[25] K Inoue S Noda N Ogonuki et al ldquoDifferential developmen-tal ability of embryos cloned from tissue-specific stem cellsrdquoStem Cells vol 25 no 5 pp 1279ndash1285 2007
[26] S L Lee E J Kang G H Maeng et al ldquoDevelopmental abilityof miniature pig embryos cloned withmesenchymal stem cellsrdquoThe Journal of Reproduction and Development vol 56 no 2 pp256ndash262 2010
[27] Z Li X He L Chen et al ldquoBone marrow mesenchymal stemcells are an attractive donor cell type for production of clonedpigs as well as genetically modified cloned pigs by somatic cellnuclear transferrdquo Cellular Reprogramming vol 15 no 5 pp459ndash470 2013
[28] B M Kumar H-F Jin J-G Kim et al ldquoDifferential geneexpression patterns in porcine nuclear transfer embryos recon-structed with fetal fibroblasts and mesenchymal stem cellsrdquoDevelopmental Dynamics vol 236 no 2 pp 435ndash446 2007
[29] J Su Y Wang Y Li et al ldquoOxamflatin significantly improvesnuclear reprogramming blastocyst quality and in vitro devel-opment of bovine SCNT embryosrdquo PLoS ONE vol 6 no 8Article ID e23805 2011
[30] Y Song THai YWang et al ldquoEpigenetic reprogramming geneexpression and in vitro development of porcine SCNT embryosare significantly improved by a histone deacetylase inhibitor-m-carboxycinnamic acid bishydroxamide (CBHA)rdquo Protein ampCell vol 5 no 5 pp 382ndash393 2014
[31] S Colleoni G Donofrio I Lagutina R Duchi C Galli andG Lazzari ldquoEstablishment differentiation electroporationviral transduction and nuclear transfer of bovine and porcinemesenchymal stem cellsrdquo Cloning and Stem Cells vol 7 no 3pp 154ndash166 2005
[32] H F Jin B M Kumar J G Kim et al ldquoEnhanced developmentof porcine embryos cloned from bone marrow mesenchymalstem cellsrdquoThe International Journal of Developmental Biologyvol 51 no 1 pp 85ndash90 2007
[33] J Opiela and M Samiec ldquoCharacterization of mesenchymalstem cells and their application in experimental embryologyrdquoPolish Journal of Veterinary Sciences vol 16 no 3 pp 593ndash5992013
[34] X Yang S L Smith X C Tian H A Lewin J-P Renard and TWakayama ldquoNuclear reprogramming of cloned embryos and itsimplications for therapeutic cloningrdquo Nature Genetics vol 39no 3 pp 295ndash302 2007
[35] XWu Y Li G-P Li et al ldquoTrichostatin A improved epigeneticmodifications of transfected cells but did not improve subse-quent cloned embryo developmentrdquo Animal Biotechnology vol19 no 4 pp 211ndash224 2008
[36] F Bo L Di F Qing-Chang et al ldquoEffect of trichostatin A ontransfected donor cells and subsequent development of porcinecloned embryosrdquo Zygote vol 19 no 3 pp 237ndash243 2011
[37] X Ding Y Wang D Zhang Z Guo and Y Zhang ldquoIncreasedpre-implantation development of cloned bovine embryostreated with 5-aza-21015840-deoxycytidine and trichostatin ArdquoTheri-ogenology vol 70 no 4 pp 622ndash630 2008
[38] H S Lee X F Yu J I Bang et al ldquoEnhanced histone acetylationin somatic cells induced by a histone deacetylase inhibitorimproved inter-generic cloned leopard cat blastocystsrdquo Theri-ogenology vol 74 no 8 pp 1439ndash1449 2010
[39] S-F Ning Q-Y Li M-M Liang et al ldquoMethylation charac-teristics and developmental potential of Guangxi Bamaminipig(Sus scrofa domestica) cloned embryos from donor cells treatedwith trichostatin A and 5-aza-21015840-deoxycytidinerdquo Zygote vol 21no 2 pp 178ndash186 2013
[40] B P Enright L-Y Sung C-C Chang X Yang and X C TianldquoMethylation and acetylation characteristics of cloned bovineembryos from donor cells treated with 5-aza-21015840-deoxycytidinerdquoBiology of Reproduction vol 72 no 4 pp 944ndash948 2005
[41] Y J Huan J Zhu B T Xie et al ldquoTreating cloned embryos butnot donor cells with 5-aza-21015840-deoxycytidine enhances thedevelopmental competence of porcine cloned embryosrdquo TheJournal of Reproduction andDevelopment vol 59 no 5 pp 442ndash449 2013
[42] B-G JeonG Coppola S D Perrault G-J RhoDH Betts andWAKing ldquoS-adenosylhomocysteine treatment of adult female
12 BioMed Research International
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
matured giltsow oocytes were incubated in the matura-tion medium supplemented with 04 120583gmL demecolcine(DMCC) for 50 to 60min The DMCC-treated oocyteswere subsequently transferred into a micromanipulationchamber filled with TC 199 medium containing 4mgmLbovine serum albumin (fraction V BSA-V) and 75 120583gmLcytochalasin B (CB) Metaphase chromosomes which hadbeen allocated into the chemically induced protrusion ofthe plasma membrane were removed microsurgically Thechemically assisted enucleation was accomplished by gentlyaspirating the ooplasmic cone which contained the con-densed chromosome cluster with the aid of a bevelledmicropipette The reconstruction of enucleated oocytes wasachieved by their electrofusion with TSA-treated or TSA-untreated MSCs Single nuclear donor cells were insertedinto the perivitelline space of previously enucleated oocytes(ie ooplastscytoplasts) The resulting somatic cell-ooplastcouplets were placed into a fusionactivation chamber filledwith electroporation medium (dielectric solution) SCNT-derived oocytes were artificially stimulated using the protocolof simultaneous fusion and electrical activation (SF-EA) Inthe SF-EA protocol electric pulses that induced a fusion ofMSC-cytoplast couplets were simultaneously the stimuli ini-tiating the activation of reconstructed oocytesThe complexesof ooplasts and MSCs were subjected to plasma membraneelectroporation by application of two successive DC pulsesof 12 kVcm for 60120583s The electropermeabilization of cellplasma membranes was performed in an isotonic dielectricsolution (03M D-mannitol) with concentration of CaCl
2
increased up to 10mM Following SF-EA porcine clonedembryos were exposed to 5120583gmL CB for 2 h and subse-quently cultured in vitro in North Carolina State University-23 (NCSU-23) medium supplemented with 4mgmL BSA-V1 Eaglersquos Minimum Essential Medium-nonessential aminoacid solution (MEM-NEAA) and 2 MEM-essential aminoacid solution (MEM-EAA) for 72ndash96 h Afterwards cleavedembryos were cultured in NCSU-23BSA-V + MEM-NEAA+ MEM-EAA medium supplemented with 10 FBS for anadditional 72 h up to morula and blastocyst stages
27 Assessment of Cloned Blastocyst Quality by TUNELAssay The blastocysts developed from NT oocytes recon-structed with TSA-treated and -untreated MSCs wereanalyzed using a Deadend Fluorometric TUNEL System(Promega Warsaw Poland) according to the protocol ofterminal deoxynucleotidyl transferase- (TdT-) mediateddUTP (21015840-deoxyuridine-51015840-triphosphate) nick-end labelling(TUNEL) that was dependent on the fluorescein isothio-cyanate (FITC) conjugated with dUTP This protocol wasthoroughly described by Opiela et al [61 62] Briefly afterfixationwith 1paraformaldehyde (PFA) diluted inPBS solu-tion the embryos were rinsed extensively (3 times) Subse-quently they were exposed to 02 Triton X-100 solution for5min followed by 1 h incubation in the reactionmixture con-sisting of equilibration buffer a cocktail of nucleotides andTdT enzyme at a maximum humidity Finally the embryoswere placed into a 2x concentrated solution of salinesodiumchloride-sodiumtrisodium citrate (SSC) buffer for 15min
After all the incubations carried out cloned blastocystswere washed three times in PBSpolyvinylpyrrolidone (PVP)solution for at least 5min and lastly transferred into a dropof VECTASHIELD mounting medium This medium wassupplemented with 410158406-diamidino-2-phenylindole (DAPI)counterstain and has been found to be unsurpassed inpreventing photobleaching of DAPI fluorochrome in orderto retain antifading ability during long-term storage of dyedembryos The evaluation of embryos that had been subjectedto TUNEL analysis was performed under a fluorescencemicroscope (Nicon Eclipse E600 Tokyo Japan) The assess-ment of blastocyst cytological quality enabled visualizingthe total number of blastomere nuclei in each embryo andthe number of cell nuclei exhibiting internucleosomallyfragmented DNA into which a FITC-conjugated dUTP wasincorporated The fluorescence filters adjusted to excitationwavelength higher than 460 nm and the filters adapted toemission wavelengths ranging from 480 nm to 540 nm wereapplied to detect the blue fluorescence emitted by cell nucleiof all the DAPI-tagged blastocyst blastomeres and the FITC-derived green fluorescence emitted by cell nuclei of late-apoptotic blastomeres
28 Determination of Apoptotic Index (DCI) in Cloned Blasto-cysts All the blastocysts generated were subjected to TUNELanalysisThenumber of all DAPI-dyed blastomere nuclei (iethe total number of inner cell massICM and trophoblastcell nuclei) and the number of all TUNEL-positiveFITC-stained (ie apoptotic) cell nuclei were estimated for eachembryo To assess the apoptotic index (dead cell index DCI)in the single blastocyst the sum of all apoptotic cell nucleidetected in the analyzed blastocyst was divided by the sumof all ICMtrophoblast cell nuclei identified in this blastocystand subsequently multiplied by 100 [62]
29 Analysis of Porcine Cloned Embryos for Oct4 Nanog andNestin mRNA Expression Profile Using Quantitative ReverseTranscriptase Real-Time PCR (qRT-PCR) The qRT-PCR wasapplied to estimate the expression levels of mRNA tran-scripts for pluripotency-related target genes (Oct4 Nanog)and multipotent stemness-associated target gene (Nestin) intwo groups of SCNT-derived blastocysts encompassing theembryos developed from oocytes reconstructed with eitherTSA-treated or -untreated MSC nuclei Briefly 40 120583L ofSideStep Lysis amp Stabilization Buffer (Agilent Technologies)was added to 10 120583L of blastocysts sample (10 blastocysts)in PBS The SideStep Buffer was used to lyse the cells ofblastocysts and ensure RNA stabilization The lysed embryosamples were vortexed at room temperature for 1min Theone-step Brilliant II SYBR Green QRT-PCR Master MixKit (Agilent Technologies) was used to perform relativequantification of gene expression Each PCR probe (totalvolume of 25 120583L) was comprised of 1 120583L of embryo cell lysateand 24120583L of reaction mixture which consisted of 125 120583L 2xSYBRGreen QRT-PCRmaster mix (containing an optimizedRT-PCR buffer MgCl
2 nucleotides (GAUC) SureStart Taq
DNA polymerase daggerSYBR Green and stabilizers) and 04 120583Lof each 200 nM forward and reverse primer (Table 1) 1 120583L
BioMed Research International 5
Table 1 Primers used in quantitative reverse transcriptase real-time PCR (qRT-PCR)
of RTRNase block enzyme mixture and 97 120583L RNase-freewater Thermal cycling conditions were as follows 30min at50∘C (for the first-strand synthesis) 10min at 95∘C 40 cyclesof 30 s at 95∘C for denaturing 60 s at 57∘C for annealing and30 s at 72∘C for extension
The glyceraldehyde-3-phosphate dehydrogenase(GAPDH) housekeeping gene was used as an endogenousstandard The results for expression levelsprofiles of indi-vidual target genes (Oct4 Nanog and Nestin) were normal-ized according to the relative concentration of the endog-enous standard Each reaction was run in triplicate and theobtained results were averaged By sample we mean mRNAderived from a pool of 10 blastocysts We made six PCR runsfrom one mRNA sample 3 for three analyzed genes (Oct4Nanog and Nestin) and 3 for the GAPDH control gene EachcDNA synthesis and PCR amplification were achieved inone tube and one buffer All data are presented as the meanvalues of the relative abundance (RA) of Oct4 Nanog andNestin mRNA transcripts Expression of these transcriptsisolated from blastocysts that originated from TSA-treatedand -untreated groups was compared to that for controlblastocysts which served as a calibrator Experiments werecarried out on a Mastercycler Realplex (Eppendorf UK Lim-ited Cambridge)
210 Statistical Analysis The 1205942 test was applied to esti-mate the differences in the in vitro developmental capa-bilities of porcine NT embryos originating from TSA-treated and -untreated MSCs The statistical analysis of vari-ance (ANOVA) and Tukeyrsquos Honestly Significant Difference(HSD) post hoc test for multiple ranges were used for com-parison of the levels of Oct4 Nanog and Nestin transcriptsbetween blastocysts derived from oocytes reconstituted withthe cell nuclei of TSA-exposed and -unexposed MSCs TheANOVA and subsequent Tukeyrsquos HSD post hoc test werealso applied to compare the mean values calculated fortotal number of DAPI-labelled cell nuclei total number ofTUNEL-positiveFITC-labelled (ie apoptotic) cell nucleiand apoptotic index (DCI) between blastocysts developedfrom NT oocytes generated using epigenetically modulatedand nonmodulated MSCs The differences with a probability(P) less than 005 were considered to be significant
3 Results
31 The In Vitro Developmental Potential of Cloned EmbryosDerived from the Cell Nuclei of MSCs Undergoing or NotUndergoing TSA-Based Epigenomic Modulation Althoughthe cleavage activity did not vary between cultured nuclear-transferred embryos reconstituted fromMSCs subjected andnot subjected to TSA-dependent epigenetic transformation(119875 ge 005) the morula and blastocyst formation rates werecharacterized by statistically significant differences A signif-icant variability in the in vitro developmental competencesto reach the morula stage (119875 lt 005) and a very highlysignificant variability in the developmental competences toreach the blastocyst stage (119875 lt 0001 Figure 1) were observedbetween these two groups of cloned embryos respectivelyThe detailed data that are focused on the assessment ofextracorporeal developmental capability of NT embryosreconstituted with epigenetically nonmodulated or modu-lated MSCs are presented in Table 2 As compared to thepresent investigation in our previous studies in which the invitro developmental potential of cloned pig embryos derivedfrom the cell nuclei of fetal fibroblast cells (being currentlyused source of nuclear donor cells) and generated applyingthe same experimental protocols of SF-EA for artificialstimulation of nuclear-transferred oocytes was explored thepercentages of the obtained morulae and blastocysts rangedfrom 582 to 613 and from 296 to 332 respectively[16 18 20] It is worth noting that all these percentagesturned out to be considerably lower than themorula and blas-tocyst formation rates achieved for NT embryos producedusing epigenomically nontransformed or transformed MSCs(Table 2)
32 Dependence of the Cloned Blastocyst Quality Evaluated byTUNEL Assay on the TSA-Based Epigenomic Modulation ofMSCs The quality of nuclear-transferred embryos derivedfrom TSA-exposed MSCs was considerably higher than thatof NT embryos derived from TSA-unexposed MSCs Veryhighly significant differences in the mean number of DAPI-stained cell nuclei per blastocyst were shown between thesetwo groups of embryos (119875 lt 0005 Table 3 Figure 2) Inturn no significant differences in the mean number of
6 BioMed Research International
005mm
(a)
005mm
(b)
005mm
(c)
005mm
(d)
Figure 1 Porcine cloned blastocysts developed fromnuclear-transferred oocytes reconstitutedwith adult bonemarrow-derivedmesenchymalstem cells undergoing trichostatinA- (TSA-) dependent epigenomicmodulation (photographs (a) and (b)) or not undergoingTSA-dependentepigenomic modulation (photographs (c) and (d)) Images were taken at magnification times200
Table 2 Effect of the TSA-dependent epigenomic modulation of MSCs on the in vitro developmental outcome of cloned pig embryos
TSA-mediated epigenetictransformation of MSCs
Number of oocytesembryos Development toEnucleated Electrofused () Cleaved () Morulae () Blastocysts ()
minus 293 275293 (939) 262275 (953) 216275 (785)b 125275 (455)B
TSA trichostatin A MSC mesenchymal stem cellValues with different small superscript letters (a and b) within the same column denote statistically significant differences between experimental groups (119875 lt005 1205942 test) Values with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0001 1205942 test)Number of replicates ge6
Table 3 Effect of the TSA-mediated epigenomic modulation of MSCs on the cytological quality of cloned pig embryos assessed by TUNELassay
TSA-mediated epigenetictransformation of MSCs
Number ofanalyzedblastocysts
Mean number ofDAPI-tagged cell nuclei per
blastocyst plusmn SD
Mean number ofTUNEL-positive (apoptotic) cell
nuclei per blastocyst plusmn SD
Mean apoptotic index(DCI) per blastocyst plusmn
SD ()+ 31 4797 plusmn 2743A 161 plusmn 338 495 plusmn 1398minus 26 2777 plusmn 1582B 158 plusmn 269 713 plusmn 1244TSA trichostatin A MSC mesenchymal stem cell TUNEL terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling DAPI 410158406-diamidino-2-phenylindole SD standard deviation DCI dead cell indexValues with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0005 ANOVA followed byTukeyrsquos HSD post hoc test) Number of replicates = 6
TUNEL-positive (ie late-apoptotic) cell nuclei per blasto-cyst were identified between NT embryos reconstituted withMSCs undergoing and not undergoing TSA-mediated epige-netic transformation (119875 ge 005 Table 3 Figure 2) Althoughno significant differences in the DCI per blastocyst were
found between both experimental groups (119875 ge 005) theincidence of late apoptosis-related internucleosomal DNAfragmentation trended slightly upwards for NT embryosgenerated using TSA-untreated MSCs as compared to thoseproduced using TSA-treated MSCs (Table 3 Figure 2)
BioMed Research International 7
005mm
(a)
005mm
(b)
005mm
(c)
005mm
(d)
005mm
(e)
005mm
(f)
Figure 2 Evaluation of cytological quality of porcine cloned blastocysts on the basis of simultaneous determination of total nuclear numberand detection of apoptotic cell nuclei by terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling (TUNEL) analysis Photographs (a) to (d) depict embryos originating from nuclear-transferred (NT) oocytes reconstitutedwith adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) subjected to epigenetic transformation via trichostatin A (TSA)treatment Photographs (e) and (f) depict embryos originating from NT oocytes reconstituted with ABM-MSCs not subjected to epigenetictransformation via TSA treatment In each blastocyst the cell nuclei of all the blastomeres (both inner cell mass (ICM) and trophectoderm(TE) cells) had been tagged with 410158406-diamidino-2-phenylindole (DAPI) counterstain and subsequently fluoresced in blue The cell nucleiof late-apoptotic blastomeres (ICM andor TE cells) exhibiting internucleosomal DNA fragmentation had been dyed with fluoresceinisothiocyanate (FITC) and then fluoresced in bright green In each photograph the DAPI-derived blue and FITC-derived green fluorescentsignals merge into one another Photographs (a) to (f) represent the blastocysts displaying different incidence of blastomere apoptosisand thereby varied advancement of internucleosomal DNA fragmentation ((a) (c) (d) and (e) the lack of apoptotic intranuclear DNAfragmentation (b) and (e) few apoptotic cell nuclei (f) increased extent of apoptotic intranuclear DNA fragmentation) Images were takenat magnification times200
8 BioMed Research International
0
1
2
3
4
5
6
B
A
Oct4 Nanog Nestin
MSCTSAMSC
Figure 3 Relative abundance (mean plusmn SD) of Oct4 Nanog andNestin mRNAs in blastocysts developed from nuclear-transferredpig embryos descended from MSCs undergoing or not undergoingTSA-dependent epigenomic transformation The highly significantdifferences were designated with the A and B letters (119875 lt 001ANOVA followed by Tukeyrsquos HSD post hoc test)
33 The Pluripotency Status of Porcine Nuclear-TransferredEmbryos Originating from Epigenomically Modulated or Non-modulated MSCs The total number of analyzed clonedblastocysts was 60 To assess relative abundance (RA) of threeanalyzed mRNA transcripts for pluripotency-related targetgenes (Oct4 Nanog) multipotent stemness-associated targetgene (Nestin) and control GAPDH gene transcript we used3 times 10 blastocysts from both TSA-treated and -untreatedgroups
Highly significant differences were indicated for theOct4gene transcript quantitative profile between blastocysts orig-inating from TSA-treated and -untreated groups (119875 lt 001Figure 3) Although no significant intergroup variability wasshown in the RA of Nanog and Nestin mRNAs (119875 ge 005)their expression levels tended to be higher in blastocystsderived from NT embryos reconstructed with TSA-exposedMSCs as compared to the TSA-untreated group (Figure 3)
4 Discussion
The abundance of the morulae and blastocysts of highercytological and molecular quality generated in the presentinvestigation confirms that the reprogrammability of adultbone marrow-derived mesenchymal stem cells which hadbeen epigenetically modified via exposure to trichostatinA underwent considerable improvement in a cytoplasm ofporcine nuclear-transferred oocytes and resultant in vitrocultured cloned embryos It is beyond any doubt thatthe impact of TSA-based epigenomic transformation ofbone marrow-retrieved MSCs representing multipotent andundifferentiated stem cells on their competence for SCNThas not yet been explored not only in pigs but also inother mammalian species So far TSA-mediated epigeneticmodulation has been applied only for differentiated somatic
cells that commonly provide the source of nuclear donorsfor cloning procedure in pigs The results of the study byDiao et al [55] proved that porcineNT embryos reconstitutedwith TSA-treated fetal fibroblast cells were characterized bytwofold higher developmental potential to reach the blasto-cyst stage (30) than the NT embryos reconstituted withTSA-untreated fibroblast cells (15) Similar tendency hasbeen also shown in our current study inwhich trichostatinA-mediated epigenetic transformation of bone marrow-derivedMSCs gave rise to significant enhancement of capability ofcloned embryos to complete their development to blastocyststage (65) as compared to that observed for embryosderived from TSA-unexposed MSC nuclei (46) Nonethe-less NT pig embryos originating from epigenomically trans-formed fetal fibroblast cells displayed considerably lowerblastocyst formation rate [55] than the rate indicated in ourpresent study for NT embryos reconstructed with epige-nomically nontransformed MSCs Moreover treatment ofnuclear donor MSCs with trichostatin A resulted in one anda half times to severalfold increase of blastocyst percentage(65) as compared to the blastocyst yields obtained by otherinvestigators using undifferentiated or in vitro differentiatedMSCs that were not modulated epigenetically [26ndash28 31 32]In the study by Kumar et al [28] the abilities of epige-nomically nontransformed bone marrow-descended MSCnuclei to direct the in vitro development of porcine clonedembryos to blastocyst stage decreased above three times(approximately 20) as compared to our present work Inturn nuclear-transferred embryos that had originated fromporcine undifferentiated bone marrow-retrieved MSCs andtheir derivatives along the osteogenic lineage were able tocomplete their in vitro development to blastocyst stage atthe rates ranging from 33 to 45 [31] In contrast Jin etal [32] and Li et al [27] reported that blastocyst yields ofNT embryos derived from porcine epigenetically unmodu-lated MSCs were maintained at the relatively low levels ofapproximately 18 and 16 respectively Nevertheless in thestudy by Lee et al [26] the in vitro developmental outcometo the blastocyst stage of NT embryos originating fromundifferentiated MSCs achieved the rate of approximately48 but even this blastocyst formation rate was considerablylower than that noticed in our current investigation
The results of the current study have also clearly demon-strated that the TSA-dependent epigenomic transformationof nuclear donor MSCs contributes to improvement of notonly in vitro developmental competences but also qualityand transcription level-related pluripotency extent of porcinecloned embryos The utilization of undifferentiated mes-enchymal stem cells and the modification of the molecularmechanisms of transcriptional reprogramming of the donornuclear genome via HDAC inhibitor-mediated epigeneticmodulation of MSCs before their use for SCNT led to correctand complete adaptation of MSC-inherited genomic DNA tothe cytoplasmic environment of enucleated oocytes and invitro cultured NT pig embryos Epigenetic reprogrammingof donor cell nuclei suggests that a new program for theirtranscriptional activity is loaded and reloaded immediatelyfollowing reconstruction of enucleated oocytes The successof SCNT may depend upon both genomic DNA-associated
BioMed Research International 9
reprogramming of gene expression for dedifferentiation ofthe donor somatic cell nuclei during early preimplantationdevelopment of cloned embryos and reprogramming of geneexpression for onset of somatogenic nuclear redifferentiationduring blastocyst formation [7 10 63 64] It has been ascer-tained that somatic cell nuclei should undergo the wide DNAcytosine residue demethylation changes throughout the earlydevelopment of NT embryos to erase and then reset theirown overall epigenetic as well as parental genomic imprintingmemory which has been established by remethylation ofthe nuclear genome within the framework of the specificpathway of somatic and germ cell lineage commitment anddifferentiation [2 4 13 30 65ndash67]
It appears that the enhanced cytological quality of clonedpig blastocysts that originated from enucleated oocytesreceiving epigenetically modulated MSC nuclei could beassociated with more faithful and faultless pattern for repro-gramming of transcriptional activity of genomic DNA inher-ited from undifferentiated and multipotent stem cells Thesecells exhibit increased genomic and epigenomic plasticity inrearrangements of their gene expression in the blastomeres ofpreimplanted NT embryos The fluorocytochemical analysisof overall mean nuclear counts revealed almost twofoldhigher total cells number in porcine blastocysts developedfrom NT oocytes reconstituted with TSA-exposed MSCs (48blastomeres) than that identified in blastocysts derived fromNT oocytes reconstituted with TSA-unexposed MSCs (28blastomeres) The quality of cloned blastocysts generated inour present study using epigenomically transformed MSCswas comparable to that noticed in the study by Lee et al[26] for porcine NT blastocysts derived from epigenomicallynontransformed MSCs These blastocysts were also charac-terized by a total cells number equal to 48 In contrast to theabovementioned findings in the investigations carried outby Li et al [27] Kumar et al [28] and Jin et al [32] theoverall number of ICM and trophectoderm cells in clonedpig blastocysts originating from embryos reconstituted withTSA-untreated MSCs ranged from 28 to 35 and decreasedapproximately one and a half times as compared to the totalcells number estimated for NT blastocysts obtained by usin the TSA-exposed MSC group Nevertheless the meanapoptotic index (ie DCI = 495) that was calculated by usfor blastocysts developed from cloned embryos reconstitutedwith epigenetically modified MSC nuclei was similar to theratios of TUNEL-positive cells (46 to 47) observed by Jinet al [32] andKumar et al [28] among porcineNT blastocystsderived from epigenetically nonmodulated counterparts Inturn cloned blastocysts produced in our study using TSA-untreated MSCs displayed the apoptotic index (713) thatwas comparable or slightly lower to indexes identified forcloned embryos created with the aid of fetal fibroblast cellsnot undergoing exposure to trichostatin A [28 32]The latterindexes (DCIs) oscillated between 73 and 78 Generally inour current work the proportion of TUNEL-positive cellsin relation to a total cells number tended to insignificantlydecrease among NT blastocysts generated using undifferen-tiated mesenchymal stem cells subjected to TSA treatmentcompared to those generated using nuclear donor cells not
exposed to TSA (495 versus 713) Analogous slight down-ward tendency in the ratios of TUNEL-positive cells wasindicated byDiao et al [55] comparing blastocysts developedfrom cloned embryos reconstructed with cell nuclei of fetalfibroblasts treated with TSA and the counterparts producedusing TSA-unexposed fetal fibroblast cell nuclei (nearly 3versus 45)
Improved reprogrammability of transcriptional activityfor nuclear genome of epigenetically modulated MSCs inthe cells of preimplanted NT embryos turned out to bepositively correlated with enhanced molecular quality ofporcine cloned blastocysts assessed on the basis of theirpluripotency extent whichwasmeasuredwith the expressionprofiles identified for Oct4 and Nanog genes A 38-kDaprotein Oct4 (ie octamer-binding transcription factor 4)that is a member of the family of POU- (Pit-Oct-Unc-)domain and homeodomain transcription factors acts as avital regulator of pluripotency extent playing an importantrole in not only controlling preimplantation embryonicdevelopment but also maintenance of ICM cell fate inblastocysts and pluripotency status of embryonic stem cells(ESCs) [68ndash70] A 35-kDa protein designated as Nanog fromCelticIrish mythical Tır na nOg (Tir Na Nog The Landof the Ever-Young) is another homeobox-containing tran-scription factor that represents the group of pivotal proteinsmodulating pluripotency degree [70 71] The homeoproteinNanog can act synergistically with Oct4 protein in retainingthe pluripotent status of blastocyst-descended ICM andepiblast cells as well as in sustaining the undifferentiatedstatus and ability for self-renewal of ESCs [71 72] Theaberrant (ie downregulated) expression levels and patternsof Oct4 and Nanog transcripts in both bovine NT blastocystsproduced using epigenetically nonmodulated calf dermalfibroblast cells [73] and porcine NT blastocysts producedusing either epigenetically nonmodulated adult MSCs orfetal fibroblast cells [28] have been found to be a majorcause of not only their declined cytological and molecularquality but also decreased in vitro developmental potentialof cloned embryos However in our current study it seemsthat TSA-dependent epigenomic transformation of nucleardonor MSCs biases correctingly the expression profile ofOct4 andNanog mRNAs in cloned pig blastocysts triggeringboth significant enhancement of relative abundance (RA)of Oct4 transcripts and slight (nonsignificant) increase inNanog transcript RA as compared to the TSA-untreatedMSCgroup On the one hand this influence could be exerted bydirect diminishment in the deacetylation level within thenucleosomal core-derived histone lysine moieties that wasevoked by trichostatin A-mediated nonspecific inhibition ofHDACs On the other hand it could be probably elicitedvia indirect nonselective suppression of cytosine residuemethylation processes within the Oct4 and Nanog gene pro-moters andor enhancers leading to upregulated expressionof these crucial pluripotency-related genes Similar resultshave been shown in the study by Wang et al [73] in whichthe sequential treatment of bovine differentiated nucleardonor fibroblast cells and resultant cloned embryos by bothnonspecific inhibitors of DNMTs and HDACs (ie 5-aza-dC
10 BioMed Research International
and TSA resp) contributed to increase of Oct4 transcriptRA in the blastocysts obtained simultaneously sustainingthe expression of Nanog mRNAs on the unchanged levelas compared to the 5-aza-dC- and TSA-unexposed groupIn this case 5-aza-21015840-deoxycytidine- and trichostatin A-mediated epigenetic modulation of both nuclear donor cellsand cloned embryos gave rise presumably to direct down-regulation in the methylation of cytosine residues withinthe Oct4 gene promoter andor enhancer that resultedfrom 5-aza-dC-dependent nonspecific inhibition of DNMTsFurthermore this two-factor epigenetic modulation couldaffect the enhanced incidence of acetylation level within thenucleosomal core-derived histone lysine moieties that wastriggered by trichostatin A-mediated nonselective inhibitionof HDACs consequently leading to indirect upregulatedexpression of the Oct4 gene Taking into consideration allthe abovementioned findings considerable transcriptionalupregulation in the expression of Oct4 gene (confirmedamong porcine NT blastocysts in our present study andamong bovineNT blastocysts in the study byWang et al [73])can be found to be pivotal indicator of increased pluripotencyextent of cloned embryos This process appears to be alsoassociated with improved cytological quality of NT blasto-cysts that was measured with their total cell counts In thecurrent investigation we have shown that the overall numberof ICM and trophoblast cells in blastocysts was significantlyhigher following the reconstruction of NT pig embryos withepigenomically transformed mesenchymal stem cells
5 Conclusions and Future Goals
Summing up artificial epigenomic modulation of in vitrocultured MSCs using the nonspecific HDAC inhibitor desig-nated as TSA seems to facilitate much more the reprogram-ming process for epigenetically determined transcriptionalactivity of somatic cell-inherited nuclear genome in theNT pig embryos The use of ectopic HDAC inhibitors forepigenetic transformation of mesenchymal stem cells whosecell nuclei were transferred into enucleated oocytes is thecompletely new approach in the studies involving somaticcell cloning of pigs and other mammalian species Enhancedreprogrammability of nuclear genome descended from TSA-exposed MSCs in the blastomeres of porcine preimplantedcloned embryos resulted in the improvements of not onlytheir capacity to complete in vitro development to themorula and blastocyst stages but also cytological and molec-ular quality of the blastocysts produced Therefore furtherinvestigations are also necessary to determine whether thenovel strategy of TSA-dependent epigenetic modification ofnuclear donor MSCs which has been recently utilized in ourlaboratory for the future goals of generating cloned pigletsenables retaining the in vivo developmental competences ofthe high-quality NT-derived blastocysts to reach full term
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Marcin Samiec and Jolanta Opiela contributed equally to thepreparation of this paper
Acknowledgments
The project was funded by the Polish National ScienceCentre resources allocated on the basis of Decision no DEC-201103DNZ905537 The authors would like to thank DrJarosław Wieczorek DVM for bone marrow retrieval
References
[1] A J Bonk H-T Cheong R Li et al ldquoCorrelation of develop-mental differences of nuclear transfer embryos cells to themethylation profiles of nuclear transfer donor cells in swinerdquoEpigenetics vol 2 no 3 pp 179ndash186 2007
[2] A J Bonk R Li L Lai et al ldquoAberrant DNA methylation inporcine in vitro- parthenogenetic- and somatic cell nucleartransfer-produced blastocystsrdquo Molecular Reproduction andDevelopment vol 75 no 2 pp 250ndash264 2008
[3] M Samiec ldquoDevelopment of pig cloning studies past presentand futurerdquo Journal of Animal and Feed Sciences vol 13 no 2pp 211ndash238 2004
[4] K MWhitworth and R S Prather ldquoSomatic cell nuclear trans-fer efficiency how can it be improved through nuclear remod-eling and reprogrammingrdquoMolecular Reproduction and Devel-opment vol 77 no 12 pp 1001ndash1015 2010
[5] K-I Yamanaka S Sugimura T Wakai M Kawahara and ESato ldquoAcetylation level of histone H3 in early embryonic stagesaffects subsequent development of miniature pig somatic cellnuclear transfer embryosrdquo The Journal of Reproduction andDevelopment vol 55 no 6 pp 638ndash644 2009
[6] R P Cervera N Martı-Gutierrez E Escorihuela R Morenoand M Stojkovic ldquoTrichostatin A affects histone acetylationand gene expression in porcine somatic cell nucleus transferembryosrdquoTheriogenology vol 72 no 8 pp 1097ndash1110 2009
[7] J Zhao J Whyte and R S Prather ldquoEffect of epigeneticregulation during swine embryogenesis and on cloning bynuclear transferrdquo Cell and Tissue Research vol 341 no 1 pp13ndash21 2010
[8] WReik ldquoStability and flexibility of epigenetic gene regulation inmammalian developmentrdquo Nature vol 447 no 7143 pp 425ndash432 2007
[9] R S Prather J W Ross S C Isom and J A Green ldquoTranscrip-tional post-transcriptional and epigenetic control of porcineoocyte maturation and embryogenesisrdquo Society of Reproductionand Fertility supplement vol 66 pp 165ndash176 2009
[10] N Rodriguez-Osorio R Urrego J B Cibelli K Eilertsen andE Memili ldquoReprogramming mammalian somatic cellsrdquo Theri-ogenology vol 78 no 9 pp 1869ndash1886 2012
[11] K J Eilertsen R A Power L L Harkins and P Misica ldquoTar-geting cellular memory to reprogram the epigenome restorepotential and improve somatic cell nuclear transferrdquo AnimalReproduction Science vol 98 no 1-2 pp 129ndash146 2007
[12] M Samiec and M Skrzyszowska ldquoMolecular conditions of thecell nucleus remodellingreprogramming process and nucleartransferred embryo development in the intraooplasmic kary-oplast injection technique a reviewrdquo Czech Journal of AnimalScience vol 50 no 5 pp 185ndash195 2005
BioMed Research International 11
[13] P Narbonne K Miyamoto and J B Gurdon ldquoReprogrammingand development in nuclear transfer embryos and in interspe-cific systemsrdquo Current Opinion in Genetics amp Development vol22 no 5 pp 450ndash458 2012
[14] J-I Bang J-G Yoo M-R Park et al ldquoThe effects of artificialactivation timing on the development of SCNT-derivedembryos and newborn pigletsrdquoReproductive Biology vol 13 no2 pp 127ndash132 2013
[15] M Samiec and M Skrzyszowska ldquoHigh developmental capa-bility of porcine cloned embryos following trichostatin A-dependent epigenomic transformation during in vitro matura-tion of oocytes pre-exposed to R-roscovitinerdquo Animal SciencePapers and Reports vol 30 no 4 pp 383ndash393 2012
[16] M Samiec and M Skrzyszowska ldquoRoscovitine is a novel agentthat can be used for the activation of porcine oocytes recon-structed with adult cutaneous or fetal fibroblast cell nucleirdquoTheriogenology vol 78 no 8 pp 1855ndash1867 2012
[17] Z Li J Shi D Liu et al ldquoEffects of donor fibroblast cell typeand transferred cloned embryo number on the efficiency of pigcloningrdquo Cellular Reprogramming vol 15 no 1 pp 35ndash42 2013
[18] M Samiec and M Skrzyszowska ldquoBiological transcomple-mentary activation as a novel and effective strategy appliedto the generation of porcine somatic cell cloned embryosrdquoReproductive Biology vol 14 no 2 pp 128ndash139 2014
[19] J Li O Svarcova K Villemoes et al ldquoHigh in vitro developmentafter somatic cell nuclear transfer and trichostatin A treatmentof reconstructed porcine embryosrdquoTheriogenology vol 70 no5 pp 800ndash808 2008
[20] M Skrzyszowska M Samiec R Słomski D Lipinski and EMały ldquoDevelopment of porcine transgenic nuclear-transferredembryos derived from fibroblast cells transfected by the noveltechnique of nucleofection or standard lipofectionrdquo Theri-ogenology vol 70 no 2 pp 248ndash259 2008
[21] Y-W Pang L An P Wang et al ldquoTreatment of porcine donorcells and reconstructed embryoswith the antioxidantmelatoninenhances cloning efficiencyrdquo Journal of Pineal Research vol 54no 4 pp 389ndash397 2013
[22] M Samiec M Skrzyszowska and J Opiela ldquoCreation of clonedpig embryos using contact-inhibited or serum-starved fibrob-last cells analysed intra vitam for apoptosis occurrencerdquo Annalsof Animal Science vol 13 no 2 pp 275ndash293 2013
[23] H Wei Y Qing W Pan et al ldquoComparison of the efficiency ofBannaminiature inbred pig somatic cell nuclear transfer amongdifferent donor cellsrdquo PLoSONE vol 8 no 2 Article ID e577282013
[24] K Inoue N Ogonuki H Miki et al ldquoInefficient reprogram-ming of the hematopoietic stem cell genome following nucleartransferrdquo Journal of Cell Science vol 119 no 10 pp 1985ndash19912006
[25] K Inoue S Noda N Ogonuki et al ldquoDifferential developmen-tal ability of embryos cloned from tissue-specific stem cellsrdquoStem Cells vol 25 no 5 pp 1279ndash1285 2007
[26] S L Lee E J Kang G H Maeng et al ldquoDevelopmental abilityof miniature pig embryos cloned withmesenchymal stem cellsrdquoThe Journal of Reproduction and Development vol 56 no 2 pp256ndash262 2010
[27] Z Li X He L Chen et al ldquoBone marrow mesenchymal stemcells are an attractive donor cell type for production of clonedpigs as well as genetically modified cloned pigs by somatic cellnuclear transferrdquo Cellular Reprogramming vol 15 no 5 pp459ndash470 2013
[28] B M Kumar H-F Jin J-G Kim et al ldquoDifferential geneexpression patterns in porcine nuclear transfer embryos recon-structed with fetal fibroblasts and mesenchymal stem cellsrdquoDevelopmental Dynamics vol 236 no 2 pp 435ndash446 2007
[29] J Su Y Wang Y Li et al ldquoOxamflatin significantly improvesnuclear reprogramming blastocyst quality and in vitro devel-opment of bovine SCNT embryosrdquo PLoS ONE vol 6 no 8Article ID e23805 2011
[30] Y Song THai YWang et al ldquoEpigenetic reprogramming geneexpression and in vitro development of porcine SCNT embryosare significantly improved by a histone deacetylase inhibitor-m-carboxycinnamic acid bishydroxamide (CBHA)rdquo Protein ampCell vol 5 no 5 pp 382ndash393 2014
[31] S Colleoni G Donofrio I Lagutina R Duchi C Galli andG Lazzari ldquoEstablishment differentiation electroporationviral transduction and nuclear transfer of bovine and porcinemesenchymal stem cellsrdquo Cloning and Stem Cells vol 7 no 3pp 154ndash166 2005
[32] H F Jin B M Kumar J G Kim et al ldquoEnhanced developmentof porcine embryos cloned from bone marrow mesenchymalstem cellsrdquoThe International Journal of Developmental Biologyvol 51 no 1 pp 85ndash90 2007
[33] J Opiela and M Samiec ldquoCharacterization of mesenchymalstem cells and their application in experimental embryologyrdquoPolish Journal of Veterinary Sciences vol 16 no 3 pp 593ndash5992013
[34] X Yang S L Smith X C Tian H A Lewin J-P Renard and TWakayama ldquoNuclear reprogramming of cloned embryos and itsimplications for therapeutic cloningrdquo Nature Genetics vol 39no 3 pp 295ndash302 2007
[35] XWu Y Li G-P Li et al ldquoTrichostatin A improved epigeneticmodifications of transfected cells but did not improve subse-quent cloned embryo developmentrdquo Animal Biotechnology vol19 no 4 pp 211ndash224 2008
[36] F Bo L Di F Qing-Chang et al ldquoEffect of trichostatin A ontransfected donor cells and subsequent development of porcinecloned embryosrdquo Zygote vol 19 no 3 pp 237ndash243 2011
[37] X Ding Y Wang D Zhang Z Guo and Y Zhang ldquoIncreasedpre-implantation development of cloned bovine embryostreated with 5-aza-21015840-deoxycytidine and trichostatin ArdquoTheri-ogenology vol 70 no 4 pp 622ndash630 2008
[38] H S Lee X F Yu J I Bang et al ldquoEnhanced histone acetylationin somatic cells induced by a histone deacetylase inhibitorimproved inter-generic cloned leopard cat blastocystsrdquo Theri-ogenology vol 74 no 8 pp 1439ndash1449 2010
[39] S-F Ning Q-Y Li M-M Liang et al ldquoMethylation charac-teristics and developmental potential of Guangxi Bamaminipig(Sus scrofa domestica) cloned embryos from donor cells treatedwith trichostatin A and 5-aza-21015840-deoxycytidinerdquo Zygote vol 21no 2 pp 178ndash186 2013
[40] B P Enright L-Y Sung C-C Chang X Yang and X C TianldquoMethylation and acetylation characteristics of cloned bovineembryos from donor cells treated with 5-aza-21015840-deoxycytidinerdquoBiology of Reproduction vol 72 no 4 pp 944ndash948 2005
[41] Y J Huan J Zhu B T Xie et al ldquoTreating cloned embryos butnot donor cells with 5-aza-21015840-deoxycytidine enhances thedevelopmental competence of porcine cloned embryosrdquo TheJournal of Reproduction andDevelopment vol 59 no 5 pp 442ndash449 2013
[42] B-G JeonG Coppola S D Perrault G-J RhoDH Betts andWAKing ldquoS-adenosylhomocysteine treatment of adult female
12 BioMed Research International
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
of RTRNase block enzyme mixture and 97 120583L RNase-freewater Thermal cycling conditions were as follows 30min at50∘C (for the first-strand synthesis) 10min at 95∘C 40 cyclesof 30 s at 95∘C for denaturing 60 s at 57∘C for annealing and30 s at 72∘C for extension
The glyceraldehyde-3-phosphate dehydrogenase(GAPDH) housekeeping gene was used as an endogenousstandard The results for expression levelsprofiles of indi-vidual target genes (Oct4 Nanog and Nestin) were normal-ized according to the relative concentration of the endog-enous standard Each reaction was run in triplicate and theobtained results were averaged By sample we mean mRNAderived from a pool of 10 blastocysts We made six PCR runsfrom one mRNA sample 3 for three analyzed genes (Oct4Nanog and Nestin) and 3 for the GAPDH control gene EachcDNA synthesis and PCR amplification were achieved inone tube and one buffer All data are presented as the meanvalues of the relative abundance (RA) of Oct4 Nanog andNestin mRNA transcripts Expression of these transcriptsisolated from blastocysts that originated from TSA-treatedand -untreated groups was compared to that for controlblastocysts which served as a calibrator Experiments werecarried out on a Mastercycler Realplex (Eppendorf UK Lim-ited Cambridge)
210 Statistical Analysis The 1205942 test was applied to esti-mate the differences in the in vitro developmental capa-bilities of porcine NT embryos originating from TSA-treated and -untreated MSCs The statistical analysis of vari-ance (ANOVA) and Tukeyrsquos Honestly Significant Difference(HSD) post hoc test for multiple ranges were used for com-parison of the levels of Oct4 Nanog and Nestin transcriptsbetween blastocysts derived from oocytes reconstituted withthe cell nuclei of TSA-exposed and -unexposed MSCs TheANOVA and subsequent Tukeyrsquos HSD post hoc test werealso applied to compare the mean values calculated fortotal number of DAPI-labelled cell nuclei total number ofTUNEL-positiveFITC-labelled (ie apoptotic) cell nucleiand apoptotic index (DCI) between blastocysts developedfrom NT oocytes generated using epigenetically modulatedand nonmodulated MSCs The differences with a probability(P) less than 005 were considered to be significant
3 Results
31 The In Vitro Developmental Potential of Cloned EmbryosDerived from the Cell Nuclei of MSCs Undergoing or NotUndergoing TSA-Based Epigenomic Modulation Althoughthe cleavage activity did not vary between cultured nuclear-transferred embryos reconstituted fromMSCs subjected andnot subjected to TSA-dependent epigenetic transformation(119875 ge 005) the morula and blastocyst formation rates werecharacterized by statistically significant differences A signif-icant variability in the in vitro developmental competencesto reach the morula stage (119875 lt 005) and a very highlysignificant variability in the developmental competences toreach the blastocyst stage (119875 lt 0001 Figure 1) were observedbetween these two groups of cloned embryos respectivelyThe detailed data that are focused on the assessment ofextracorporeal developmental capability of NT embryosreconstituted with epigenetically nonmodulated or modu-lated MSCs are presented in Table 2 As compared to thepresent investigation in our previous studies in which the invitro developmental potential of cloned pig embryos derivedfrom the cell nuclei of fetal fibroblast cells (being currentlyused source of nuclear donor cells) and generated applyingthe same experimental protocols of SF-EA for artificialstimulation of nuclear-transferred oocytes was explored thepercentages of the obtained morulae and blastocysts rangedfrom 582 to 613 and from 296 to 332 respectively[16 18 20] It is worth noting that all these percentagesturned out to be considerably lower than themorula and blas-tocyst formation rates achieved for NT embryos producedusing epigenomically nontransformed or transformed MSCs(Table 2)
32 Dependence of the Cloned Blastocyst Quality Evaluated byTUNEL Assay on the TSA-Based Epigenomic Modulation ofMSCs The quality of nuclear-transferred embryos derivedfrom TSA-exposed MSCs was considerably higher than thatof NT embryos derived from TSA-unexposed MSCs Veryhighly significant differences in the mean number of DAPI-stained cell nuclei per blastocyst were shown between thesetwo groups of embryos (119875 lt 0005 Table 3 Figure 2) Inturn no significant differences in the mean number of
6 BioMed Research International
005mm
(a)
005mm
(b)
005mm
(c)
005mm
(d)
Figure 1 Porcine cloned blastocysts developed fromnuclear-transferred oocytes reconstitutedwith adult bonemarrow-derivedmesenchymalstem cells undergoing trichostatinA- (TSA-) dependent epigenomicmodulation (photographs (a) and (b)) or not undergoingTSA-dependentepigenomic modulation (photographs (c) and (d)) Images were taken at magnification times200
Table 2 Effect of the TSA-dependent epigenomic modulation of MSCs on the in vitro developmental outcome of cloned pig embryos
TSA-mediated epigenetictransformation of MSCs
Number of oocytesembryos Development toEnucleated Electrofused () Cleaved () Morulae () Blastocysts ()
minus 293 275293 (939) 262275 (953) 216275 (785)b 125275 (455)B
TSA trichostatin A MSC mesenchymal stem cellValues with different small superscript letters (a and b) within the same column denote statistically significant differences between experimental groups (119875 lt005 1205942 test) Values with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0001 1205942 test)Number of replicates ge6
Table 3 Effect of the TSA-mediated epigenomic modulation of MSCs on the cytological quality of cloned pig embryos assessed by TUNELassay
TSA-mediated epigenetictransformation of MSCs
Number ofanalyzedblastocysts
Mean number ofDAPI-tagged cell nuclei per
blastocyst plusmn SD
Mean number ofTUNEL-positive (apoptotic) cell
nuclei per blastocyst plusmn SD
Mean apoptotic index(DCI) per blastocyst plusmn
SD ()+ 31 4797 plusmn 2743A 161 plusmn 338 495 plusmn 1398minus 26 2777 plusmn 1582B 158 plusmn 269 713 plusmn 1244TSA trichostatin A MSC mesenchymal stem cell TUNEL terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling DAPI 410158406-diamidino-2-phenylindole SD standard deviation DCI dead cell indexValues with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0005 ANOVA followed byTukeyrsquos HSD post hoc test) Number of replicates = 6
TUNEL-positive (ie late-apoptotic) cell nuclei per blasto-cyst were identified between NT embryos reconstituted withMSCs undergoing and not undergoing TSA-mediated epige-netic transformation (119875 ge 005 Table 3 Figure 2) Althoughno significant differences in the DCI per blastocyst were
found between both experimental groups (119875 ge 005) theincidence of late apoptosis-related internucleosomal DNAfragmentation trended slightly upwards for NT embryosgenerated using TSA-untreated MSCs as compared to thoseproduced using TSA-treated MSCs (Table 3 Figure 2)
BioMed Research International 7
005mm
(a)
005mm
(b)
005mm
(c)
005mm
(d)
005mm
(e)
005mm
(f)
Figure 2 Evaluation of cytological quality of porcine cloned blastocysts on the basis of simultaneous determination of total nuclear numberand detection of apoptotic cell nuclei by terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling (TUNEL) analysis Photographs (a) to (d) depict embryos originating from nuclear-transferred (NT) oocytes reconstitutedwith adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) subjected to epigenetic transformation via trichostatin A (TSA)treatment Photographs (e) and (f) depict embryos originating from NT oocytes reconstituted with ABM-MSCs not subjected to epigenetictransformation via TSA treatment In each blastocyst the cell nuclei of all the blastomeres (both inner cell mass (ICM) and trophectoderm(TE) cells) had been tagged with 410158406-diamidino-2-phenylindole (DAPI) counterstain and subsequently fluoresced in blue The cell nucleiof late-apoptotic blastomeres (ICM andor TE cells) exhibiting internucleosomal DNA fragmentation had been dyed with fluoresceinisothiocyanate (FITC) and then fluoresced in bright green In each photograph the DAPI-derived blue and FITC-derived green fluorescentsignals merge into one another Photographs (a) to (f) represent the blastocysts displaying different incidence of blastomere apoptosisand thereby varied advancement of internucleosomal DNA fragmentation ((a) (c) (d) and (e) the lack of apoptotic intranuclear DNAfragmentation (b) and (e) few apoptotic cell nuclei (f) increased extent of apoptotic intranuclear DNA fragmentation) Images were takenat magnification times200
8 BioMed Research International
0
1
2
3
4
5
6
B
A
Oct4 Nanog Nestin
MSCTSAMSC
Figure 3 Relative abundance (mean plusmn SD) of Oct4 Nanog andNestin mRNAs in blastocysts developed from nuclear-transferredpig embryos descended from MSCs undergoing or not undergoingTSA-dependent epigenomic transformation The highly significantdifferences were designated with the A and B letters (119875 lt 001ANOVA followed by Tukeyrsquos HSD post hoc test)
33 The Pluripotency Status of Porcine Nuclear-TransferredEmbryos Originating from Epigenomically Modulated or Non-modulated MSCs The total number of analyzed clonedblastocysts was 60 To assess relative abundance (RA) of threeanalyzed mRNA transcripts for pluripotency-related targetgenes (Oct4 Nanog) multipotent stemness-associated targetgene (Nestin) and control GAPDH gene transcript we used3 times 10 blastocysts from both TSA-treated and -untreatedgroups
Highly significant differences were indicated for theOct4gene transcript quantitative profile between blastocysts orig-inating from TSA-treated and -untreated groups (119875 lt 001Figure 3) Although no significant intergroup variability wasshown in the RA of Nanog and Nestin mRNAs (119875 ge 005)their expression levels tended to be higher in blastocystsderived from NT embryos reconstructed with TSA-exposedMSCs as compared to the TSA-untreated group (Figure 3)
4 Discussion
The abundance of the morulae and blastocysts of highercytological and molecular quality generated in the presentinvestigation confirms that the reprogrammability of adultbone marrow-derived mesenchymal stem cells which hadbeen epigenetically modified via exposure to trichostatinA underwent considerable improvement in a cytoplasm ofporcine nuclear-transferred oocytes and resultant in vitrocultured cloned embryos It is beyond any doubt thatthe impact of TSA-based epigenomic transformation ofbone marrow-retrieved MSCs representing multipotent andundifferentiated stem cells on their competence for SCNThas not yet been explored not only in pigs but also inother mammalian species So far TSA-mediated epigeneticmodulation has been applied only for differentiated somatic
cells that commonly provide the source of nuclear donorsfor cloning procedure in pigs The results of the study byDiao et al [55] proved that porcineNT embryos reconstitutedwith TSA-treated fetal fibroblast cells were characterized bytwofold higher developmental potential to reach the blasto-cyst stage (30) than the NT embryos reconstituted withTSA-untreated fibroblast cells (15) Similar tendency hasbeen also shown in our current study inwhich trichostatinA-mediated epigenetic transformation of bone marrow-derivedMSCs gave rise to significant enhancement of capability ofcloned embryos to complete their development to blastocyststage (65) as compared to that observed for embryosderived from TSA-unexposed MSC nuclei (46) Nonethe-less NT pig embryos originating from epigenomically trans-formed fetal fibroblast cells displayed considerably lowerblastocyst formation rate [55] than the rate indicated in ourpresent study for NT embryos reconstructed with epige-nomically nontransformed MSCs Moreover treatment ofnuclear donor MSCs with trichostatin A resulted in one anda half times to severalfold increase of blastocyst percentage(65) as compared to the blastocyst yields obtained by otherinvestigators using undifferentiated or in vitro differentiatedMSCs that were not modulated epigenetically [26ndash28 31 32]In the study by Kumar et al [28] the abilities of epige-nomically nontransformed bone marrow-descended MSCnuclei to direct the in vitro development of porcine clonedembryos to blastocyst stage decreased above three times(approximately 20) as compared to our present work Inturn nuclear-transferred embryos that had originated fromporcine undifferentiated bone marrow-retrieved MSCs andtheir derivatives along the osteogenic lineage were able tocomplete their in vitro development to blastocyst stage atthe rates ranging from 33 to 45 [31] In contrast Jin etal [32] and Li et al [27] reported that blastocyst yields ofNT embryos derived from porcine epigenetically unmodu-lated MSCs were maintained at the relatively low levels ofapproximately 18 and 16 respectively Nevertheless in thestudy by Lee et al [26] the in vitro developmental outcometo the blastocyst stage of NT embryos originating fromundifferentiated MSCs achieved the rate of approximately48 but even this blastocyst formation rate was considerablylower than that noticed in our current investigation
The results of the current study have also clearly demon-strated that the TSA-dependent epigenomic transformationof nuclear donor MSCs contributes to improvement of notonly in vitro developmental competences but also qualityand transcription level-related pluripotency extent of porcinecloned embryos The utilization of undifferentiated mes-enchymal stem cells and the modification of the molecularmechanisms of transcriptional reprogramming of the donornuclear genome via HDAC inhibitor-mediated epigeneticmodulation of MSCs before their use for SCNT led to correctand complete adaptation of MSC-inherited genomic DNA tothe cytoplasmic environment of enucleated oocytes and invitro cultured NT pig embryos Epigenetic reprogrammingof donor cell nuclei suggests that a new program for theirtranscriptional activity is loaded and reloaded immediatelyfollowing reconstruction of enucleated oocytes The successof SCNT may depend upon both genomic DNA-associated
BioMed Research International 9
reprogramming of gene expression for dedifferentiation ofthe donor somatic cell nuclei during early preimplantationdevelopment of cloned embryos and reprogramming of geneexpression for onset of somatogenic nuclear redifferentiationduring blastocyst formation [7 10 63 64] It has been ascer-tained that somatic cell nuclei should undergo the wide DNAcytosine residue demethylation changes throughout the earlydevelopment of NT embryos to erase and then reset theirown overall epigenetic as well as parental genomic imprintingmemory which has been established by remethylation ofthe nuclear genome within the framework of the specificpathway of somatic and germ cell lineage commitment anddifferentiation [2 4 13 30 65ndash67]
It appears that the enhanced cytological quality of clonedpig blastocysts that originated from enucleated oocytesreceiving epigenetically modulated MSC nuclei could beassociated with more faithful and faultless pattern for repro-gramming of transcriptional activity of genomic DNA inher-ited from undifferentiated and multipotent stem cells Thesecells exhibit increased genomic and epigenomic plasticity inrearrangements of their gene expression in the blastomeres ofpreimplanted NT embryos The fluorocytochemical analysisof overall mean nuclear counts revealed almost twofoldhigher total cells number in porcine blastocysts developedfrom NT oocytes reconstituted with TSA-exposed MSCs (48blastomeres) than that identified in blastocysts derived fromNT oocytes reconstituted with TSA-unexposed MSCs (28blastomeres) The quality of cloned blastocysts generated inour present study using epigenomically transformed MSCswas comparable to that noticed in the study by Lee et al[26] for porcine NT blastocysts derived from epigenomicallynontransformed MSCs These blastocysts were also charac-terized by a total cells number equal to 48 In contrast to theabovementioned findings in the investigations carried outby Li et al [27] Kumar et al [28] and Jin et al [32] theoverall number of ICM and trophectoderm cells in clonedpig blastocysts originating from embryos reconstituted withTSA-untreated MSCs ranged from 28 to 35 and decreasedapproximately one and a half times as compared to the totalcells number estimated for NT blastocysts obtained by usin the TSA-exposed MSC group Nevertheless the meanapoptotic index (ie DCI = 495) that was calculated by usfor blastocysts developed from cloned embryos reconstitutedwith epigenetically modified MSC nuclei was similar to theratios of TUNEL-positive cells (46 to 47) observed by Jinet al [32] andKumar et al [28] among porcineNT blastocystsderived from epigenetically nonmodulated counterparts Inturn cloned blastocysts produced in our study using TSA-untreated MSCs displayed the apoptotic index (713) thatwas comparable or slightly lower to indexes identified forcloned embryos created with the aid of fetal fibroblast cellsnot undergoing exposure to trichostatin A [28 32]The latterindexes (DCIs) oscillated between 73 and 78 Generally inour current work the proportion of TUNEL-positive cellsin relation to a total cells number tended to insignificantlydecrease among NT blastocysts generated using undifferen-tiated mesenchymal stem cells subjected to TSA treatmentcompared to those generated using nuclear donor cells not
exposed to TSA (495 versus 713) Analogous slight down-ward tendency in the ratios of TUNEL-positive cells wasindicated byDiao et al [55] comparing blastocysts developedfrom cloned embryos reconstructed with cell nuclei of fetalfibroblasts treated with TSA and the counterparts producedusing TSA-unexposed fetal fibroblast cell nuclei (nearly 3versus 45)
Improved reprogrammability of transcriptional activityfor nuclear genome of epigenetically modulated MSCs inthe cells of preimplanted NT embryos turned out to bepositively correlated with enhanced molecular quality ofporcine cloned blastocysts assessed on the basis of theirpluripotency extent whichwasmeasuredwith the expressionprofiles identified for Oct4 and Nanog genes A 38-kDaprotein Oct4 (ie octamer-binding transcription factor 4)that is a member of the family of POU- (Pit-Oct-Unc-)domain and homeodomain transcription factors acts as avital regulator of pluripotency extent playing an importantrole in not only controlling preimplantation embryonicdevelopment but also maintenance of ICM cell fate inblastocysts and pluripotency status of embryonic stem cells(ESCs) [68ndash70] A 35-kDa protein designated as Nanog fromCelticIrish mythical Tır na nOg (Tir Na Nog The Landof the Ever-Young) is another homeobox-containing tran-scription factor that represents the group of pivotal proteinsmodulating pluripotency degree [70 71] The homeoproteinNanog can act synergistically with Oct4 protein in retainingthe pluripotent status of blastocyst-descended ICM andepiblast cells as well as in sustaining the undifferentiatedstatus and ability for self-renewal of ESCs [71 72] Theaberrant (ie downregulated) expression levels and patternsof Oct4 and Nanog transcripts in both bovine NT blastocystsproduced using epigenetically nonmodulated calf dermalfibroblast cells [73] and porcine NT blastocysts producedusing either epigenetically nonmodulated adult MSCs orfetal fibroblast cells [28] have been found to be a majorcause of not only their declined cytological and molecularquality but also decreased in vitro developmental potentialof cloned embryos However in our current study it seemsthat TSA-dependent epigenomic transformation of nucleardonor MSCs biases correctingly the expression profile ofOct4 andNanog mRNAs in cloned pig blastocysts triggeringboth significant enhancement of relative abundance (RA)of Oct4 transcripts and slight (nonsignificant) increase inNanog transcript RA as compared to the TSA-untreatedMSCgroup On the one hand this influence could be exerted bydirect diminishment in the deacetylation level within thenucleosomal core-derived histone lysine moieties that wasevoked by trichostatin A-mediated nonspecific inhibition ofHDACs On the other hand it could be probably elicitedvia indirect nonselective suppression of cytosine residuemethylation processes within the Oct4 and Nanog gene pro-moters andor enhancers leading to upregulated expressionof these crucial pluripotency-related genes Similar resultshave been shown in the study by Wang et al [73] in whichthe sequential treatment of bovine differentiated nucleardonor fibroblast cells and resultant cloned embryos by bothnonspecific inhibitors of DNMTs and HDACs (ie 5-aza-dC
10 BioMed Research International
and TSA resp) contributed to increase of Oct4 transcriptRA in the blastocysts obtained simultaneously sustainingthe expression of Nanog mRNAs on the unchanged levelas compared to the 5-aza-dC- and TSA-unexposed groupIn this case 5-aza-21015840-deoxycytidine- and trichostatin A-mediated epigenetic modulation of both nuclear donor cellsand cloned embryos gave rise presumably to direct down-regulation in the methylation of cytosine residues withinthe Oct4 gene promoter andor enhancer that resultedfrom 5-aza-dC-dependent nonspecific inhibition of DNMTsFurthermore this two-factor epigenetic modulation couldaffect the enhanced incidence of acetylation level within thenucleosomal core-derived histone lysine moieties that wastriggered by trichostatin A-mediated nonselective inhibitionof HDACs consequently leading to indirect upregulatedexpression of the Oct4 gene Taking into consideration allthe abovementioned findings considerable transcriptionalupregulation in the expression of Oct4 gene (confirmedamong porcine NT blastocysts in our present study andamong bovineNT blastocysts in the study byWang et al [73])can be found to be pivotal indicator of increased pluripotencyextent of cloned embryos This process appears to be alsoassociated with improved cytological quality of NT blasto-cysts that was measured with their total cell counts In thecurrent investigation we have shown that the overall numberof ICM and trophoblast cells in blastocysts was significantlyhigher following the reconstruction of NT pig embryos withepigenomically transformed mesenchymal stem cells
5 Conclusions and Future Goals
Summing up artificial epigenomic modulation of in vitrocultured MSCs using the nonspecific HDAC inhibitor desig-nated as TSA seems to facilitate much more the reprogram-ming process for epigenetically determined transcriptionalactivity of somatic cell-inherited nuclear genome in theNT pig embryos The use of ectopic HDAC inhibitors forepigenetic transformation of mesenchymal stem cells whosecell nuclei were transferred into enucleated oocytes is thecompletely new approach in the studies involving somaticcell cloning of pigs and other mammalian species Enhancedreprogrammability of nuclear genome descended from TSA-exposed MSCs in the blastomeres of porcine preimplantedcloned embryos resulted in the improvements of not onlytheir capacity to complete in vitro development to themorula and blastocyst stages but also cytological and molec-ular quality of the blastocysts produced Therefore furtherinvestigations are also necessary to determine whether thenovel strategy of TSA-dependent epigenetic modification ofnuclear donor MSCs which has been recently utilized in ourlaboratory for the future goals of generating cloned pigletsenables retaining the in vivo developmental competences ofthe high-quality NT-derived blastocysts to reach full term
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Marcin Samiec and Jolanta Opiela contributed equally to thepreparation of this paper
Acknowledgments
The project was funded by the Polish National ScienceCentre resources allocated on the basis of Decision no DEC-201103DNZ905537 The authors would like to thank DrJarosław Wieczorek DVM for bone marrow retrieval
References
[1] A J Bonk H-T Cheong R Li et al ldquoCorrelation of develop-mental differences of nuclear transfer embryos cells to themethylation profiles of nuclear transfer donor cells in swinerdquoEpigenetics vol 2 no 3 pp 179ndash186 2007
[2] A J Bonk R Li L Lai et al ldquoAberrant DNA methylation inporcine in vitro- parthenogenetic- and somatic cell nucleartransfer-produced blastocystsrdquo Molecular Reproduction andDevelopment vol 75 no 2 pp 250ndash264 2008
[3] M Samiec ldquoDevelopment of pig cloning studies past presentand futurerdquo Journal of Animal and Feed Sciences vol 13 no 2pp 211ndash238 2004
[4] K MWhitworth and R S Prather ldquoSomatic cell nuclear trans-fer efficiency how can it be improved through nuclear remod-eling and reprogrammingrdquoMolecular Reproduction and Devel-opment vol 77 no 12 pp 1001ndash1015 2010
[5] K-I Yamanaka S Sugimura T Wakai M Kawahara and ESato ldquoAcetylation level of histone H3 in early embryonic stagesaffects subsequent development of miniature pig somatic cellnuclear transfer embryosrdquo The Journal of Reproduction andDevelopment vol 55 no 6 pp 638ndash644 2009
[6] R P Cervera N Martı-Gutierrez E Escorihuela R Morenoand M Stojkovic ldquoTrichostatin A affects histone acetylationand gene expression in porcine somatic cell nucleus transferembryosrdquoTheriogenology vol 72 no 8 pp 1097ndash1110 2009
[7] J Zhao J Whyte and R S Prather ldquoEffect of epigeneticregulation during swine embryogenesis and on cloning bynuclear transferrdquo Cell and Tissue Research vol 341 no 1 pp13ndash21 2010
[8] WReik ldquoStability and flexibility of epigenetic gene regulation inmammalian developmentrdquo Nature vol 447 no 7143 pp 425ndash432 2007
[9] R S Prather J W Ross S C Isom and J A Green ldquoTranscrip-tional post-transcriptional and epigenetic control of porcineoocyte maturation and embryogenesisrdquo Society of Reproductionand Fertility supplement vol 66 pp 165ndash176 2009
[10] N Rodriguez-Osorio R Urrego J B Cibelli K Eilertsen andE Memili ldquoReprogramming mammalian somatic cellsrdquo Theri-ogenology vol 78 no 9 pp 1869ndash1886 2012
[11] K J Eilertsen R A Power L L Harkins and P Misica ldquoTar-geting cellular memory to reprogram the epigenome restorepotential and improve somatic cell nuclear transferrdquo AnimalReproduction Science vol 98 no 1-2 pp 129ndash146 2007
[12] M Samiec and M Skrzyszowska ldquoMolecular conditions of thecell nucleus remodellingreprogramming process and nucleartransferred embryo development in the intraooplasmic kary-oplast injection technique a reviewrdquo Czech Journal of AnimalScience vol 50 no 5 pp 185ndash195 2005
BioMed Research International 11
[13] P Narbonne K Miyamoto and J B Gurdon ldquoReprogrammingand development in nuclear transfer embryos and in interspe-cific systemsrdquo Current Opinion in Genetics amp Development vol22 no 5 pp 450ndash458 2012
[14] J-I Bang J-G Yoo M-R Park et al ldquoThe effects of artificialactivation timing on the development of SCNT-derivedembryos and newborn pigletsrdquoReproductive Biology vol 13 no2 pp 127ndash132 2013
[15] M Samiec and M Skrzyszowska ldquoHigh developmental capa-bility of porcine cloned embryos following trichostatin A-dependent epigenomic transformation during in vitro matura-tion of oocytes pre-exposed to R-roscovitinerdquo Animal SciencePapers and Reports vol 30 no 4 pp 383ndash393 2012
[16] M Samiec and M Skrzyszowska ldquoRoscovitine is a novel agentthat can be used for the activation of porcine oocytes recon-structed with adult cutaneous or fetal fibroblast cell nucleirdquoTheriogenology vol 78 no 8 pp 1855ndash1867 2012
[17] Z Li J Shi D Liu et al ldquoEffects of donor fibroblast cell typeand transferred cloned embryo number on the efficiency of pigcloningrdquo Cellular Reprogramming vol 15 no 1 pp 35ndash42 2013
[18] M Samiec and M Skrzyszowska ldquoBiological transcomple-mentary activation as a novel and effective strategy appliedto the generation of porcine somatic cell cloned embryosrdquoReproductive Biology vol 14 no 2 pp 128ndash139 2014
[19] J Li O Svarcova K Villemoes et al ldquoHigh in vitro developmentafter somatic cell nuclear transfer and trichostatin A treatmentof reconstructed porcine embryosrdquoTheriogenology vol 70 no5 pp 800ndash808 2008
[20] M Skrzyszowska M Samiec R Słomski D Lipinski and EMały ldquoDevelopment of porcine transgenic nuclear-transferredembryos derived from fibroblast cells transfected by the noveltechnique of nucleofection or standard lipofectionrdquo Theri-ogenology vol 70 no 2 pp 248ndash259 2008
[21] Y-W Pang L An P Wang et al ldquoTreatment of porcine donorcells and reconstructed embryoswith the antioxidantmelatoninenhances cloning efficiencyrdquo Journal of Pineal Research vol 54no 4 pp 389ndash397 2013
[22] M Samiec M Skrzyszowska and J Opiela ldquoCreation of clonedpig embryos using contact-inhibited or serum-starved fibrob-last cells analysed intra vitam for apoptosis occurrencerdquo Annalsof Animal Science vol 13 no 2 pp 275ndash293 2013
[23] H Wei Y Qing W Pan et al ldquoComparison of the efficiency ofBannaminiature inbred pig somatic cell nuclear transfer amongdifferent donor cellsrdquo PLoSONE vol 8 no 2 Article ID e577282013
[24] K Inoue N Ogonuki H Miki et al ldquoInefficient reprogram-ming of the hematopoietic stem cell genome following nucleartransferrdquo Journal of Cell Science vol 119 no 10 pp 1985ndash19912006
[25] K Inoue S Noda N Ogonuki et al ldquoDifferential developmen-tal ability of embryos cloned from tissue-specific stem cellsrdquoStem Cells vol 25 no 5 pp 1279ndash1285 2007
[26] S L Lee E J Kang G H Maeng et al ldquoDevelopmental abilityof miniature pig embryos cloned withmesenchymal stem cellsrdquoThe Journal of Reproduction and Development vol 56 no 2 pp256ndash262 2010
[27] Z Li X He L Chen et al ldquoBone marrow mesenchymal stemcells are an attractive donor cell type for production of clonedpigs as well as genetically modified cloned pigs by somatic cellnuclear transferrdquo Cellular Reprogramming vol 15 no 5 pp459ndash470 2013
[28] B M Kumar H-F Jin J-G Kim et al ldquoDifferential geneexpression patterns in porcine nuclear transfer embryos recon-structed with fetal fibroblasts and mesenchymal stem cellsrdquoDevelopmental Dynamics vol 236 no 2 pp 435ndash446 2007
[29] J Su Y Wang Y Li et al ldquoOxamflatin significantly improvesnuclear reprogramming blastocyst quality and in vitro devel-opment of bovine SCNT embryosrdquo PLoS ONE vol 6 no 8Article ID e23805 2011
[30] Y Song THai YWang et al ldquoEpigenetic reprogramming geneexpression and in vitro development of porcine SCNT embryosare significantly improved by a histone deacetylase inhibitor-m-carboxycinnamic acid bishydroxamide (CBHA)rdquo Protein ampCell vol 5 no 5 pp 382ndash393 2014
[31] S Colleoni G Donofrio I Lagutina R Duchi C Galli andG Lazzari ldquoEstablishment differentiation electroporationviral transduction and nuclear transfer of bovine and porcinemesenchymal stem cellsrdquo Cloning and Stem Cells vol 7 no 3pp 154ndash166 2005
[32] H F Jin B M Kumar J G Kim et al ldquoEnhanced developmentof porcine embryos cloned from bone marrow mesenchymalstem cellsrdquoThe International Journal of Developmental Biologyvol 51 no 1 pp 85ndash90 2007
[33] J Opiela and M Samiec ldquoCharacterization of mesenchymalstem cells and their application in experimental embryologyrdquoPolish Journal of Veterinary Sciences vol 16 no 3 pp 593ndash5992013
[34] X Yang S L Smith X C Tian H A Lewin J-P Renard and TWakayama ldquoNuclear reprogramming of cloned embryos and itsimplications for therapeutic cloningrdquo Nature Genetics vol 39no 3 pp 295ndash302 2007
[35] XWu Y Li G-P Li et al ldquoTrichostatin A improved epigeneticmodifications of transfected cells but did not improve subse-quent cloned embryo developmentrdquo Animal Biotechnology vol19 no 4 pp 211ndash224 2008
[36] F Bo L Di F Qing-Chang et al ldquoEffect of trichostatin A ontransfected donor cells and subsequent development of porcinecloned embryosrdquo Zygote vol 19 no 3 pp 237ndash243 2011
[37] X Ding Y Wang D Zhang Z Guo and Y Zhang ldquoIncreasedpre-implantation development of cloned bovine embryostreated with 5-aza-21015840-deoxycytidine and trichostatin ArdquoTheri-ogenology vol 70 no 4 pp 622ndash630 2008
[38] H S Lee X F Yu J I Bang et al ldquoEnhanced histone acetylationin somatic cells induced by a histone deacetylase inhibitorimproved inter-generic cloned leopard cat blastocystsrdquo Theri-ogenology vol 74 no 8 pp 1439ndash1449 2010
[39] S-F Ning Q-Y Li M-M Liang et al ldquoMethylation charac-teristics and developmental potential of Guangxi Bamaminipig(Sus scrofa domestica) cloned embryos from donor cells treatedwith trichostatin A and 5-aza-21015840-deoxycytidinerdquo Zygote vol 21no 2 pp 178ndash186 2013
[40] B P Enright L-Y Sung C-C Chang X Yang and X C TianldquoMethylation and acetylation characteristics of cloned bovineembryos from donor cells treated with 5-aza-21015840-deoxycytidinerdquoBiology of Reproduction vol 72 no 4 pp 944ndash948 2005
[41] Y J Huan J Zhu B T Xie et al ldquoTreating cloned embryos butnot donor cells with 5-aza-21015840-deoxycytidine enhances thedevelopmental competence of porcine cloned embryosrdquo TheJournal of Reproduction andDevelopment vol 59 no 5 pp 442ndash449 2013
[42] B-G JeonG Coppola S D Perrault G-J RhoDH Betts andWAKing ldquoS-adenosylhomocysteine treatment of adult female
12 BioMed Research International
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
Figure 1 Porcine cloned blastocysts developed fromnuclear-transferred oocytes reconstitutedwith adult bonemarrow-derivedmesenchymalstem cells undergoing trichostatinA- (TSA-) dependent epigenomicmodulation (photographs (a) and (b)) or not undergoingTSA-dependentepigenomic modulation (photographs (c) and (d)) Images were taken at magnification times200
Table 2 Effect of the TSA-dependent epigenomic modulation of MSCs on the in vitro developmental outcome of cloned pig embryos
TSA-mediated epigenetictransformation of MSCs
Number of oocytesembryos Development toEnucleated Electrofused () Cleaved () Morulae () Blastocysts ()
minus 293 275293 (939) 262275 (953) 216275 (785)b 125275 (455)B
TSA trichostatin A MSC mesenchymal stem cellValues with different small superscript letters (a and b) within the same column denote statistically significant differences between experimental groups (119875 lt005 1205942 test) Values with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0001 1205942 test)Number of replicates ge6
Table 3 Effect of the TSA-mediated epigenomic modulation of MSCs on the cytological quality of cloned pig embryos assessed by TUNELassay
TSA-mediated epigenetictransformation of MSCs
Number ofanalyzedblastocysts
Mean number ofDAPI-tagged cell nuclei per
blastocyst plusmn SD
Mean number ofTUNEL-positive (apoptotic) cell
nuclei per blastocyst plusmn SD
Mean apoptotic index(DCI) per blastocyst plusmn
SD ()+ 31 4797 plusmn 2743A 161 plusmn 338 495 plusmn 1398minus 26 2777 plusmn 1582B 158 plusmn 269 713 plusmn 1244TSA trichostatin A MSC mesenchymal stem cell TUNEL terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling DAPI 410158406-diamidino-2-phenylindole SD standard deviation DCI dead cell indexValues with different large superscript letters (A and B) within the same column denote very highly significant differences (119875 lt 0005 ANOVA followed byTukeyrsquos HSD post hoc test) Number of replicates = 6
TUNEL-positive (ie late-apoptotic) cell nuclei per blasto-cyst were identified between NT embryos reconstituted withMSCs undergoing and not undergoing TSA-mediated epige-netic transformation (119875 ge 005 Table 3 Figure 2) Althoughno significant differences in the DCI per blastocyst were
found between both experimental groups (119875 ge 005) theincidence of late apoptosis-related internucleosomal DNAfragmentation trended slightly upwards for NT embryosgenerated using TSA-untreated MSCs as compared to thoseproduced using TSA-treated MSCs (Table 3 Figure 2)
BioMed Research International 7
005mm
(a)
005mm
(b)
005mm
(c)
005mm
(d)
005mm
(e)
005mm
(f)
Figure 2 Evaluation of cytological quality of porcine cloned blastocysts on the basis of simultaneous determination of total nuclear numberand detection of apoptotic cell nuclei by terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling (TUNEL) analysis Photographs (a) to (d) depict embryos originating from nuclear-transferred (NT) oocytes reconstitutedwith adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) subjected to epigenetic transformation via trichostatin A (TSA)treatment Photographs (e) and (f) depict embryos originating from NT oocytes reconstituted with ABM-MSCs not subjected to epigenetictransformation via TSA treatment In each blastocyst the cell nuclei of all the blastomeres (both inner cell mass (ICM) and trophectoderm(TE) cells) had been tagged with 410158406-diamidino-2-phenylindole (DAPI) counterstain and subsequently fluoresced in blue The cell nucleiof late-apoptotic blastomeres (ICM andor TE cells) exhibiting internucleosomal DNA fragmentation had been dyed with fluoresceinisothiocyanate (FITC) and then fluoresced in bright green In each photograph the DAPI-derived blue and FITC-derived green fluorescentsignals merge into one another Photographs (a) to (f) represent the blastocysts displaying different incidence of blastomere apoptosisand thereby varied advancement of internucleosomal DNA fragmentation ((a) (c) (d) and (e) the lack of apoptotic intranuclear DNAfragmentation (b) and (e) few apoptotic cell nuclei (f) increased extent of apoptotic intranuclear DNA fragmentation) Images were takenat magnification times200
8 BioMed Research International
0
1
2
3
4
5
6
B
A
Oct4 Nanog Nestin
MSCTSAMSC
Figure 3 Relative abundance (mean plusmn SD) of Oct4 Nanog andNestin mRNAs in blastocysts developed from nuclear-transferredpig embryos descended from MSCs undergoing or not undergoingTSA-dependent epigenomic transformation The highly significantdifferences were designated with the A and B letters (119875 lt 001ANOVA followed by Tukeyrsquos HSD post hoc test)
33 The Pluripotency Status of Porcine Nuclear-TransferredEmbryos Originating from Epigenomically Modulated or Non-modulated MSCs The total number of analyzed clonedblastocysts was 60 To assess relative abundance (RA) of threeanalyzed mRNA transcripts for pluripotency-related targetgenes (Oct4 Nanog) multipotent stemness-associated targetgene (Nestin) and control GAPDH gene transcript we used3 times 10 blastocysts from both TSA-treated and -untreatedgroups
Highly significant differences were indicated for theOct4gene transcript quantitative profile between blastocysts orig-inating from TSA-treated and -untreated groups (119875 lt 001Figure 3) Although no significant intergroup variability wasshown in the RA of Nanog and Nestin mRNAs (119875 ge 005)their expression levels tended to be higher in blastocystsderived from NT embryos reconstructed with TSA-exposedMSCs as compared to the TSA-untreated group (Figure 3)
4 Discussion
The abundance of the morulae and blastocysts of highercytological and molecular quality generated in the presentinvestigation confirms that the reprogrammability of adultbone marrow-derived mesenchymal stem cells which hadbeen epigenetically modified via exposure to trichostatinA underwent considerable improvement in a cytoplasm ofporcine nuclear-transferred oocytes and resultant in vitrocultured cloned embryos It is beyond any doubt thatthe impact of TSA-based epigenomic transformation ofbone marrow-retrieved MSCs representing multipotent andundifferentiated stem cells on their competence for SCNThas not yet been explored not only in pigs but also inother mammalian species So far TSA-mediated epigeneticmodulation has been applied only for differentiated somatic
cells that commonly provide the source of nuclear donorsfor cloning procedure in pigs The results of the study byDiao et al [55] proved that porcineNT embryos reconstitutedwith TSA-treated fetal fibroblast cells were characterized bytwofold higher developmental potential to reach the blasto-cyst stage (30) than the NT embryos reconstituted withTSA-untreated fibroblast cells (15) Similar tendency hasbeen also shown in our current study inwhich trichostatinA-mediated epigenetic transformation of bone marrow-derivedMSCs gave rise to significant enhancement of capability ofcloned embryos to complete their development to blastocyststage (65) as compared to that observed for embryosderived from TSA-unexposed MSC nuclei (46) Nonethe-less NT pig embryos originating from epigenomically trans-formed fetal fibroblast cells displayed considerably lowerblastocyst formation rate [55] than the rate indicated in ourpresent study for NT embryos reconstructed with epige-nomically nontransformed MSCs Moreover treatment ofnuclear donor MSCs with trichostatin A resulted in one anda half times to severalfold increase of blastocyst percentage(65) as compared to the blastocyst yields obtained by otherinvestigators using undifferentiated or in vitro differentiatedMSCs that were not modulated epigenetically [26ndash28 31 32]In the study by Kumar et al [28] the abilities of epige-nomically nontransformed bone marrow-descended MSCnuclei to direct the in vitro development of porcine clonedembryos to blastocyst stage decreased above three times(approximately 20) as compared to our present work Inturn nuclear-transferred embryos that had originated fromporcine undifferentiated bone marrow-retrieved MSCs andtheir derivatives along the osteogenic lineage were able tocomplete their in vitro development to blastocyst stage atthe rates ranging from 33 to 45 [31] In contrast Jin etal [32] and Li et al [27] reported that blastocyst yields ofNT embryos derived from porcine epigenetically unmodu-lated MSCs were maintained at the relatively low levels ofapproximately 18 and 16 respectively Nevertheless in thestudy by Lee et al [26] the in vitro developmental outcometo the blastocyst stage of NT embryos originating fromundifferentiated MSCs achieved the rate of approximately48 but even this blastocyst formation rate was considerablylower than that noticed in our current investigation
The results of the current study have also clearly demon-strated that the TSA-dependent epigenomic transformationof nuclear donor MSCs contributes to improvement of notonly in vitro developmental competences but also qualityand transcription level-related pluripotency extent of porcinecloned embryos The utilization of undifferentiated mes-enchymal stem cells and the modification of the molecularmechanisms of transcriptional reprogramming of the donornuclear genome via HDAC inhibitor-mediated epigeneticmodulation of MSCs before their use for SCNT led to correctand complete adaptation of MSC-inherited genomic DNA tothe cytoplasmic environment of enucleated oocytes and invitro cultured NT pig embryos Epigenetic reprogrammingof donor cell nuclei suggests that a new program for theirtranscriptional activity is loaded and reloaded immediatelyfollowing reconstruction of enucleated oocytes The successof SCNT may depend upon both genomic DNA-associated
BioMed Research International 9
reprogramming of gene expression for dedifferentiation ofthe donor somatic cell nuclei during early preimplantationdevelopment of cloned embryos and reprogramming of geneexpression for onset of somatogenic nuclear redifferentiationduring blastocyst formation [7 10 63 64] It has been ascer-tained that somatic cell nuclei should undergo the wide DNAcytosine residue demethylation changes throughout the earlydevelopment of NT embryos to erase and then reset theirown overall epigenetic as well as parental genomic imprintingmemory which has been established by remethylation ofthe nuclear genome within the framework of the specificpathway of somatic and germ cell lineage commitment anddifferentiation [2 4 13 30 65ndash67]
It appears that the enhanced cytological quality of clonedpig blastocysts that originated from enucleated oocytesreceiving epigenetically modulated MSC nuclei could beassociated with more faithful and faultless pattern for repro-gramming of transcriptional activity of genomic DNA inher-ited from undifferentiated and multipotent stem cells Thesecells exhibit increased genomic and epigenomic plasticity inrearrangements of their gene expression in the blastomeres ofpreimplanted NT embryos The fluorocytochemical analysisof overall mean nuclear counts revealed almost twofoldhigher total cells number in porcine blastocysts developedfrom NT oocytes reconstituted with TSA-exposed MSCs (48blastomeres) than that identified in blastocysts derived fromNT oocytes reconstituted with TSA-unexposed MSCs (28blastomeres) The quality of cloned blastocysts generated inour present study using epigenomically transformed MSCswas comparable to that noticed in the study by Lee et al[26] for porcine NT blastocysts derived from epigenomicallynontransformed MSCs These blastocysts were also charac-terized by a total cells number equal to 48 In contrast to theabovementioned findings in the investigations carried outby Li et al [27] Kumar et al [28] and Jin et al [32] theoverall number of ICM and trophectoderm cells in clonedpig blastocysts originating from embryos reconstituted withTSA-untreated MSCs ranged from 28 to 35 and decreasedapproximately one and a half times as compared to the totalcells number estimated for NT blastocysts obtained by usin the TSA-exposed MSC group Nevertheless the meanapoptotic index (ie DCI = 495) that was calculated by usfor blastocysts developed from cloned embryos reconstitutedwith epigenetically modified MSC nuclei was similar to theratios of TUNEL-positive cells (46 to 47) observed by Jinet al [32] andKumar et al [28] among porcineNT blastocystsderived from epigenetically nonmodulated counterparts Inturn cloned blastocysts produced in our study using TSA-untreated MSCs displayed the apoptotic index (713) thatwas comparable or slightly lower to indexes identified forcloned embryos created with the aid of fetal fibroblast cellsnot undergoing exposure to trichostatin A [28 32]The latterindexes (DCIs) oscillated between 73 and 78 Generally inour current work the proportion of TUNEL-positive cellsin relation to a total cells number tended to insignificantlydecrease among NT blastocysts generated using undifferen-tiated mesenchymal stem cells subjected to TSA treatmentcompared to those generated using nuclear donor cells not
exposed to TSA (495 versus 713) Analogous slight down-ward tendency in the ratios of TUNEL-positive cells wasindicated byDiao et al [55] comparing blastocysts developedfrom cloned embryos reconstructed with cell nuclei of fetalfibroblasts treated with TSA and the counterparts producedusing TSA-unexposed fetal fibroblast cell nuclei (nearly 3versus 45)
Improved reprogrammability of transcriptional activityfor nuclear genome of epigenetically modulated MSCs inthe cells of preimplanted NT embryos turned out to bepositively correlated with enhanced molecular quality ofporcine cloned blastocysts assessed on the basis of theirpluripotency extent whichwasmeasuredwith the expressionprofiles identified for Oct4 and Nanog genes A 38-kDaprotein Oct4 (ie octamer-binding transcription factor 4)that is a member of the family of POU- (Pit-Oct-Unc-)domain and homeodomain transcription factors acts as avital regulator of pluripotency extent playing an importantrole in not only controlling preimplantation embryonicdevelopment but also maintenance of ICM cell fate inblastocysts and pluripotency status of embryonic stem cells(ESCs) [68ndash70] A 35-kDa protein designated as Nanog fromCelticIrish mythical Tır na nOg (Tir Na Nog The Landof the Ever-Young) is another homeobox-containing tran-scription factor that represents the group of pivotal proteinsmodulating pluripotency degree [70 71] The homeoproteinNanog can act synergistically with Oct4 protein in retainingthe pluripotent status of blastocyst-descended ICM andepiblast cells as well as in sustaining the undifferentiatedstatus and ability for self-renewal of ESCs [71 72] Theaberrant (ie downregulated) expression levels and patternsof Oct4 and Nanog transcripts in both bovine NT blastocystsproduced using epigenetically nonmodulated calf dermalfibroblast cells [73] and porcine NT blastocysts producedusing either epigenetically nonmodulated adult MSCs orfetal fibroblast cells [28] have been found to be a majorcause of not only their declined cytological and molecularquality but also decreased in vitro developmental potentialof cloned embryos However in our current study it seemsthat TSA-dependent epigenomic transformation of nucleardonor MSCs biases correctingly the expression profile ofOct4 andNanog mRNAs in cloned pig blastocysts triggeringboth significant enhancement of relative abundance (RA)of Oct4 transcripts and slight (nonsignificant) increase inNanog transcript RA as compared to the TSA-untreatedMSCgroup On the one hand this influence could be exerted bydirect diminishment in the deacetylation level within thenucleosomal core-derived histone lysine moieties that wasevoked by trichostatin A-mediated nonspecific inhibition ofHDACs On the other hand it could be probably elicitedvia indirect nonselective suppression of cytosine residuemethylation processes within the Oct4 and Nanog gene pro-moters andor enhancers leading to upregulated expressionof these crucial pluripotency-related genes Similar resultshave been shown in the study by Wang et al [73] in whichthe sequential treatment of bovine differentiated nucleardonor fibroblast cells and resultant cloned embryos by bothnonspecific inhibitors of DNMTs and HDACs (ie 5-aza-dC
10 BioMed Research International
and TSA resp) contributed to increase of Oct4 transcriptRA in the blastocysts obtained simultaneously sustainingthe expression of Nanog mRNAs on the unchanged levelas compared to the 5-aza-dC- and TSA-unexposed groupIn this case 5-aza-21015840-deoxycytidine- and trichostatin A-mediated epigenetic modulation of both nuclear donor cellsand cloned embryos gave rise presumably to direct down-regulation in the methylation of cytosine residues withinthe Oct4 gene promoter andor enhancer that resultedfrom 5-aza-dC-dependent nonspecific inhibition of DNMTsFurthermore this two-factor epigenetic modulation couldaffect the enhanced incidence of acetylation level within thenucleosomal core-derived histone lysine moieties that wastriggered by trichostatin A-mediated nonselective inhibitionof HDACs consequently leading to indirect upregulatedexpression of the Oct4 gene Taking into consideration allthe abovementioned findings considerable transcriptionalupregulation in the expression of Oct4 gene (confirmedamong porcine NT blastocysts in our present study andamong bovineNT blastocysts in the study byWang et al [73])can be found to be pivotal indicator of increased pluripotencyextent of cloned embryos This process appears to be alsoassociated with improved cytological quality of NT blasto-cysts that was measured with their total cell counts In thecurrent investigation we have shown that the overall numberof ICM and trophoblast cells in blastocysts was significantlyhigher following the reconstruction of NT pig embryos withepigenomically transformed mesenchymal stem cells
5 Conclusions and Future Goals
Summing up artificial epigenomic modulation of in vitrocultured MSCs using the nonspecific HDAC inhibitor desig-nated as TSA seems to facilitate much more the reprogram-ming process for epigenetically determined transcriptionalactivity of somatic cell-inherited nuclear genome in theNT pig embryos The use of ectopic HDAC inhibitors forepigenetic transformation of mesenchymal stem cells whosecell nuclei were transferred into enucleated oocytes is thecompletely new approach in the studies involving somaticcell cloning of pigs and other mammalian species Enhancedreprogrammability of nuclear genome descended from TSA-exposed MSCs in the blastomeres of porcine preimplantedcloned embryos resulted in the improvements of not onlytheir capacity to complete in vitro development to themorula and blastocyst stages but also cytological and molec-ular quality of the blastocysts produced Therefore furtherinvestigations are also necessary to determine whether thenovel strategy of TSA-dependent epigenetic modification ofnuclear donor MSCs which has been recently utilized in ourlaboratory for the future goals of generating cloned pigletsenables retaining the in vivo developmental competences ofthe high-quality NT-derived blastocysts to reach full term
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Marcin Samiec and Jolanta Opiela contributed equally to thepreparation of this paper
Acknowledgments
The project was funded by the Polish National ScienceCentre resources allocated on the basis of Decision no DEC-201103DNZ905537 The authors would like to thank DrJarosław Wieczorek DVM for bone marrow retrieval
References
[1] A J Bonk H-T Cheong R Li et al ldquoCorrelation of develop-mental differences of nuclear transfer embryos cells to themethylation profiles of nuclear transfer donor cells in swinerdquoEpigenetics vol 2 no 3 pp 179ndash186 2007
[2] A J Bonk R Li L Lai et al ldquoAberrant DNA methylation inporcine in vitro- parthenogenetic- and somatic cell nucleartransfer-produced blastocystsrdquo Molecular Reproduction andDevelopment vol 75 no 2 pp 250ndash264 2008
[3] M Samiec ldquoDevelopment of pig cloning studies past presentand futurerdquo Journal of Animal and Feed Sciences vol 13 no 2pp 211ndash238 2004
[4] K MWhitworth and R S Prather ldquoSomatic cell nuclear trans-fer efficiency how can it be improved through nuclear remod-eling and reprogrammingrdquoMolecular Reproduction and Devel-opment vol 77 no 12 pp 1001ndash1015 2010
[5] K-I Yamanaka S Sugimura T Wakai M Kawahara and ESato ldquoAcetylation level of histone H3 in early embryonic stagesaffects subsequent development of miniature pig somatic cellnuclear transfer embryosrdquo The Journal of Reproduction andDevelopment vol 55 no 6 pp 638ndash644 2009
[6] R P Cervera N Martı-Gutierrez E Escorihuela R Morenoand M Stojkovic ldquoTrichostatin A affects histone acetylationand gene expression in porcine somatic cell nucleus transferembryosrdquoTheriogenology vol 72 no 8 pp 1097ndash1110 2009
[7] J Zhao J Whyte and R S Prather ldquoEffect of epigeneticregulation during swine embryogenesis and on cloning bynuclear transferrdquo Cell and Tissue Research vol 341 no 1 pp13ndash21 2010
[8] WReik ldquoStability and flexibility of epigenetic gene regulation inmammalian developmentrdquo Nature vol 447 no 7143 pp 425ndash432 2007
[9] R S Prather J W Ross S C Isom and J A Green ldquoTranscrip-tional post-transcriptional and epigenetic control of porcineoocyte maturation and embryogenesisrdquo Society of Reproductionand Fertility supplement vol 66 pp 165ndash176 2009
[10] N Rodriguez-Osorio R Urrego J B Cibelli K Eilertsen andE Memili ldquoReprogramming mammalian somatic cellsrdquo Theri-ogenology vol 78 no 9 pp 1869ndash1886 2012
[11] K J Eilertsen R A Power L L Harkins and P Misica ldquoTar-geting cellular memory to reprogram the epigenome restorepotential and improve somatic cell nuclear transferrdquo AnimalReproduction Science vol 98 no 1-2 pp 129ndash146 2007
[12] M Samiec and M Skrzyszowska ldquoMolecular conditions of thecell nucleus remodellingreprogramming process and nucleartransferred embryo development in the intraooplasmic kary-oplast injection technique a reviewrdquo Czech Journal of AnimalScience vol 50 no 5 pp 185ndash195 2005
BioMed Research International 11
[13] P Narbonne K Miyamoto and J B Gurdon ldquoReprogrammingand development in nuclear transfer embryos and in interspe-cific systemsrdquo Current Opinion in Genetics amp Development vol22 no 5 pp 450ndash458 2012
[14] J-I Bang J-G Yoo M-R Park et al ldquoThe effects of artificialactivation timing on the development of SCNT-derivedembryos and newborn pigletsrdquoReproductive Biology vol 13 no2 pp 127ndash132 2013
[15] M Samiec and M Skrzyszowska ldquoHigh developmental capa-bility of porcine cloned embryos following trichostatin A-dependent epigenomic transformation during in vitro matura-tion of oocytes pre-exposed to R-roscovitinerdquo Animal SciencePapers and Reports vol 30 no 4 pp 383ndash393 2012
[16] M Samiec and M Skrzyszowska ldquoRoscovitine is a novel agentthat can be used for the activation of porcine oocytes recon-structed with adult cutaneous or fetal fibroblast cell nucleirdquoTheriogenology vol 78 no 8 pp 1855ndash1867 2012
[17] Z Li J Shi D Liu et al ldquoEffects of donor fibroblast cell typeand transferred cloned embryo number on the efficiency of pigcloningrdquo Cellular Reprogramming vol 15 no 1 pp 35ndash42 2013
[18] M Samiec and M Skrzyszowska ldquoBiological transcomple-mentary activation as a novel and effective strategy appliedto the generation of porcine somatic cell cloned embryosrdquoReproductive Biology vol 14 no 2 pp 128ndash139 2014
[19] J Li O Svarcova K Villemoes et al ldquoHigh in vitro developmentafter somatic cell nuclear transfer and trichostatin A treatmentof reconstructed porcine embryosrdquoTheriogenology vol 70 no5 pp 800ndash808 2008
[20] M Skrzyszowska M Samiec R Słomski D Lipinski and EMały ldquoDevelopment of porcine transgenic nuclear-transferredembryos derived from fibroblast cells transfected by the noveltechnique of nucleofection or standard lipofectionrdquo Theri-ogenology vol 70 no 2 pp 248ndash259 2008
[21] Y-W Pang L An P Wang et al ldquoTreatment of porcine donorcells and reconstructed embryoswith the antioxidantmelatoninenhances cloning efficiencyrdquo Journal of Pineal Research vol 54no 4 pp 389ndash397 2013
[22] M Samiec M Skrzyszowska and J Opiela ldquoCreation of clonedpig embryos using contact-inhibited or serum-starved fibrob-last cells analysed intra vitam for apoptosis occurrencerdquo Annalsof Animal Science vol 13 no 2 pp 275ndash293 2013
[23] H Wei Y Qing W Pan et al ldquoComparison of the efficiency ofBannaminiature inbred pig somatic cell nuclear transfer amongdifferent donor cellsrdquo PLoSONE vol 8 no 2 Article ID e577282013
[24] K Inoue N Ogonuki H Miki et al ldquoInefficient reprogram-ming of the hematopoietic stem cell genome following nucleartransferrdquo Journal of Cell Science vol 119 no 10 pp 1985ndash19912006
[25] K Inoue S Noda N Ogonuki et al ldquoDifferential developmen-tal ability of embryos cloned from tissue-specific stem cellsrdquoStem Cells vol 25 no 5 pp 1279ndash1285 2007
[26] S L Lee E J Kang G H Maeng et al ldquoDevelopmental abilityof miniature pig embryos cloned withmesenchymal stem cellsrdquoThe Journal of Reproduction and Development vol 56 no 2 pp256ndash262 2010
[27] Z Li X He L Chen et al ldquoBone marrow mesenchymal stemcells are an attractive donor cell type for production of clonedpigs as well as genetically modified cloned pigs by somatic cellnuclear transferrdquo Cellular Reprogramming vol 15 no 5 pp459ndash470 2013
[28] B M Kumar H-F Jin J-G Kim et al ldquoDifferential geneexpression patterns in porcine nuclear transfer embryos recon-structed with fetal fibroblasts and mesenchymal stem cellsrdquoDevelopmental Dynamics vol 236 no 2 pp 435ndash446 2007
[29] J Su Y Wang Y Li et al ldquoOxamflatin significantly improvesnuclear reprogramming blastocyst quality and in vitro devel-opment of bovine SCNT embryosrdquo PLoS ONE vol 6 no 8Article ID e23805 2011
[30] Y Song THai YWang et al ldquoEpigenetic reprogramming geneexpression and in vitro development of porcine SCNT embryosare significantly improved by a histone deacetylase inhibitor-m-carboxycinnamic acid bishydroxamide (CBHA)rdquo Protein ampCell vol 5 no 5 pp 382ndash393 2014
[31] S Colleoni G Donofrio I Lagutina R Duchi C Galli andG Lazzari ldquoEstablishment differentiation electroporationviral transduction and nuclear transfer of bovine and porcinemesenchymal stem cellsrdquo Cloning and Stem Cells vol 7 no 3pp 154ndash166 2005
[32] H F Jin B M Kumar J G Kim et al ldquoEnhanced developmentof porcine embryos cloned from bone marrow mesenchymalstem cellsrdquoThe International Journal of Developmental Biologyvol 51 no 1 pp 85ndash90 2007
[33] J Opiela and M Samiec ldquoCharacterization of mesenchymalstem cells and their application in experimental embryologyrdquoPolish Journal of Veterinary Sciences vol 16 no 3 pp 593ndash5992013
[34] X Yang S L Smith X C Tian H A Lewin J-P Renard and TWakayama ldquoNuclear reprogramming of cloned embryos and itsimplications for therapeutic cloningrdquo Nature Genetics vol 39no 3 pp 295ndash302 2007
[35] XWu Y Li G-P Li et al ldquoTrichostatin A improved epigeneticmodifications of transfected cells but did not improve subse-quent cloned embryo developmentrdquo Animal Biotechnology vol19 no 4 pp 211ndash224 2008
[36] F Bo L Di F Qing-Chang et al ldquoEffect of trichostatin A ontransfected donor cells and subsequent development of porcinecloned embryosrdquo Zygote vol 19 no 3 pp 237ndash243 2011
[37] X Ding Y Wang D Zhang Z Guo and Y Zhang ldquoIncreasedpre-implantation development of cloned bovine embryostreated with 5-aza-21015840-deoxycytidine and trichostatin ArdquoTheri-ogenology vol 70 no 4 pp 622ndash630 2008
[38] H S Lee X F Yu J I Bang et al ldquoEnhanced histone acetylationin somatic cells induced by a histone deacetylase inhibitorimproved inter-generic cloned leopard cat blastocystsrdquo Theri-ogenology vol 74 no 8 pp 1439ndash1449 2010
[39] S-F Ning Q-Y Li M-M Liang et al ldquoMethylation charac-teristics and developmental potential of Guangxi Bamaminipig(Sus scrofa domestica) cloned embryos from donor cells treatedwith trichostatin A and 5-aza-21015840-deoxycytidinerdquo Zygote vol 21no 2 pp 178ndash186 2013
[40] B P Enright L-Y Sung C-C Chang X Yang and X C TianldquoMethylation and acetylation characteristics of cloned bovineembryos from donor cells treated with 5-aza-21015840-deoxycytidinerdquoBiology of Reproduction vol 72 no 4 pp 944ndash948 2005
[41] Y J Huan J Zhu B T Xie et al ldquoTreating cloned embryos butnot donor cells with 5-aza-21015840-deoxycytidine enhances thedevelopmental competence of porcine cloned embryosrdquo TheJournal of Reproduction andDevelopment vol 59 no 5 pp 442ndash449 2013
[42] B-G JeonG Coppola S D Perrault G-J RhoDH Betts andWAKing ldquoS-adenosylhomocysteine treatment of adult female
12 BioMed Research International
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
Figure 2 Evaluation of cytological quality of porcine cloned blastocysts on the basis of simultaneous determination of total nuclear numberand detection of apoptotic cell nuclei by terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (21015840-deoxyuridine-51015840-triphosphate)nick-end labelling (TUNEL) analysis Photographs (a) to (d) depict embryos originating from nuclear-transferred (NT) oocytes reconstitutedwith adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) subjected to epigenetic transformation via trichostatin A (TSA)treatment Photographs (e) and (f) depict embryos originating from NT oocytes reconstituted with ABM-MSCs not subjected to epigenetictransformation via TSA treatment In each blastocyst the cell nuclei of all the blastomeres (both inner cell mass (ICM) and trophectoderm(TE) cells) had been tagged with 410158406-diamidino-2-phenylindole (DAPI) counterstain and subsequently fluoresced in blue The cell nucleiof late-apoptotic blastomeres (ICM andor TE cells) exhibiting internucleosomal DNA fragmentation had been dyed with fluoresceinisothiocyanate (FITC) and then fluoresced in bright green In each photograph the DAPI-derived blue and FITC-derived green fluorescentsignals merge into one another Photographs (a) to (f) represent the blastocysts displaying different incidence of blastomere apoptosisand thereby varied advancement of internucleosomal DNA fragmentation ((a) (c) (d) and (e) the lack of apoptotic intranuclear DNAfragmentation (b) and (e) few apoptotic cell nuclei (f) increased extent of apoptotic intranuclear DNA fragmentation) Images were takenat magnification times200
8 BioMed Research International
0
1
2
3
4
5
6
B
A
Oct4 Nanog Nestin
MSCTSAMSC
Figure 3 Relative abundance (mean plusmn SD) of Oct4 Nanog andNestin mRNAs in blastocysts developed from nuclear-transferredpig embryos descended from MSCs undergoing or not undergoingTSA-dependent epigenomic transformation The highly significantdifferences were designated with the A and B letters (119875 lt 001ANOVA followed by Tukeyrsquos HSD post hoc test)
33 The Pluripotency Status of Porcine Nuclear-TransferredEmbryos Originating from Epigenomically Modulated or Non-modulated MSCs The total number of analyzed clonedblastocysts was 60 To assess relative abundance (RA) of threeanalyzed mRNA transcripts for pluripotency-related targetgenes (Oct4 Nanog) multipotent stemness-associated targetgene (Nestin) and control GAPDH gene transcript we used3 times 10 blastocysts from both TSA-treated and -untreatedgroups
Highly significant differences were indicated for theOct4gene transcript quantitative profile between blastocysts orig-inating from TSA-treated and -untreated groups (119875 lt 001Figure 3) Although no significant intergroup variability wasshown in the RA of Nanog and Nestin mRNAs (119875 ge 005)their expression levels tended to be higher in blastocystsderived from NT embryos reconstructed with TSA-exposedMSCs as compared to the TSA-untreated group (Figure 3)
4 Discussion
The abundance of the morulae and blastocysts of highercytological and molecular quality generated in the presentinvestigation confirms that the reprogrammability of adultbone marrow-derived mesenchymal stem cells which hadbeen epigenetically modified via exposure to trichostatinA underwent considerable improvement in a cytoplasm ofporcine nuclear-transferred oocytes and resultant in vitrocultured cloned embryos It is beyond any doubt thatthe impact of TSA-based epigenomic transformation ofbone marrow-retrieved MSCs representing multipotent andundifferentiated stem cells on their competence for SCNThas not yet been explored not only in pigs but also inother mammalian species So far TSA-mediated epigeneticmodulation has been applied only for differentiated somatic
cells that commonly provide the source of nuclear donorsfor cloning procedure in pigs The results of the study byDiao et al [55] proved that porcineNT embryos reconstitutedwith TSA-treated fetal fibroblast cells were characterized bytwofold higher developmental potential to reach the blasto-cyst stage (30) than the NT embryos reconstituted withTSA-untreated fibroblast cells (15) Similar tendency hasbeen also shown in our current study inwhich trichostatinA-mediated epigenetic transformation of bone marrow-derivedMSCs gave rise to significant enhancement of capability ofcloned embryos to complete their development to blastocyststage (65) as compared to that observed for embryosderived from TSA-unexposed MSC nuclei (46) Nonethe-less NT pig embryos originating from epigenomically trans-formed fetal fibroblast cells displayed considerably lowerblastocyst formation rate [55] than the rate indicated in ourpresent study for NT embryos reconstructed with epige-nomically nontransformed MSCs Moreover treatment ofnuclear donor MSCs with trichostatin A resulted in one anda half times to severalfold increase of blastocyst percentage(65) as compared to the blastocyst yields obtained by otherinvestigators using undifferentiated or in vitro differentiatedMSCs that were not modulated epigenetically [26ndash28 31 32]In the study by Kumar et al [28] the abilities of epige-nomically nontransformed bone marrow-descended MSCnuclei to direct the in vitro development of porcine clonedembryos to blastocyst stage decreased above three times(approximately 20) as compared to our present work Inturn nuclear-transferred embryos that had originated fromporcine undifferentiated bone marrow-retrieved MSCs andtheir derivatives along the osteogenic lineage were able tocomplete their in vitro development to blastocyst stage atthe rates ranging from 33 to 45 [31] In contrast Jin etal [32] and Li et al [27] reported that blastocyst yields ofNT embryos derived from porcine epigenetically unmodu-lated MSCs were maintained at the relatively low levels ofapproximately 18 and 16 respectively Nevertheless in thestudy by Lee et al [26] the in vitro developmental outcometo the blastocyst stage of NT embryos originating fromundifferentiated MSCs achieved the rate of approximately48 but even this blastocyst formation rate was considerablylower than that noticed in our current investigation
The results of the current study have also clearly demon-strated that the TSA-dependent epigenomic transformationof nuclear donor MSCs contributes to improvement of notonly in vitro developmental competences but also qualityand transcription level-related pluripotency extent of porcinecloned embryos The utilization of undifferentiated mes-enchymal stem cells and the modification of the molecularmechanisms of transcriptional reprogramming of the donornuclear genome via HDAC inhibitor-mediated epigeneticmodulation of MSCs before their use for SCNT led to correctand complete adaptation of MSC-inherited genomic DNA tothe cytoplasmic environment of enucleated oocytes and invitro cultured NT pig embryos Epigenetic reprogrammingof donor cell nuclei suggests that a new program for theirtranscriptional activity is loaded and reloaded immediatelyfollowing reconstruction of enucleated oocytes The successof SCNT may depend upon both genomic DNA-associated
BioMed Research International 9
reprogramming of gene expression for dedifferentiation ofthe donor somatic cell nuclei during early preimplantationdevelopment of cloned embryos and reprogramming of geneexpression for onset of somatogenic nuclear redifferentiationduring blastocyst formation [7 10 63 64] It has been ascer-tained that somatic cell nuclei should undergo the wide DNAcytosine residue demethylation changes throughout the earlydevelopment of NT embryos to erase and then reset theirown overall epigenetic as well as parental genomic imprintingmemory which has been established by remethylation ofthe nuclear genome within the framework of the specificpathway of somatic and germ cell lineage commitment anddifferentiation [2 4 13 30 65ndash67]
It appears that the enhanced cytological quality of clonedpig blastocysts that originated from enucleated oocytesreceiving epigenetically modulated MSC nuclei could beassociated with more faithful and faultless pattern for repro-gramming of transcriptional activity of genomic DNA inher-ited from undifferentiated and multipotent stem cells Thesecells exhibit increased genomic and epigenomic plasticity inrearrangements of their gene expression in the blastomeres ofpreimplanted NT embryos The fluorocytochemical analysisof overall mean nuclear counts revealed almost twofoldhigher total cells number in porcine blastocysts developedfrom NT oocytes reconstituted with TSA-exposed MSCs (48blastomeres) than that identified in blastocysts derived fromNT oocytes reconstituted with TSA-unexposed MSCs (28blastomeres) The quality of cloned blastocysts generated inour present study using epigenomically transformed MSCswas comparable to that noticed in the study by Lee et al[26] for porcine NT blastocysts derived from epigenomicallynontransformed MSCs These blastocysts were also charac-terized by a total cells number equal to 48 In contrast to theabovementioned findings in the investigations carried outby Li et al [27] Kumar et al [28] and Jin et al [32] theoverall number of ICM and trophectoderm cells in clonedpig blastocysts originating from embryos reconstituted withTSA-untreated MSCs ranged from 28 to 35 and decreasedapproximately one and a half times as compared to the totalcells number estimated for NT blastocysts obtained by usin the TSA-exposed MSC group Nevertheless the meanapoptotic index (ie DCI = 495) that was calculated by usfor blastocysts developed from cloned embryos reconstitutedwith epigenetically modified MSC nuclei was similar to theratios of TUNEL-positive cells (46 to 47) observed by Jinet al [32] andKumar et al [28] among porcineNT blastocystsderived from epigenetically nonmodulated counterparts Inturn cloned blastocysts produced in our study using TSA-untreated MSCs displayed the apoptotic index (713) thatwas comparable or slightly lower to indexes identified forcloned embryos created with the aid of fetal fibroblast cellsnot undergoing exposure to trichostatin A [28 32]The latterindexes (DCIs) oscillated between 73 and 78 Generally inour current work the proportion of TUNEL-positive cellsin relation to a total cells number tended to insignificantlydecrease among NT blastocysts generated using undifferen-tiated mesenchymal stem cells subjected to TSA treatmentcompared to those generated using nuclear donor cells not
exposed to TSA (495 versus 713) Analogous slight down-ward tendency in the ratios of TUNEL-positive cells wasindicated byDiao et al [55] comparing blastocysts developedfrom cloned embryos reconstructed with cell nuclei of fetalfibroblasts treated with TSA and the counterparts producedusing TSA-unexposed fetal fibroblast cell nuclei (nearly 3versus 45)
Improved reprogrammability of transcriptional activityfor nuclear genome of epigenetically modulated MSCs inthe cells of preimplanted NT embryos turned out to bepositively correlated with enhanced molecular quality ofporcine cloned blastocysts assessed on the basis of theirpluripotency extent whichwasmeasuredwith the expressionprofiles identified for Oct4 and Nanog genes A 38-kDaprotein Oct4 (ie octamer-binding transcription factor 4)that is a member of the family of POU- (Pit-Oct-Unc-)domain and homeodomain transcription factors acts as avital regulator of pluripotency extent playing an importantrole in not only controlling preimplantation embryonicdevelopment but also maintenance of ICM cell fate inblastocysts and pluripotency status of embryonic stem cells(ESCs) [68ndash70] A 35-kDa protein designated as Nanog fromCelticIrish mythical Tır na nOg (Tir Na Nog The Landof the Ever-Young) is another homeobox-containing tran-scription factor that represents the group of pivotal proteinsmodulating pluripotency degree [70 71] The homeoproteinNanog can act synergistically with Oct4 protein in retainingthe pluripotent status of blastocyst-descended ICM andepiblast cells as well as in sustaining the undifferentiatedstatus and ability for self-renewal of ESCs [71 72] Theaberrant (ie downregulated) expression levels and patternsof Oct4 and Nanog transcripts in both bovine NT blastocystsproduced using epigenetically nonmodulated calf dermalfibroblast cells [73] and porcine NT blastocysts producedusing either epigenetically nonmodulated adult MSCs orfetal fibroblast cells [28] have been found to be a majorcause of not only their declined cytological and molecularquality but also decreased in vitro developmental potentialof cloned embryos However in our current study it seemsthat TSA-dependent epigenomic transformation of nucleardonor MSCs biases correctingly the expression profile ofOct4 andNanog mRNAs in cloned pig blastocysts triggeringboth significant enhancement of relative abundance (RA)of Oct4 transcripts and slight (nonsignificant) increase inNanog transcript RA as compared to the TSA-untreatedMSCgroup On the one hand this influence could be exerted bydirect diminishment in the deacetylation level within thenucleosomal core-derived histone lysine moieties that wasevoked by trichostatin A-mediated nonspecific inhibition ofHDACs On the other hand it could be probably elicitedvia indirect nonselective suppression of cytosine residuemethylation processes within the Oct4 and Nanog gene pro-moters andor enhancers leading to upregulated expressionof these crucial pluripotency-related genes Similar resultshave been shown in the study by Wang et al [73] in whichthe sequential treatment of bovine differentiated nucleardonor fibroblast cells and resultant cloned embryos by bothnonspecific inhibitors of DNMTs and HDACs (ie 5-aza-dC
10 BioMed Research International
and TSA resp) contributed to increase of Oct4 transcriptRA in the blastocysts obtained simultaneously sustainingthe expression of Nanog mRNAs on the unchanged levelas compared to the 5-aza-dC- and TSA-unexposed groupIn this case 5-aza-21015840-deoxycytidine- and trichostatin A-mediated epigenetic modulation of both nuclear donor cellsand cloned embryos gave rise presumably to direct down-regulation in the methylation of cytosine residues withinthe Oct4 gene promoter andor enhancer that resultedfrom 5-aza-dC-dependent nonspecific inhibition of DNMTsFurthermore this two-factor epigenetic modulation couldaffect the enhanced incidence of acetylation level within thenucleosomal core-derived histone lysine moieties that wastriggered by trichostatin A-mediated nonselective inhibitionof HDACs consequently leading to indirect upregulatedexpression of the Oct4 gene Taking into consideration allthe abovementioned findings considerable transcriptionalupregulation in the expression of Oct4 gene (confirmedamong porcine NT blastocysts in our present study andamong bovineNT blastocysts in the study byWang et al [73])can be found to be pivotal indicator of increased pluripotencyextent of cloned embryos This process appears to be alsoassociated with improved cytological quality of NT blasto-cysts that was measured with their total cell counts In thecurrent investigation we have shown that the overall numberof ICM and trophoblast cells in blastocysts was significantlyhigher following the reconstruction of NT pig embryos withepigenomically transformed mesenchymal stem cells
5 Conclusions and Future Goals
Summing up artificial epigenomic modulation of in vitrocultured MSCs using the nonspecific HDAC inhibitor desig-nated as TSA seems to facilitate much more the reprogram-ming process for epigenetically determined transcriptionalactivity of somatic cell-inherited nuclear genome in theNT pig embryos The use of ectopic HDAC inhibitors forepigenetic transformation of mesenchymal stem cells whosecell nuclei were transferred into enucleated oocytes is thecompletely new approach in the studies involving somaticcell cloning of pigs and other mammalian species Enhancedreprogrammability of nuclear genome descended from TSA-exposed MSCs in the blastomeres of porcine preimplantedcloned embryos resulted in the improvements of not onlytheir capacity to complete in vitro development to themorula and blastocyst stages but also cytological and molec-ular quality of the blastocysts produced Therefore furtherinvestigations are also necessary to determine whether thenovel strategy of TSA-dependent epigenetic modification ofnuclear donor MSCs which has been recently utilized in ourlaboratory for the future goals of generating cloned pigletsenables retaining the in vivo developmental competences ofthe high-quality NT-derived blastocysts to reach full term
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Marcin Samiec and Jolanta Opiela contributed equally to thepreparation of this paper
Acknowledgments
The project was funded by the Polish National ScienceCentre resources allocated on the basis of Decision no DEC-201103DNZ905537 The authors would like to thank DrJarosław Wieczorek DVM for bone marrow retrieval
References
[1] A J Bonk H-T Cheong R Li et al ldquoCorrelation of develop-mental differences of nuclear transfer embryos cells to themethylation profiles of nuclear transfer donor cells in swinerdquoEpigenetics vol 2 no 3 pp 179ndash186 2007
[2] A J Bonk R Li L Lai et al ldquoAberrant DNA methylation inporcine in vitro- parthenogenetic- and somatic cell nucleartransfer-produced blastocystsrdquo Molecular Reproduction andDevelopment vol 75 no 2 pp 250ndash264 2008
[3] M Samiec ldquoDevelopment of pig cloning studies past presentand futurerdquo Journal of Animal and Feed Sciences vol 13 no 2pp 211ndash238 2004
[4] K MWhitworth and R S Prather ldquoSomatic cell nuclear trans-fer efficiency how can it be improved through nuclear remod-eling and reprogrammingrdquoMolecular Reproduction and Devel-opment vol 77 no 12 pp 1001ndash1015 2010
[5] K-I Yamanaka S Sugimura T Wakai M Kawahara and ESato ldquoAcetylation level of histone H3 in early embryonic stagesaffects subsequent development of miniature pig somatic cellnuclear transfer embryosrdquo The Journal of Reproduction andDevelopment vol 55 no 6 pp 638ndash644 2009
[6] R P Cervera N Martı-Gutierrez E Escorihuela R Morenoand M Stojkovic ldquoTrichostatin A affects histone acetylationand gene expression in porcine somatic cell nucleus transferembryosrdquoTheriogenology vol 72 no 8 pp 1097ndash1110 2009
[7] J Zhao J Whyte and R S Prather ldquoEffect of epigeneticregulation during swine embryogenesis and on cloning bynuclear transferrdquo Cell and Tissue Research vol 341 no 1 pp13ndash21 2010
[8] WReik ldquoStability and flexibility of epigenetic gene regulation inmammalian developmentrdquo Nature vol 447 no 7143 pp 425ndash432 2007
[9] R S Prather J W Ross S C Isom and J A Green ldquoTranscrip-tional post-transcriptional and epigenetic control of porcineoocyte maturation and embryogenesisrdquo Society of Reproductionand Fertility supplement vol 66 pp 165ndash176 2009
[10] N Rodriguez-Osorio R Urrego J B Cibelli K Eilertsen andE Memili ldquoReprogramming mammalian somatic cellsrdquo Theri-ogenology vol 78 no 9 pp 1869ndash1886 2012
[11] K J Eilertsen R A Power L L Harkins and P Misica ldquoTar-geting cellular memory to reprogram the epigenome restorepotential and improve somatic cell nuclear transferrdquo AnimalReproduction Science vol 98 no 1-2 pp 129ndash146 2007
[12] M Samiec and M Skrzyszowska ldquoMolecular conditions of thecell nucleus remodellingreprogramming process and nucleartransferred embryo development in the intraooplasmic kary-oplast injection technique a reviewrdquo Czech Journal of AnimalScience vol 50 no 5 pp 185ndash195 2005
BioMed Research International 11
[13] P Narbonne K Miyamoto and J B Gurdon ldquoReprogrammingand development in nuclear transfer embryos and in interspe-cific systemsrdquo Current Opinion in Genetics amp Development vol22 no 5 pp 450ndash458 2012
[14] J-I Bang J-G Yoo M-R Park et al ldquoThe effects of artificialactivation timing on the development of SCNT-derivedembryos and newborn pigletsrdquoReproductive Biology vol 13 no2 pp 127ndash132 2013
[15] M Samiec and M Skrzyszowska ldquoHigh developmental capa-bility of porcine cloned embryos following trichostatin A-dependent epigenomic transformation during in vitro matura-tion of oocytes pre-exposed to R-roscovitinerdquo Animal SciencePapers and Reports vol 30 no 4 pp 383ndash393 2012
[16] M Samiec and M Skrzyszowska ldquoRoscovitine is a novel agentthat can be used for the activation of porcine oocytes recon-structed with adult cutaneous or fetal fibroblast cell nucleirdquoTheriogenology vol 78 no 8 pp 1855ndash1867 2012
[17] Z Li J Shi D Liu et al ldquoEffects of donor fibroblast cell typeand transferred cloned embryo number on the efficiency of pigcloningrdquo Cellular Reprogramming vol 15 no 1 pp 35ndash42 2013
[18] M Samiec and M Skrzyszowska ldquoBiological transcomple-mentary activation as a novel and effective strategy appliedto the generation of porcine somatic cell cloned embryosrdquoReproductive Biology vol 14 no 2 pp 128ndash139 2014
[19] J Li O Svarcova K Villemoes et al ldquoHigh in vitro developmentafter somatic cell nuclear transfer and trichostatin A treatmentof reconstructed porcine embryosrdquoTheriogenology vol 70 no5 pp 800ndash808 2008
[20] M Skrzyszowska M Samiec R Słomski D Lipinski and EMały ldquoDevelopment of porcine transgenic nuclear-transferredembryos derived from fibroblast cells transfected by the noveltechnique of nucleofection or standard lipofectionrdquo Theri-ogenology vol 70 no 2 pp 248ndash259 2008
[21] Y-W Pang L An P Wang et al ldquoTreatment of porcine donorcells and reconstructed embryoswith the antioxidantmelatoninenhances cloning efficiencyrdquo Journal of Pineal Research vol 54no 4 pp 389ndash397 2013
[22] M Samiec M Skrzyszowska and J Opiela ldquoCreation of clonedpig embryos using contact-inhibited or serum-starved fibrob-last cells analysed intra vitam for apoptosis occurrencerdquo Annalsof Animal Science vol 13 no 2 pp 275ndash293 2013
[23] H Wei Y Qing W Pan et al ldquoComparison of the efficiency ofBannaminiature inbred pig somatic cell nuclear transfer amongdifferent donor cellsrdquo PLoSONE vol 8 no 2 Article ID e577282013
[24] K Inoue N Ogonuki H Miki et al ldquoInefficient reprogram-ming of the hematopoietic stem cell genome following nucleartransferrdquo Journal of Cell Science vol 119 no 10 pp 1985ndash19912006
[25] K Inoue S Noda N Ogonuki et al ldquoDifferential developmen-tal ability of embryos cloned from tissue-specific stem cellsrdquoStem Cells vol 25 no 5 pp 1279ndash1285 2007
[26] S L Lee E J Kang G H Maeng et al ldquoDevelopmental abilityof miniature pig embryos cloned withmesenchymal stem cellsrdquoThe Journal of Reproduction and Development vol 56 no 2 pp256ndash262 2010
[27] Z Li X He L Chen et al ldquoBone marrow mesenchymal stemcells are an attractive donor cell type for production of clonedpigs as well as genetically modified cloned pigs by somatic cellnuclear transferrdquo Cellular Reprogramming vol 15 no 5 pp459ndash470 2013
[28] B M Kumar H-F Jin J-G Kim et al ldquoDifferential geneexpression patterns in porcine nuclear transfer embryos recon-structed with fetal fibroblasts and mesenchymal stem cellsrdquoDevelopmental Dynamics vol 236 no 2 pp 435ndash446 2007
[29] J Su Y Wang Y Li et al ldquoOxamflatin significantly improvesnuclear reprogramming blastocyst quality and in vitro devel-opment of bovine SCNT embryosrdquo PLoS ONE vol 6 no 8Article ID e23805 2011
[30] Y Song THai YWang et al ldquoEpigenetic reprogramming geneexpression and in vitro development of porcine SCNT embryosare significantly improved by a histone deacetylase inhibitor-m-carboxycinnamic acid bishydroxamide (CBHA)rdquo Protein ampCell vol 5 no 5 pp 382ndash393 2014
[31] S Colleoni G Donofrio I Lagutina R Duchi C Galli andG Lazzari ldquoEstablishment differentiation electroporationviral transduction and nuclear transfer of bovine and porcinemesenchymal stem cellsrdquo Cloning and Stem Cells vol 7 no 3pp 154ndash166 2005
[32] H F Jin B M Kumar J G Kim et al ldquoEnhanced developmentof porcine embryos cloned from bone marrow mesenchymalstem cellsrdquoThe International Journal of Developmental Biologyvol 51 no 1 pp 85ndash90 2007
[33] J Opiela and M Samiec ldquoCharacterization of mesenchymalstem cells and their application in experimental embryologyrdquoPolish Journal of Veterinary Sciences vol 16 no 3 pp 593ndash5992013
[34] X Yang S L Smith X C Tian H A Lewin J-P Renard and TWakayama ldquoNuclear reprogramming of cloned embryos and itsimplications for therapeutic cloningrdquo Nature Genetics vol 39no 3 pp 295ndash302 2007
[35] XWu Y Li G-P Li et al ldquoTrichostatin A improved epigeneticmodifications of transfected cells but did not improve subse-quent cloned embryo developmentrdquo Animal Biotechnology vol19 no 4 pp 211ndash224 2008
[36] F Bo L Di F Qing-Chang et al ldquoEffect of trichostatin A ontransfected donor cells and subsequent development of porcinecloned embryosrdquo Zygote vol 19 no 3 pp 237ndash243 2011
[37] X Ding Y Wang D Zhang Z Guo and Y Zhang ldquoIncreasedpre-implantation development of cloned bovine embryostreated with 5-aza-21015840-deoxycytidine and trichostatin ArdquoTheri-ogenology vol 70 no 4 pp 622ndash630 2008
[38] H S Lee X F Yu J I Bang et al ldquoEnhanced histone acetylationin somatic cells induced by a histone deacetylase inhibitorimproved inter-generic cloned leopard cat blastocystsrdquo Theri-ogenology vol 74 no 8 pp 1439ndash1449 2010
[39] S-F Ning Q-Y Li M-M Liang et al ldquoMethylation charac-teristics and developmental potential of Guangxi Bamaminipig(Sus scrofa domestica) cloned embryos from donor cells treatedwith trichostatin A and 5-aza-21015840-deoxycytidinerdquo Zygote vol 21no 2 pp 178ndash186 2013
[40] B P Enright L-Y Sung C-C Chang X Yang and X C TianldquoMethylation and acetylation characteristics of cloned bovineembryos from donor cells treated with 5-aza-21015840-deoxycytidinerdquoBiology of Reproduction vol 72 no 4 pp 944ndash948 2005
[41] Y J Huan J Zhu B T Xie et al ldquoTreating cloned embryos butnot donor cells with 5-aza-21015840-deoxycytidine enhances thedevelopmental competence of porcine cloned embryosrdquo TheJournal of Reproduction andDevelopment vol 59 no 5 pp 442ndash449 2013
[42] B-G JeonG Coppola S D Perrault G-J RhoDH Betts andWAKing ldquoS-adenosylhomocysteine treatment of adult female
12 BioMed Research International
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
Figure 3 Relative abundance (mean plusmn SD) of Oct4 Nanog andNestin mRNAs in blastocysts developed from nuclear-transferredpig embryos descended from MSCs undergoing or not undergoingTSA-dependent epigenomic transformation The highly significantdifferences were designated with the A and B letters (119875 lt 001ANOVA followed by Tukeyrsquos HSD post hoc test)
33 The Pluripotency Status of Porcine Nuclear-TransferredEmbryos Originating from Epigenomically Modulated or Non-modulated MSCs The total number of analyzed clonedblastocysts was 60 To assess relative abundance (RA) of threeanalyzed mRNA transcripts for pluripotency-related targetgenes (Oct4 Nanog) multipotent stemness-associated targetgene (Nestin) and control GAPDH gene transcript we used3 times 10 blastocysts from both TSA-treated and -untreatedgroups
Highly significant differences were indicated for theOct4gene transcript quantitative profile between blastocysts orig-inating from TSA-treated and -untreated groups (119875 lt 001Figure 3) Although no significant intergroup variability wasshown in the RA of Nanog and Nestin mRNAs (119875 ge 005)their expression levels tended to be higher in blastocystsderived from NT embryos reconstructed with TSA-exposedMSCs as compared to the TSA-untreated group (Figure 3)
4 Discussion
The abundance of the morulae and blastocysts of highercytological and molecular quality generated in the presentinvestigation confirms that the reprogrammability of adultbone marrow-derived mesenchymal stem cells which hadbeen epigenetically modified via exposure to trichostatinA underwent considerable improvement in a cytoplasm ofporcine nuclear-transferred oocytes and resultant in vitrocultured cloned embryos It is beyond any doubt thatthe impact of TSA-based epigenomic transformation ofbone marrow-retrieved MSCs representing multipotent andundifferentiated stem cells on their competence for SCNThas not yet been explored not only in pigs but also inother mammalian species So far TSA-mediated epigeneticmodulation has been applied only for differentiated somatic
cells that commonly provide the source of nuclear donorsfor cloning procedure in pigs The results of the study byDiao et al [55] proved that porcineNT embryos reconstitutedwith TSA-treated fetal fibroblast cells were characterized bytwofold higher developmental potential to reach the blasto-cyst stage (30) than the NT embryos reconstituted withTSA-untreated fibroblast cells (15) Similar tendency hasbeen also shown in our current study inwhich trichostatinA-mediated epigenetic transformation of bone marrow-derivedMSCs gave rise to significant enhancement of capability ofcloned embryos to complete their development to blastocyststage (65) as compared to that observed for embryosderived from TSA-unexposed MSC nuclei (46) Nonethe-less NT pig embryos originating from epigenomically trans-formed fetal fibroblast cells displayed considerably lowerblastocyst formation rate [55] than the rate indicated in ourpresent study for NT embryos reconstructed with epige-nomically nontransformed MSCs Moreover treatment ofnuclear donor MSCs with trichostatin A resulted in one anda half times to severalfold increase of blastocyst percentage(65) as compared to the blastocyst yields obtained by otherinvestigators using undifferentiated or in vitro differentiatedMSCs that were not modulated epigenetically [26ndash28 31 32]In the study by Kumar et al [28] the abilities of epige-nomically nontransformed bone marrow-descended MSCnuclei to direct the in vitro development of porcine clonedembryos to blastocyst stage decreased above three times(approximately 20) as compared to our present work Inturn nuclear-transferred embryos that had originated fromporcine undifferentiated bone marrow-retrieved MSCs andtheir derivatives along the osteogenic lineage were able tocomplete their in vitro development to blastocyst stage atthe rates ranging from 33 to 45 [31] In contrast Jin etal [32] and Li et al [27] reported that blastocyst yields ofNT embryos derived from porcine epigenetically unmodu-lated MSCs were maintained at the relatively low levels ofapproximately 18 and 16 respectively Nevertheless in thestudy by Lee et al [26] the in vitro developmental outcometo the blastocyst stage of NT embryos originating fromundifferentiated MSCs achieved the rate of approximately48 but even this blastocyst formation rate was considerablylower than that noticed in our current investigation
The results of the current study have also clearly demon-strated that the TSA-dependent epigenomic transformationof nuclear donor MSCs contributes to improvement of notonly in vitro developmental competences but also qualityand transcription level-related pluripotency extent of porcinecloned embryos The utilization of undifferentiated mes-enchymal stem cells and the modification of the molecularmechanisms of transcriptional reprogramming of the donornuclear genome via HDAC inhibitor-mediated epigeneticmodulation of MSCs before their use for SCNT led to correctand complete adaptation of MSC-inherited genomic DNA tothe cytoplasmic environment of enucleated oocytes and invitro cultured NT pig embryos Epigenetic reprogrammingof donor cell nuclei suggests that a new program for theirtranscriptional activity is loaded and reloaded immediatelyfollowing reconstruction of enucleated oocytes The successof SCNT may depend upon both genomic DNA-associated
BioMed Research International 9
reprogramming of gene expression for dedifferentiation ofthe donor somatic cell nuclei during early preimplantationdevelopment of cloned embryos and reprogramming of geneexpression for onset of somatogenic nuclear redifferentiationduring blastocyst formation [7 10 63 64] It has been ascer-tained that somatic cell nuclei should undergo the wide DNAcytosine residue demethylation changes throughout the earlydevelopment of NT embryos to erase and then reset theirown overall epigenetic as well as parental genomic imprintingmemory which has been established by remethylation ofthe nuclear genome within the framework of the specificpathway of somatic and germ cell lineage commitment anddifferentiation [2 4 13 30 65ndash67]
It appears that the enhanced cytological quality of clonedpig blastocysts that originated from enucleated oocytesreceiving epigenetically modulated MSC nuclei could beassociated with more faithful and faultless pattern for repro-gramming of transcriptional activity of genomic DNA inher-ited from undifferentiated and multipotent stem cells Thesecells exhibit increased genomic and epigenomic plasticity inrearrangements of their gene expression in the blastomeres ofpreimplanted NT embryos The fluorocytochemical analysisof overall mean nuclear counts revealed almost twofoldhigher total cells number in porcine blastocysts developedfrom NT oocytes reconstituted with TSA-exposed MSCs (48blastomeres) than that identified in blastocysts derived fromNT oocytes reconstituted with TSA-unexposed MSCs (28blastomeres) The quality of cloned blastocysts generated inour present study using epigenomically transformed MSCswas comparable to that noticed in the study by Lee et al[26] for porcine NT blastocysts derived from epigenomicallynontransformed MSCs These blastocysts were also charac-terized by a total cells number equal to 48 In contrast to theabovementioned findings in the investigations carried outby Li et al [27] Kumar et al [28] and Jin et al [32] theoverall number of ICM and trophectoderm cells in clonedpig blastocysts originating from embryos reconstituted withTSA-untreated MSCs ranged from 28 to 35 and decreasedapproximately one and a half times as compared to the totalcells number estimated for NT blastocysts obtained by usin the TSA-exposed MSC group Nevertheless the meanapoptotic index (ie DCI = 495) that was calculated by usfor blastocysts developed from cloned embryos reconstitutedwith epigenetically modified MSC nuclei was similar to theratios of TUNEL-positive cells (46 to 47) observed by Jinet al [32] andKumar et al [28] among porcineNT blastocystsderived from epigenetically nonmodulated counterparts Inturn cloned blastocysts produced in our study using TSA-untreated MSCs displayed the apoptotic index (713) thatwas comparable or slightly lower to indexes identified forcloned embryos created with the aid of fetal fibroblast cellsnot undergoing exposure to trichostatin A [28 32]The latterindexes (DCIs) oscillated between 73 and 78 Generally inour current work the proportion of TUNEL-positive cellsin relation to a total cells number tended to insignificantlydecrease among NT blastocysts generated using undifferen-tiated mesenchymal stem cells subjected to TSA treatmentcompared to those generated using nuclear donor cells not
exposed to TSA (495 versus 713) Analogous slight down-ward tendency in the ratios of TUNEL-positive cells wasindicated byDiao et al [55] comparing blastocysts developedfrom cloned embryos reconstructed with cell nuclei of fetalfibroblasts treated with TSA and the counterparts producedusing TSA-unexposed fetal fibroblast cell nuclei (nearly 3versus 45)
Improved reprogrammability of transcriptional activityfor nuclear genome of epigenetically modulated MSCs inthe cells of preimplanted NT embryos turned out to bepositively correlated with enhanced molecular quality ofporcine cloned blastocysts assessed on the basis of theirpluripotency extent whichwasmeasuredwith the expressionprofiles identified for Oct4 and Nanog genes A 38-kDaprotein Oct4 (ie octamer-binding transcription factor 4)that is a member of the family of POU- (Pit-Oct-Unc-)domain and homeodomain transcription factors acts as avital regulator of pluripotency extent playing an importantrole in not only controlling preimplantation embryonicdevelopment but also maintenance of ICM cell fate inblastocysts and pluripotency status of embryonic stem cells(ESCs) [68ndash70] A 35-kDa protein designated as Nanog fromCelticIrish mythical Tır na nOg (Tir Na Nog The Landof the Ever-Young) is another homeobox-containing tran-scription factor that represents the group of pivotal proteinsmodulating pluripotency degree [70 71] The homeoproteinNanog can act synergistically with Oct4 protein in retainingthe pluripotent status of blastocyst-descended ICM andepiblast cells as well as in sustaining the undifferentiatedstatus and ability for self-renewal of ESCs [71 72] Theaberrant (ie downregulated) expression levels and patternsof Oct4 and Nanog transcripts in both bovine NT blastocystsproduced using epigenetically nonmodulated calf dermalfibroblast cells [73] and porcine NT blastocysts producedusing either epigenetically nonmodulated adult MSCs orfetal fibroblast cells [28] have been found to be a majorcause of not only their declined cytological and molecularquality but also decreased in vitro developmental potentialof cloned embryos However in our current study it seemsthat TSA-dependent epigenomic transformation of nucleardonor MSCs biases correctingly the expression profile ofOct4 andNanog mRNAs in cloned pig blastocysts triggeringboth significant enhancement of relative abundance (RA)of Oct4 transcripts and slight (nonsignificant) increase inNanog transcript RA as compared to the TSA-untreatedMSCgroup On the one hand this influence could be exerted bydirect diminishment in the deacetylation level within thenucleosomal core-derived histone lysine moieties that wasevoked by trichostatin A-mediated nonspecific inhibition ofHDACs On the other hand it could be probably elicitedvia indirect nonselective suppression of cytosine residuemethylation processes within the Oct4 and Nanog gene pro-moters andor enhancers leading to upregulated expressionof these crucial pluripotency-related genes Similar resultshave been shown in the study by Wang et al [73] in whichthe sequential treatment of bovine differentiated nucleardonor fibroblast cells and resultant cloned embryos by bothnonspecific inhibitors of DNMTs and HDACs (ie 5-aza-dC
10 BioMed Research International
and TSA resp) contributed to increase of Oct4 transcriptRA in the blastocysts obtained simultaneously sustainingthe expression of Nanog mRNAs on the unchanged levelas compared to the 5-aza-dC- and TSA-unexposed groupIn this case 5-aza-21015840-deoxycytidine- and trichostatin A-mediated epigenetic modulation of both nuclear donor cellsand cloned embryos gave rise presumably to direct down-regulation in the methylation of cytosine residues withinthe Oct4 gene promoter andor enhancer that resultedfrom 5-aza-dC-dependent nonspecific inhibition of DNMTsFurthermore this two-factor epigenetic modulation couldaffect the enhanced incidence of acetylation level within thenucleosomal core-derived histone lysine moieties that wastriggered by trichostatin A-mediated nonselective inhibitionof HDACs consequently leading to indirect upregulatedexpression of the Oct4 gene Taking into consideration allthe abovementioned findings considerable transcriptionalupregulation in the expression of Oct4 gene (confirmedamong porcine NT blastocysts in our present study andamong bovineNT blastocysts in the study byWang et al [73])can be found to be pivotal indicator of increased pluripotencyextent of cloned embryos This process appears to be alsoassociated with improved cytological quality of NT blasto-cysts that was measured with their total cell counts In thecurrent investigation we have shown that the overall numberof ICM and trophoblast cells in blastocysts was significantlyhigher following the reconstruction of NT pig embryos withepigenomically transformed mesenchymal stem cells
5 Conclusions and Future Goals
Summing up artificial epigenomic modulation of in vitrocultured MSCs using the nonspecific HDAC inhibitor desig-nated as TSA seems to facilitate much more the reprogram-ming process for epigenetically determined transcriptionalactivity of somatic cell-inherited nuclear genome in theNT pig embryos The use of ectopic HDAC inhibitors forepigenetic transformation of mesenchymal stem cells whosecell nuclei were transferred into enucleated oocytes is thecompletely new approach in the studies involving somaticcell cloning of pigs and other mammalian species Enhancedreprogrammability of nuclear genome descended from TSA-exposed MSCs in the blastomeres of porcine preimplantedcloned embryos resulted in the improvements of not onlytheir capacity to complete in vitro development to themorula and blastocyst stages but also cytological and molec-ular quality of the blastocysts produced Therefore furtherinvestigations are also necessary to determine whether thenovel strategy of TSA-dependent epigenetic modification ofnuclear donor MSCs which has been recently utilized in ourlaboratory for the future goals of generating cloned pigletsenables retaining the in vivo developmental competences ofthe high-quality NT-derived blastocysts to reach full term
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Marcin Samiec and Jolanta Opiela contributed equally to thepreparation of this paper
Acknowledgments
The project was funded by the Polish National ScienceCentre resources allocated on the basis of Decision no DEC-201103DNZ905537 The authors would like to thank DrJarosław Wieczorek DVM for bone marrow retrieval
References
[1] A J Bonk H-T Cheong R Li et al ldquoCorrelation of develop-mental differences of nuclear transfer embryos cells to themethylation profiles of nuclear transfer donor cells in swinerdquoEpigenetics vol 2 no 3 pp 179ndash186 2007
[2] A J Bonk R Li L Lai et al ldquoAberrant DNA methylation inporcine in vitro- parthenogenetic- and somatic cell nucleartransfer-produced blastocystsrdquo Molecular Reproduction andDevelopment vol 75 no 2 pp 250ndash264 2008
[3] M Samiec ldquoDevelopment of pig cloning studies past presentand futurerdquo Journal of Animal and Feed Sciences vol 13 no 2pp 211ndash238 2004
[4] K MWhitworth and R S Prather ldquoSomatic cell nuclear trans-fer efficiency how can it be improved through nuclear remod-eling and reprogrammingrdquoMolecular Reproduction and Devel-opment vol 77 no 12 pp 1001ndash1015 2010
[5] K-I Yamanaka S Sugimura T Wakai M Kawahara and ESato ldquoAcetylation level of histone H3 in early embryonic stagesaffects subsequent development of miniature pig somatic cellnuclear transfer embryosrdquo The Journal of Reproduction andDevelopment vol 55 no 6 pp 638ndash644 2009
[6] R P Cervera N Martı-Gutierrez E Escorihuela R Morenoand M Stojkovic ldquoTrichostatin A affects histone acetylationand gene expression in porcine somatic cell nucleus transferembryosrdquoTheriogenology vol 72 no 8 pp 1097ndash1110 2009
[7] J Zhao J Whyte and R S Prather ldquoEffect of epigeneticregulation during swine embryogenesis and on cloning bynuclear transferrdquo Cell and Tissue Research vol 341 no 1 pp13ndash21 2010
[8] WReik ldquoStability and flexibility of epigenetic gene regulation inmammalian developmentrdquo Nature vol 447 no 7143 pp 425ndash432 2007
[9] R S Prather J W Ross S C Isom and J A Green ldquoTranscrip-tional post-transcriptional and epigenetic control of porcineoocyte maturation and embryogenesisrdquo Society of Reproductionand Fertility supplement vol 66 pp 165ndash176 2009
[10] N Rodriguez-Osorio R Urrego J B Cibelli K Eilertsen andE Memili ldquoReprogramming mammalian somatic cellsrdquo Theri-ogenology vol 78 no 9 pp 1869ndash1886 2012
[11] K J Eilertsen R A Power L L Harkins and P Misica ldquoTar-geting cellular memory to reprogram the epigenome restorepotential and improve somatic cell nuclear transferrdquo AnimalReproduction Science vol 98 no 1-2 pp 129ndash146 2007
[12] M Samiec and M Skrzyszowska ldquoMolecular conditions of thecell nucleus remodellingreprogramming process and nucleartransferred embryo development in the intraooplasmic kary-oplast injection technique a reviewrdquo Czech Journal of AnimalScience vol 50 no 5 pp 185ndash195 2005
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[13] P Narbonne K Miyamoto and J B Gurdon ldquoReprogrammingand development in nuclear transfer embryos and in interspe-cific systemsrdquo Current Opinion in Genetics amp Development vol22 no 5 pp 450ndash458 2012
[14] J-I Bang J-G Yoo M-R Park et al ldquoThe effects of artificialactivation timing on the development of SCNT-derivedembryos and newborn pigletsrdquoReproductive Biology vol 13 no2 pp 127ndash132 2013
[15] M Samiec and M Skrzyszowska ldquoHigh developmental capa-bility of porcine cloned embryos following trichostatin A-dependent epigenomic transformation during in vitro matura-tion of oocytes pre-exposed to R-roscovitinerdquo Animal SciencePapers and Reports vol 30 no 4 pp 383ndash393 2012
[16] M Samiec and M Skrzyszowska ldquoRoscovitine is a novel agentthat can be used for the activation of porcine oocytes recon-structed with adult cutaneous or fetal fibroblast cell nucleirdquoTheriogenology vol 78 no 8 pp 1855ndash1867 2012
[17] Z Li J Shi D Liu et al ldquoEffects of donor fibroblast cell typeand transferred cloned embryo number on the efficiency of pigcloningrdquo Cellular Reprogramming vol 15 no 1 pp 35ndash42 2013
[18] M Samiec and M Skrzyszowska ldquoBiological transcomple-mentary activation as a novel and effective strategy appliedto the generation of porcine somatic cell cloned embryosrdquoReproductive Biology vol 14 no 2 pp 128ndash139 2014
[19] J Li O Svarcova K Villemoes et al ldquoHigh in vitro developmentafter somatic cell nuclear transfer and trichostatin A treatmentof reconstructed porcine embryosrdquoTheriogenology vol 70 no5 pp 800ndash808 2008
[20] M Skrzyszowska M Samiec R Słomski D Lipinski and EMały ldquoDevelopment of porcine transgenic nuclear-transferredembryos derived from fibroblast cells transfected by the noveltechnique of nucleofection or standard lipofectionrdquo Theri-ogenology vol 70 no 2 pp 248ndash259 2008
[21] Y-W Pang L An P Wang et al ldquoTreatment of porcine donorcells and reconstructed embryoswith the antioxidantmelatoninenhances cloning efficiencyrdquo Journal of Pineal Research vol 54no 4 pp 389ndash397 2013
[22] M Samiec M Skrzyszowska and J Opiela ldquoCreation of clonedpig embryos using contact-inhibited or serum-starved fibrob-last cells analysed intra vitam for apoptosis occurrencerdquo Annalsof Animal Science vol 13 no 2 pp 275ndash293 2013
[23] H Wei Y Qing W Pan et al ldquoComparison of the efficiency ofBannaminiature inbred pig somatic cell nuclear transfer amongdifferent donor cellsrdquo PLoSONE vol 8 no 2 Article ID e577282013
[24] K Inoue N Ogonuki H Miki et al ldquoInefficient reprogram-ming of the hematopoietic stem cell genome following nucleartransferrdquo Journal of Cell Science vol 119 no 10 pp 1985ndash19912006
[25] K Inoue S Noda N Ogonuki et al ldquoDifferential developmen-tal ability of embryos cloned from tissue-specific stem cellsrdquoStem Cells vol 25 no 5 pp 1279ndash1285 2007
[26] S L Lee E J Kang G H Maeng et al ldquoDevelopmental abilityof miniature pig embryos cloned withmesenchymal stem cellsrdquoThe Journal of Reproduction and Development vol 56 no 2 pp256ndash262 2010
[27] Z Li X He L Chen et al ldquoBone marrow mesenchymal stemcells are an attractive donor cell type for production of clonedpigs as well as genetically modified cloned pigs by somatic cellnuclear transferrdquo Cellular Reprogramming vol 15 no 5 pp459ndash470 2013
[28] B M Kumar H-F Jin J-G Kim et al ldquoDifferential geneexpression patterns in porcine nuclear transfer embryos recon-structed with fetal fibroblasts and mesenchymal stem cellsrdquoDevelopmental Dynamics vol 236 no 2 pp 435ndash446 2007
[29] J Su Y Wang Y Li et al ldquoOxamflatin significantly improvesnuclear reprogramming blastocyst quality and in vitro devel-opment of bovine SCNT embryosrdquo PLoS ONE vol 6 no 8Article ID e23805 2011
[30] Y Song THai YWang et al ldquoEpigenetic reprogramming geneexpression and in vitro development of porcine SCNT embryosare significantly improved by a histone deacetylase inhibitor-m-carboxycinnamic acid bishydroxamide (CBHA)rdquo Protein ampCell vol 5 no 5 pp 382ndash393 2014
[31] S Colleoni G Donofrio I Lagutina R Duchi C Galli andG Lazzari ldquoEstablishment differentiation electroporationviral transduction and nuclear transfer of bovine and porcinemesenchymal stem cellsrdquo Cloning and Stem Cells vol 7 no 3pp 154ndash166 2005
[32] H F Jin B M Kumar J G Kim et al ldquoEnhanced developmentof porcine embryos cloned from bone marrow mesenchymalstem cellsrdquoThe International Journal of Developmental Biologyvol 51 no 1 pp 85ndash90 2007
[33] J Opiela and M Samiec ldquoCharacterization of mesenchymalstem cells and their application in experimental embryologyrdquoPolish Journal of Veterinary Sciences vol 16 no 3 pp 593ndash5992013
[34] X Yang S L Smith X C Tian H A Lewin J-P Renard and TWakayama ldquoNuclear reprogramming of cloned embryos and itsimplications for therapeutic cloningrdquo Nature Genetics vol 39no 3 pp 295ndash302 2007
[35] XWu Y Li G-P Li et al ldquoTrichostatin A improved epigeneticmodifications of transfected cells but did not improve subse-quent cloned embryo developmentrdquo Animal Biotechnology vol19 no 4 pp 211ndash224 2008
[36] F Bo L Di F Qing-Chang et al ldquoEffect of trichostatin A ontransfected donor cells and subsequent development of porcinecloned embryosrdquo Zygote vol 19 no 3 pp 237ndash243 2011
[37] X Ding Y Wang D Zhang Z Guo and Y Zhang ldquoIncreasedpre-implantation development of cloned bovine embryostreated with 5-aza-21015840-deoxycytidine and trichostatin ArdquoTheri-ogenology vol 70 no 4 pp 622ndash630 2008
[38] H S Lee X F Yu J I Bang et al ldquoEnhanced histone acetylationin somatic cells induced by a histone deacetylase inhibitorimproved inter-generic cloned leopard cat blastocystsrdquo Theri-ogenology vol 74 no 8 pp 1439ndash1449 2010
[39] S-F Ning Q-Y Li M-M Liang et al ldquoMethylation charac-teristics and developmental potential of Guangxi Bamaminipig(Sus scrofa domestica) cloned embryos from donor cells treatedwith trichostatin A and 5-aza-21015840-deoxycytidinerdquo Zygote vol 21no 2 pp 178ndash186 2013
[40] B P Enright L-Y Sung C-C Chang X Yang and X C TianldquoMethylation and acetylation characteristics of cloned bovineembryos from donor cells treated with 5-aza-21015840-deoxycytidinerdquoBiology of Reproduction vol 72 no 4 pp 944ndash948 2005
[41] Y J Huan J Zhu B T Xie et al ldquoTreating cloned embryos butnot donor cells with 5-aza-21015840-deoxycytidine enhances thedevelopmental competence of porcine cloned embryosrdquo TheJournal of Reproduction andDevelopment vol 59 no 5 pp 442ndash449 2013
[42] B-G JeonG Coppola S D Perrault G-J RhoDH Betts andWAKing ldquoS-adenosylhomocysteine treatment of adult female
12 BioMed Research International
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
reprogramming of gene expression for dedifferentiation ofthe donor somatic cell nuclei during early preimplantationdevelopment of cloned embryos and reprogramming of geneexpression for onset of somatogenic nuclear redifferentiationduring blastocyst formation [7 10 63 64] It has been ascer-tained that somatic cell nuclei should undergo the wide DNAcytosine residue demethylation changes throughout the earlydevelopment of NT embryos to erase and then reset theirown overall epigenetic as well as parental genomic imprintingmemory which has been established by remethylation ofthe nuclear genome within the framework of the specificpathway of somatic and germ cell lineage commitment anddifferentiation [2 4 13 30 65ndash67]
It appears that the enhanced cytological quality of clonedpig blastocysts that originated from enucleated oocytesreceiving epigenetically modulated MSC nuclei could beassociated with more faithful and faultless pattern for repro-gramming of transcriptional activity of genomic DNA inher-ited from undifferentiated and multipotent stem cells Thesecells exhibit increased genomic and epigenomic plasticity inrearrangements of their gene expression in the blastomeres ofpreimplanted NT embryos The fluorocytochemical analysisof overall mean nuclear counts revealed almost twofoldhigher total cells number in porcine blastocysts developedfrom NT oocytes reconstituted with TSA-exposed MSCs (48blastomeres) than that identified in blastocysts derived fromNT oocytes reconstituted with TSA-unexposed MSCs (28blastomeres) The quality of cloned blastocysts generated inour present study using epigenomically transformed MSCswas comparable to that noticed in the study by Lee et al[26] for porcine NT blastocysts derived from epigenomicallynontransformed MSCs These blastocysts were also charac-terized by a total cells number equal to 48 In contrast to theabovementioned findings in the investigations carried outby Li et al [27] Kumar et al [28] and Jin et al [32] theoverall number of ICM and trophectoderm cells in clonedpig blastocysts originating from embryos reconstituted withTSA-untreated MSCs ranged from 28 to 35 and decreasedapproximately one and a half times as compared to the totalcells number estimated for NT blastocysts obtained by usin the TSA-exposed MSC group Nevertheless the meanapoptotic index (ie DCI = 495) that was calculated by usfor blastocysts developed from cloned embryos reconstitutedwith epigenetically modified MSC nuclei was similar to theratios of TUNEL-positive cells (46 to 47) observed by Jinet al [32] andKumar et al [28] among porcineNT blastocystsderived from epigenetically nonmodulated counterparts Inturn cloned blastocysts produced in our study using TSA-untreated MSCs displayed the apoptotic index (713) thatwas comparable or slightly lower to indexes identified forcloned embryos created with the aid of fetal fibroblast cellsnot undergoing exposure to trichostatin A [28 32]The latterindexes (DCIs) oscillated between 73 and 78 Generally inour current work the proportion of TUNEL-positive cellsin relation to a total cells number tended to insignificantlydecrease among NT blastocysts generated using undifferen-tiated mesenchymal stem cells subjected to TSA treatmentcompared to those generated using nuclear donor cells not
exposed to TSA (495 versus 713) Analogous slight down-ward tendency in the ratios of TUNEL-positive cells wasindicated byDiao et al [55] comparing blastocysts developedfrom cloned embryos reconstructed with cell nuclei of fetalfibroblasts treated with TSA and the counterparts producedusing TSA-unexposed fetal fibroblast cell nuclei (nearly 3versus 45)
Improved reprogrammability of transcriptional activityfor nuclear genome of epigenetically modulated MSCs inthe cells of preimplanted NT embryos turned out to bepositively correlated with enhanced molecular quality ofporcine cloned blastocysts assessed on the basis of theirpluripotency extent whichwasmeasuredwith the expressionprofiles identified for Oct4 and Nanog genes A 38-kDaprotein Oct4 (ie octamer-binding transcription factor 4)that is a member of the family of POU- (Pit-Oct-Unc-)domain and homeodomain transcription factors acts as avital regulator of pluripotency extent playing an importantrole in not only controlling preimplantation embryonicdevelopment but also maintenance of ICM cell fate inblastocysts and pluripotency status of embryonic stem cells(ESCs) [68ndash70] A 35-kDa protein designated as Nanog fromCelticIrish mythical Tır na nOg (Tir Na Nog The Landof the Ever-Young) is another homeobox-containing tran-scription factor that represents the group of pivotal proteinsmodulating pluripotency degree [70 71] The homeoproteinNanog can act synergistically with Oct4 protein in retainingthe pluripotent status of blastocyst-descended ICM andepiblast cells as well as in sustaining the undifferentiatedstatus and ability for self-renewal of ESCs [71 72] Theaberrant (ie downregulated) expression levels and patternsof Oct4 and Nanog transcripts in both bovine NT blastocystsproduced using epigenetically nonmodulated calf dermalfibroblast cells [73] and porcine NT blastocysts producedusing either epigenetically nonmodulated adult MSCs orfetal fibroblast cells [28] have been found to be a majorcause of not only their declined cytological and molecularquality but also decreased in vitro developmental potentialof cloned embryos However in our current study it seemsthat TSA-dependent epigenomic transformation of nucleardonor MSCs biases correctingly the expression profile ofOct4 andNanog mRNAs in cloned pig blastocysts triggeringboth significant enhancement of relative abundance (RA)of Oct4 transcripts and slight (nonsignificant) increase inNanog transcript RA as compared to the TSA-untreatedMSCgroup On the one hand this influence could be exerted bydirect diminishment in the deacetylation level within thenucleosomal core-derived histone lysine moieties that wasevoked by trichostatin A-mediated nonspecific inhibition ofHDACs On the other hand it could be probably elicitedvia indirect nonselective suppression of cytosine residuemethylation processes within the Oct4 and Nanog gene pro-moters andor enhancers leading to upregulated expressionof these crucial pluripotency-related genes Similar resultshave been shown in the study by Wang et al [73] in whichthe sequential treatment of bovine differentiated nucleardonor fibroblast cells and resultant cloned embryos by bothnonspecific inhibitors of DNMTs and HDACs (ie 5-aza-dC
10 BioMed Research International
and TSA resp) contributed to increase of Oct4 transcriptRA in the blastocysts obtained simultaneously sustainingthe expression of Nanog mRNAs on the unchanged levelas compared to the 5-aza-dC- and TSA-unexposed groupIn this case 5-aza-21015840-deoxycytidine- and trichostatin A-mediated epigenetic modulation of both nuclear donor cellsand cloned embryos gave rise presumably to direct down-regulation in the methylation of cytosine residues withinthe Oct4 gene promoter andor enhancer that resultedfrom 5-aza-dC-dependent nonspecific inhibition of DNMTsFurthermore this two-factor epigenetic modulation couldaffect the enhanced incidence of acetylation level within thenucleosomal core-derived histone lysine moieties that wastriggered by trichostatin A-mediated nonselective inhibitionof HDACs consequently leading to indirect upregulatedexpression of the Oct4 gene Taking into consideration allthe abovementioned findings considerable transcriptionalupregulation in the expression of Oct4 gene (confirmedamong porcine NT blastocysts in our present study andamong bovineNT blastocysts in the study byWang et al [73])can be found to be pivotal indicator of increased pluripotencyextent of cloned embryos This process appears to be alsoassociated with improved cytological quality of NT blasto-cysts that was measured with their total cell counts In thecurrent investigation we have shown that the overall numberof ICM and trophoblast cells in blastocysts was significantlyhigher following the reconstruction of NT pig embryos withepigenomically transformed mesenchymal stem cells
5 Conclusions and Future Goals
Summing up artificial epigenomic modulation of in vitrocultured MSCs using the nonspecific HDAC inhibitor desig-nated as TSA seems to facilitate much more the reprogram-ming process for epigenetically determined transcriptionalactivity of somatic cell-inherited nuclear genome in theNT pig embryos The use of ectopic HDAC inhibitors forepigenetic transformation of mesenchymal stem cells whosecell nuclei were transferred into enucleated oocytes is thecompletely new approach in the studies involving somaticcell cloning of pigs and other mammalian species Enhancedreprogrammability of nuclear genome descended from TSA-exposed MSCs in the blastomeres of porcine preimplantedcloned embryos resulted in the improvements of not onlytheir capacity to complete in vitro development to themorula and blastocyst stages but also cytological and molec-ular quality of the blastocysts produced Therefore furtherinvestigations are also necessary to determine whether thenovel strategy of TSA-dependent epigenetic modification ofnuclear donor MSCs which has been recently utilized in ourlaboratory for the future goals of generating cloned pigletsenables retaining the in vivo developmental competences ofthe high-quality NT-derived blastocysts to reach full term
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Marcin Samiec and Jolanta Opiela contributed equally to thepreparation of this paper
Acknowledgments
The project was funded by the Polish National ScienceCentre resources allocated on the basis of Decision no DEC-201103DNZ905537 The authors would like to thank DrJarosław Wieczorek DVM for bone marrow retrieval
References
[1] A J Bonk H-T Cheong R Li et al ldquoCorrelation of develop-mental differences of nuclear transfer embryos cells to themethylation profiles of nuclear transfer donor cells in swinerdquoEpigenetics vol 2 no 3 pp 179ndash186 2007
[2] A J Bonk R Li L Lai et al ldquoAberrant DNA methylation inporcine in vitro- parthenogenetic- and somatic cell nucleartransfer-produced blastocystsrdquo Molecular Reproduction andDevelopment vol 75 no 2 pp 250ndash264 2008
[3] M Samiec ldquoDevelopment of pig cloning studies past presentand futurerdquo Journal of Animal and Feed Sciences vol 13 no 2pp 211ndash238 2004
[4] K MWhitworth and R S Prather ldquoSomatic cell nuclear trans-fer efficiency how can it be improved through nuclear remod-eling and reprogrammingrdquoMolecular Reproduction and Devel-opment vol 77 no 12 pp 1001ndash1015 2010
[5] K-I Yamanaka S Sugimura T Wakai M Kawahara and ESato ldquoAcetylation level of histone H3 in early embryonic stagesaffects subsequent development of miniature pig somatic cellnuclear transfer embryosrdquo The Journal of Reproduction andDevelopment vol 55 no 6 pp 638ndash644 2009
[6] R P Cervera N Martı-Gutierrez E Escorihuela R Morenoand M Stojkovic ldquoTrichostatin A affects histone acetylationand gene expression in porcine somatic cell nucleus transferembryosrdquoTheriogenology vol 72 no 8 pp 1097ndash1110 2009
[7] J Zhao J Whyte and R S Prather ldquoEffect of epigeneticregulation during swine embryogenesis and on cloning bynuclear transferrdquo Cell and Tissue Research vol 341 no 1 pp13ndash21 2010
[8] WReik ldquoStability and flexibility of epigenetic gene regulation inmammalian developmentrdquo Nature vol 447 no 7143 pp 425ndash432 2007
[9] R S Prather J W Ross S C Isom and J A Green ldquoTranscrip-tional post-transcriptional and epigenetic control of porcineoocyte maturation and embryogenesisrdquo Society of Reproductionand Fertility supplement vol 66 pp 165ndash176 2009
[10] N Rodriguez-Osorio R Urrego J B Cibelli K Eilertsen andE Memili ldquoReprogramming mammalian somatic cellsrdquo Theri-ogenology vol 78 no 9 pp 1869ndash1886 2012
[11] K J Eilertsen R A Power L L Harkins and P Misica ldquoTar-geting cellular memory to reprogram the epigenome restorepotential and improve somatic cell nuclear transferrdquo AnimalReproduction Science vol 98 no 1-2 pp 129ndash146 2007
[12] M Samiec and M Skrzyszowska ldquoMolecular conditions of thecell nucleus remodellingreprogramming process and nucleartransferred embryo development in the intraooplasmic kary-oplast injection technique a reviewrdquo Czech Journal of AnimalScience vol 50 no 5 pp 185ndash195 2005
BioMed Research International 11
[13] P Narbonne K Miyamoto and J B Gurdon ldquoReprogrammingand development in nuclear transfer embryos and in interspe-cific systemsrdquo Current Opinion in Genetics amp Development vol22 no 5 pp 450ndash458 2012
[14] J-I Bang J-G Yoo M-R Park et al ldquoThe effects of artificialactivation timing on the development of SCNT-derivedembryos and newborn pigletsrdquoReproductive Biology vol 13 no2 pp 127ndash132 2013
[15] M Samiec and M Skrzyszowska ldquoHigh developmental capa-bility of porcine cloned embryos following trichostatin A-dependent epigenomic transformation during in vitro matura-tion of oocytes pre-exposed to R-roscovitinerdquo Animal SciencePapers and Reports vol 30 no 4 pp 383ndash393 2012
[16] M Samiec and M Skrzyszowska ldquoRoscovitine is a novel agentthat can be used for the activation of porcine oocytes recon-structed with adult cutaneous or fetal fibroblast cell nucleirdquoTheriogenology vol 78 no 8 pp 1855ndash1867 2012
[17] Z Li J Shi D Liu et al ldquoEffects of donor fibroblast cell typeand transferred cloned embryo number on the efficiency of pigcloningrdquo Cellular Reprogramming vol 15 no 1 pp 35ndash42 2013
[18] M Samiec and M Skrzyszowska ldquoBiological transcomple-mentary activation as a novel and effective strategy appliedto the generation of porcine somatic cell cloned embryosrdquoReproductive Biology vol 14 no 2 pp 128ndash139 2014
[19] J Li O Svarcova K Villemoes et al ldquoHigh in vitro developmentafter somatic cell nuclear transfer and trichostatin A treatmentof reconstructed porcine embryosrdquoTheriogenology vol 70 no5 pp 800ndash808 2008
[20] M Skrzyszowska M Samiec R Słomski D Lipinski and EMały ldquoDevelopment of porcine transgenic nuclear-transferredembryos derived from fibroblast cells transfected by the noveltechnique of nucleofection or standard lipofectionrdquo Theri-ogenology vol 70 no 2 pp 248ndash259 2008
[21] Y-W Pang L An P Wang et al ldquoTreatment of porcine donorcells and reconstructed embryoswith the antioxidantmelatoninenhances cloning efficiencyrdquo Journal of Pineal Research vol 54no 4 pp 389ndash397 2013
[22] M Samiec M Skrzyszowska and J Opiela ldquoCreation of clonedpig embryos using contact-inhibited or serum-starved fibrob-last cells analysed intra vitam for apoptosis occurrencerdquo Annalsof Animal Science vol 13 no 2 pp 275ndash293 2013
[23] H Wei Y Qing W Pan et al ldquoComparison of the efficiency ofBannaminiature inbred pig somatic cell nuclear transfer amongdifferent donor cellsrdquo PLoSONE vol 8 no 2 Article ID e577282013
[24] K Inoue N Ogonuki H Miki et al ldquoInefficient reprogram-ming of the hematopoietic stem cell genome following nucleartransferrdquo Journal of Cell Science vol 119 no 10 pp 1985ndash19912006
[25] K Inoue S Noda N Ogonuki et al ldquoDifferential developmen-tal ability of embryos cloned from tissue-specific stem cellsrdquoStem Cells vol 25 no 5 pp 1279ndash1285 2007
[26] S L Lee E J Kang G H Maeng et al ldquoDevelopmental abilityof miniature pig embryos cloned withmesenchymal stem cellsrdquoThe Journal of Reproduction and Development vol 56 no 2 pp256ndash262 2010
[27] Z Li X He L Chen et al ldquoBone marrow mesenchymal stemcells are an attractive donor cell type for production of clonedpigs as well as genetically modified cloned pigs by somatic cellnuclear transferrdquo Cellular Reprogramming vol 15 no 5 pp459ndash470 2013
[28] B M Kumar H-F Jin J-G Kim et al ldquoDifferential geneexpression patterns in porcine nuclear transfer embryos recon-structed with fetal fibroblasts and mesenchymal stem cellsrdquoDevelopmental Dynamics vol 236 no 2 pp 435ndash446 2007
[29] J Su Y Wang Y Li et al ldquoOxamflatin significantly improvesnuclear reprogramming blastocyst quality and in vitro devel-opment of bovine SCNT embryosrdquo PLoS ONE vol 6 no 8Article ID e23805 2011
[30] Y Song THai YWang et al ldquoEpigenetic reprogramming geneexpression and in vitro development of porcine SCNT embryosare significantly improved by a histone deacetylase inhibitor-m-carboxycinnamic acid bishydroxamide (CBHA)rdquo Protein ampCell vol 5 no 5 pp 382ndash393 2014
[31] S Colleoni G Donofrio I Lagutina R Duchi C Galli andG Lazzari ldquoEstablishment differentiation electroporationviral transduction and nuclear transfer of bovine and porcinemesenchymal stem cellsrdquo Cloning and Stem Cells vol 7 no 3pp 154ndash166 2005
[32] H F Jin B M Kumar J G Kim et al ldquoEnhanced developmentof porcine embryos cloned from bone marrow mesenchymalstem cellsrdquoThe International Journal of Developmental Biologyvol 51 no 1 pp 85ndash90 2007
[33] J Opiela and M Samiec ldquoCharacterization of mesenchymalstem cells and their application in experimental embryologyrdquoPolish Journal of Veterinary Sciences vol 16 no 3 pp 593ndash5992013
[34] X Yang S L Smith X C Tian H A Lewin J-P Renard and TWakayama ldquoNuclear reprogramming of cloned embryos and itsimplications for therapeutic cloningrdquo Nature Genetics vol 39no 3 pp 295ndash302 2007
[35] XWu Y Li G-P Li et al ldquoTrichostatin A improved epigeneticmodifications of transfected cells but did not improve subse-quent cloned embryo developmentrdquo Animal Biotechnology vol19 no 4 pp 211ndash224 2008
[36] F Bo L Di F Qing-Chang et al ldquoEffect of trichostatin A ontransfected donor cells and subsequent development of porcinecloned embryosrdquo Zygote vol 19 no 3 pp 237ndash243 2011
[37] X Ding Y Wang D Zhang Z Guo and Y Zhang ldquoIncreasedpre-implantation development of cloned bovine embryostreated with 5-aza-21015840-deoxycytidine and trichostatin ArdquoTheri-ogenology vol 70 no 4 pp 622ndash630 2008
[38] H S Lee X F Yu J I Bang et al ldquoEnhanced histone acetylationin somatic cells induced by a histone deacetylase inhibitorimproved inter-generic cloned leopard cat blastocystsrdquo Theri-ogenology vol 74 no 8 pp 1439ndash1449 2010
[39] S-F Ning Q-Y Li M-M Liang et al ldquoMethylation charac-teristics and developmental potential of Guangxi Bamaminipig(Sus scrofa domestica) cloned embryos from donor cells treatedwith trichostatin A and 5-aza-21015840-deoxycytidinerdquo Zygote vol 21no 2 pp 178ndash186 2013
[40] B P Enright L-Y Sung C-C Chang X Yang and X C TianldquoMethylation and acetylation characteristics of cloned bovineembryos from donor cells treated with 5-aza-21015840-deoxycytidinerdquoBiology of Reproduction vol 72 no 4 pp 944ndash948 2005
[41] Y J Huan J Zhu B T Xie et al ldquoTreating cloned embryos butnot donor cells with 5-aza-21015840-deoxycytidine enhances thedevelopmental competence of porcine cloned embryosrdquo TheJournal of Reproduction andDevelopment vol 59 no 5 pp 442ndash449 2013
[42] B-G JeonG Coppola S D Perrault G-J RhoDH Betts andWAKing ldquoS-adenosylhomocysteine treatment of adult female
12 BioMed Research International
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
and TSA resp) contributed to increase of Oct4 transcriptRA in the blastocysts obtained simultaneously sustainingthe expression of Nanog mRNAs on the unchanged levelas compared to the 5-aza-dC- and TSA-unexposed groupIn this case 5-aza-21015840-deoxycytidine- and trichostatin A-mediated epigenetic modulation of both nuclear donor cellsand cloned embryos gave rise presumably to direct down-regulation in the methylation of cytosine residues withinthe Oct4 gene promoter andor enhancer that resultedfrom 5-aza-dC-dependent nonspecific inhibition of DNMTsFurthermore this two-factor epigenetic modulation couldaffect the enhanced incidence of acetylation level within thenucleosomal core-derived histone lysine moieties that wastriggered by trichostatin A-mediated nonselective inhibitionof HDACs consequently leading to indirect upregulatedexpression of the Oct4 gene Taking into consideration allthe abovementioned findings considerable transcriptionalupregulation in the expression of Oct4 gene (confirmedamong porcine NT blastocysts in our present study andamong bovineNT blastocysts in the study byWang et al [73])can be found to be pivotal indicator of increased pluripotencyextent of cloned embryos This process appears to be alsoassociated with improved cytological quality of NT blasto-cysts that was measured with their total cell counts In thecurrent investigation we have shown that the overall numberof ICM and trophoblast cells in blastocysts was significantlyhigher following the reconstruction of NT pig embryos withepigenomically transformed mesenchymal stem cells
5 Conclusions and Future Goals
Summing up artificial epigenomic modulation of in vitrocultured MSCs using the nonspecific HDAC inhibitor desig-nated as TSA seems to facilitate much more the reprogram-ming process for epigenetically determined transcriptionalactivity of somatic cell-inherited nuclear genome in theNT pig embryos The use of ectopic HDAC inhibitors forepigenetic transformation of mesenchymal stem cells whosecell nuclei were transferred into enucleated oocytes is thecompletely new approach in the studies involving somaticcell cloning of pigs and other mammalian species Enhancedreprogrammability of nuclear genome descended from TSA-exposed MSCs in the blastomeres of porcine preimplantedcloned embryos resulted in the improvements of not onlytheir capacity to complete in vitro development to themorula and blastocyst stages but also cytological and molec-ular quality of the blastocysts produced Therefore furtherinvestigations are also necessary to determine whether thenovel strategy of TSA-dependent epigenetic modification ofnuclear donor MSCs which has been recently utilized in ourlaboratory for the future goals of generating cloned pigletsenables retaining the in vivo developmental competences ofthe high-quality NT-derived blastocysts to reach full term
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
Marcin Samiec and Jolanta Opiela contributed equally to thepreparation of this paper
Acknowledgments
The project was funded by the Polish National ScienceCentre resources allocated on the basis of Decision no DEC-201103DNZ905537 The authors would like to thank DrJarosław Wieczorek DVM for bone marrow retrieval
References
[1] A J Bonk H-T Cheong R Li et al ldquoCorrelation of develop-mental differences of nuclear transfer embryos cells to themethylation profiles of nuclear transfer donor cells in swinerdquoEpigenetics vol 2 no 3 pp 179ndash186 2007
[2] A J Bonk R Li L Lai et al ldquoAberrant DNA methylation inporcine in vitro- parthenogenetic- and somatic cell nucleartransfer-produced blastocystsrdquo Molecular Reproduction andDevelopment vol 75 no 2 pp 250ndash264 2008
[3] M Samiec ldquoDevelopment of pig cloning studies past presentand futurerdquo Journal of Animal and Feed Sciences vol 13 no 2pp 211ndash238 2004
[4] K MWhitworth and R S Prather ldquoSomatic cell nuclear trans-fer efficiency how can it be improved through nuclear remod-eling and reprogrammingrdquoMolecular Reproduction and Devel-opment vol 77 no 12 pp 1001ndash1015 2010
[5] K-I Yamanaka S Sugimura T Wakai M Kawahara and ESato ldquoAcetylation level of histone H3 in early embryonic stagesaffects subsequent development of miniature pig somatic cellnuclear transfer embryosrdquo The Journal of Reproduction andDevelopment vol 55 no 6 pp 638ndash644 2009
[6] R P Cervera N Martı-Gutierrez E Escorihuela R Morenoand M Stojkovic ldquoTrichostatin A affects histone acetylationand gene expression in porcine somatic cell nucleus transferembryosrdquoTheriogenology vol 72 no 8 pp 1097ndash1110 2009
[7] J Zhao J Whyte and R S Prather ldquoEffect of epigeneticregulation during swine embryogenesis and on cloning bynuclear transferrdquo Cell and Tissue Research vol 341 no 1 pp13ndash21 2010
[8] WReik ldquoStability and flexibility of epigenetic gene regulation inmammalian developmentrdquo Nature vol 447 no 7143 pp 425ndash432 2007
[9] R S Prather J W Ross S C Isom and J A Green ldquoTranscrip-tional post-transcriptional and epigenetic control of porcineoocyte maturation and embryogenesisrdquo Society of Reproductionand Fertility supplement vol 66 pp 165ndash176 2009
[10] N Rodriguez-Osorio R Urrego J B Cibelli K Eilertsen andE Memili ldquoReprogramming mammalian somatic cellsrdquo Theri-ogenology vol 78 no 9 pp 1869ndash1886 2012
[11] K J Eilertsen R A Power L L Harkins and P Misica ldquoTar-geting cellular memory to reprogram the epigenome restorepotential and improve somatic cell nuclear transferrdquo AnimalReproduction Science vol 98 no 1-2 pp 129ndash146 2007
[12] M Samiec and M Skrzyszowska ldquoMolecular conditions of thecell nucleus remodellingreprogramming process and nucleartransferred embryo development in the intraooplasmic kary-oplast injection technique a reviewrdquo Czech Journal of AnimalScience vol 50 no 5 pp 185ndash195 2005
BioMed Research International 11
[13] P Narbonne K Miyamoto and J B Gurdon ldquoReprogrammingand development in nuclear transfer embryos and in interspe-cific systemsrdquo Current Opinion in Genetics amp Development vol22 no 5 pp 450ndash458 2012
[14] J-I Bang J-G Yoo M-R Park et al ldquoThe effects of artificialactivation timing on the development of SCNT-derivedembryos and newborn pigletsrdquoReproductive Biology vol 13 no2 pp 127ndash132 2013
[15] M Samiec and M Skrzyszowska ldquoHigh developmental capa-bility of porcine cloned embryos following trichostatin A-dependent epigenomic transformation during in vitro matura-tion of oocytes pre-exposed to R-roscovitinerdquo Animal SciencePapers and Reports vol 30 no 4 pp 383ndash393 2012
[16] M Samiec and M Skrzyszowska ldquoRoscovitine is a novel agentthat can be used for the activation of porcine oocytes recon-structed with adult cutaneous or fetal fibroblast cell nucleirdquoTheriogenology vol 78 no 8 pp 1855ndash1867 2012
[17] Z Li J Shi D Liu et al ldquoEffects of donor fibroblast cell typeand transferred cloned embryo number on the efficiency of pigcloningrdquo Cellular Reprogramming vol 15 no 1 pp 35ndash42 2013
[18] M Samiec and M Skrzyszowska ldquoBiological transcomple-mentary activation as a novel and effective strategy appliedto the generation of porcine somatic cell cloned embryosrdquoReproductive Biology vol 14 no 2 pp 128ndash139 2014
[19] J Li O Svarcova K Villemoes et al ldquoHigh in vitro developmentafter somatic cell nuclear transfer and trichostatin A treatmentof reconstructed porcine embryosrdquoTheriogenology vol 70 no5 pp 800ndash808 2008
[20] M Skrzyszowska M Samiec R Słomski D Lipinski and EMały ldquoDevelopment of porcine transgenic nuclear-transferredembryos derived from fibroblast cells transfected by the noveltechnique of nucleofection or standard lipofectionrdquo Theri-ogenology vol 70 no 2 pp 248ndash259 2008
[21] Y-W Pang L An P Wang et al ldquoTreatment of porcine donorcells and reconstructed embryoswith the antioxidantmelatoninenhances cloning efficiencyrdquo Journal of Pineal Research vol 54no 4 pp 389ndash397 2013
[22] M Samiec M Skrzyszowska and J Opiela ldquoCreation of clonedpig embryos using contact-inhibited or serum-starved fibrob-last cells analysed intra vitam for apoptosis occurrencerdquo Annalsof Animal Science vol 13 no 2 pp 275ndash293 2013
[23] H Wei Y Qing W Pan et al ldquoComparison of the efficiency ofBannaminiature inbred pig somatic cell nuclear transfer amongdifferent donor cellsrdquo PLoSONE vol 8 no 2 Article ID e577282013
[24] K Inoue N Ogonuki H Miki et al ldquoInefficient reprogram-ming of the hematopoietic stem cell genome following nucleartransferrdquo Journal of Cell Science vol 119 no 10 pp 1985ndash19912006
[25] K Inoue S Noda N Ogonuki et al ldquoDifferential developmen-tal ability of embryos cloned from tissue-specific stem cellsrdquoStem Cells vol 25 no 5 pp 1279ndash1285 2007
[26] S L Lee E J Kang G H Maeng et al ldquoDevelopmental abilityof miniature pig embryos cloned withmesenchymal stem cellsrdquoThe Journal of Reproduction and Development vol 56 no 2 pp256ndash262 2010
[27] Z Li X He L Chen et al ldquoBone marrow mesenchymal stemcells are an attractive donor cell type for production of clonedpigs as well as genetically modified cloned pigs by somatic cellnuclear transferrdquo Cellular Reprogramming vol 15 no 5 pp459ndash470 2013
[28] B M Kumar H-F Jin J-G Kim et al ldquoDifferential geneexpression patterns in porcine nuclear transfer embryos recon-structed with fetal fibroblasts and mesenchymal stem cellsrdquoDevelopmental Dynamics vol 236 no 2 pp 435ndash446 2007
[29] J Su Y Wang Y Li et al ldquoOxamflatin significantly improvesnuclear reprogramming blastocyst quality and in vitro devel-opment of bovine SCNT embryosrdquo PLoS ONE vol 6 no 8Article ID e23805 2011
[30] Y Song THai YWang et al ldquoEpigenetic reprogramming geneexpression and in vitro development of porcine SCNT embryosare significantly improved by a histone deacetylase inhibitor-m-carboxycinnamic acid bishydroxamide (CBHA)rdquo Protein ampCell vol 5 no 5 pp 382ndash393 2014
[31] S Colleoni G Donofrio I Lagutina R Duchi C Galli andG Lazzari ldquoEstablishment differentiation electroporationviral transduction and nuclear transfer of bovine and porcinemesenchymal stem cellsrdquo Cloning and Stem Cells vol 7 no 3pp 154ndash166 2005
[32] H F Jin B M Kumar J G Kim et al ldquoEnhanced developmentof porcine embryos cloned from bone marrow mesenchymalstem cellsrdquoThe International Journal of Developmental Biologyvol 51 no 1 pp 85ndash90 2007
[33] J Opiela and M Samiec ldquoCharacterization of mesenchymalstem cells and their application in experimental embryologyrdquoPolish Journal of Veterinary Sciences vol 16 no 3 pp 593ndash5992013
[34] X Yang S L Smith X C Tian H A Lewin J-P Renard and TWakayama ldquoNuclear reprogramming of cloned embryos and itsimplications for therapeutic cloningrdquo Nature Genetics vol 39no 3 pp 295ndash302 2007
[35] XWu Y Li G-P Li et al ldquoTrichostatin A improved epigeneticmodifications of transfected cells but did not improve subse-quent cloned embryo developmentrdquo Animal Biotechnology vol19 no 4 pp 211ndash224 2008
[36] F Bo L Di F Qing-Chang et al ldquoEffect of trichostatin A ontransfected donor cells and subsequent development of porcinecloned embryosrdquo Zygote vol 19 no 3 pp 237ndash243 2011
[37] X Ding Y Wang D Zhang Z Guo and Y Zhang ldquoIncreasedpre-implantation development of cloned bovine embryostreated with 5-aza-21015840-deoxycytidine and trichostatin ArdquoTheri-ogenology vol 70 no 4 pp 622ndash630 2008
[38] H S Lee X F Yu J I Bang et al ldquoEnhanced histone acetylationin somatic cells induced by a histone deacetylase inhibitorimproved inter-generic cloned leopard cat blastocystsrdquo Theri-ogenology vol 74 no 8 pp 1439ndash1449 2010
[39] S-F Ning Q-Y Li M-M Liang et al ldquoMethylation charac-teristics and developmental potential of Guangxi Bamaminipig(Sus scrofa domestica) cloned embryos from donor cells treatedwith trichostatin A and 5-aza-21015840-deoxycytidinerdquo Zygote vol 21no 2 pp 178ndash186 2013
[40] B P Enright L-Y Sung C-C Chang X Yang and X C TianldquoMethylation and acetylation characteristics of cloned bovineembryos from donor cells treated with 5-aza-21015840-deoxycytidinerdquoBiology of Reproduction vol 72 no 4 pp 944ndash948 2005
[41] Y J Huan J Zhu B T Xie et al ldquoTreating cloned embryos butnot donor cells with 5-aza-21015840-deoxycytidine enhances thedevelopmental competence of porcine cloned embryosrdquo TheJournal of Reproduction andDevelopment vol 59 no 5 pp 442ndash449 2013
[42] B-G JeonG Coppola S D Perrault G-J RhoDH Betts andWAKing ldquoS-adenosylhomocysteine treatment of adult female
12 BioMed Research International
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
[13] P Narbonne K Miyamoto and J B Gurdon ldquoReprogrammingand development in nuclear transfer embryos and in interspe-cific systemsrdquo Current Opinion in Genetics amp Development vol22 no 5 pp 450ndash458 2012
[14] J-I Bang J-G Yoo M-R Park et al ldquoThe effects of artificialactivation timing on the development of SCNT-derivedembryos and newborn pigletsrdquoReproductive Biology vol 13 no2 pp 127ndash132 2013
[15] M Samiec and M Skrzyszowska ldquoHigh developmental capa-bility of porcine cloned embryos following trichostatin A-dependent epigenomic transformation during in vitro matura-tion of oocytes pre-exposed to R-roscovitinerdquo Animal SciencePapers and Reports vol 30 no 4 pp 383ndash393 2012
[16] M Samiec and M Skrzyszowska ldquoRoscovitine is a novel agentthat can be used for the activation of porcine oocytes recon-structed with adult cutaneous or fetal fibroblast cell nucleirdquoTheriogenology vol 78 no 8 pp 1855ndash1867 2012
[17] Z Li J Shi D Liu et al ldquoEffects of donor fibroblast cell typeand transferred cloned embryo number on the efficiency of pigcloningrdquo Cellular Reprogramming vol 15 no 1 pp 35ndash42 2013
[18] M Samiec and M Skrzyszowska ldquoBiological transcomple-mentary activation as a novel and effective strategy appliedto the generation of porcine somatic cell cloned embryosrdquoReproductive Biology vol 14 no 2 pp 128ndash139 2014
[19] J Li O Svarcova K Villemoes et al ldquoHigh in vitro developmentafter somatic cell nuclear transfer and trichostatin A treatmentof reconstructed porcine embryosrdquoTheriogenology vol 70 no5 pp 800ndash808 2008
[20] M Skrzyszowska M Samiec R Słomski D Lipinski and EMały ldquoDevelopment of porcine transgenic nuclear-transferredembryos derived from fibroblast cells transfected by the noveltechnique of nucleofection or standard lipofectionrdquo Theri-ogenology vol 70 no 2 pp 248ndash259 2008
[21] Y-W Pang L An P Wang et al ldquoTreatment of porcine donorcells and reconstructed embryoswith the antioxidantmelatoninenhances cloning efficiencyrdquo Journal of Pineal Research vol 54no 4 pp 389ndash397 2013
[22] M Samiec M Skrzyszowska and J Opiela ldquoCreation of clonedpig embryos using contact-inhibited or serum-starved fibrob-last cells analysed intra vitam for apoptosis occurrencerdquo Annalsof Animal Science vol 13 no 2 pp 275ndash293 2013
[23] H Wei Y Qing W Pan et al ldquoComparison of the efficiency ofBannaminiature inbred pig somatic cell nuclear transfer amongdifferent donor cellsrdquo PLoSONE vol 8 no 2 Article ID e577282013
[24] K Inoue N Ogonuki H Miki et al ldquoInefficient reprogram-ming of the hematopoietic stem cell genome following nucleartransferrdquo Journal of Cell Science vol 119 no 10 pp 1985ndash19912006
[25] K Inoue S Noda N Ogonuki et al ldquoDifferential developmen-tal ability of embryos cloned from tissue-specific stem cellsrdquoStem Cells vol 25 no 5 pp 1279ndash1285 2007
[26] S L Lee E J Kang G H Maeng et al ldquoDevelopmental abilityof miniature pig embryos cloned withmesenchymal stem cellsrdquoThe Journal of Reproduction and Development vol 56 no 2 pp256ndash262 2010
[27] Z Li X He L Chen et al ldquoBone marrow mesenchymal stemcells are an attractive donor cell type for production of clonedpigs as well as genetically modified cloned pigs by somatic cellnuclear transferrdquo Cellular Reprogramming vol 15 no 5 pp459ndash470 2013
[28] B M Kumar H-F Jin J-G Kim et al ldquoDifferential geneexpression patterns in porcine nuclear transfer embryos recon-structed with fetal fibroblasts and mesenchymal stem cellsrdquoDevelopmental Dynamics vol 236 no 2 pp 435ndash446 2007
[29] J Su Y Wang Y Li et al ldquoOxamflatin significantly improvesnuclear reprogramming blastocyst quality and in vitro devel-opment of bovine SCNT embryosrdquo PLoS ONE vol 6 no 8Article ID e23805 2011
[30] Y Song THai YWang et al ldquoEpigenetic reprogramming geneexpression and in vitro development of porcine SCNT embryosare significantly improved by a histone deacetylase inhibitor-m-carboxycinnamic acid bishydroxamide (CBHA)rdquo Protein ampCell vol 5 no 5 pp 382ndash393 2014
[31] S Colleoni G Donofrio I Lagutina R Duchi C Galli andG Lazzari ldquoEstablishment differentiation electroporationviral transduction and nuclear transfer of bovine and porcinemesenchymal stem cellsrdquo Cloning and Stem Cells vol 7 no 3pp 154ndash166 2005
[32] H F Jin B M Kumar J G Kim et al ldquoEnhanced developmentof porcine embryos cloned from bone marrow mesenchymalstem cellsrdquoThe International Journal of Developmental Biologyvol 51 no 1 pp 85ndash90 2007
[33] J Opiela and M Samiec ldquoCharacterization of mesenchymalstem cells and their application in experimental embryologyrdquoPolish Journal of Veterinary Sciences vol 16 no 3 pp 593ndash5992013
[34] X Yang S L Smith X C Tian H A Lewin J-P Renard and TWakayama ldquoNuclear reprogramming of cloned embryos and itsimplications for therapeutic cloningrdquo Nature Genetics vol 39no 3 pp 295ndash302 2007
[35] XWu Y Li G-P Li et al ldquoTrichostatin A improved epigeneticmodifications of transfected cells but did not improve subse-quent cloned embryo developmentrdquo Animal Biotechnology vol19 no 4 pp 211ndash224 2008
[36] F Bo L Di F Qing-Chang et al ldquoEffect of trichostatin A ontransfected donor cells and subsequent development of porcinecloned embryosrdquo Zygote vol 19 no 3 pp 237ndash243 2011
[37] X Ding Y Wang D Zhang Z Guo and Y Zhang ldquoIncreasedpre-implantation development of cloned bovine embryostreated with 5-aza-21015840-deoxycytidine and trichostatin ArdquoTheri-ogenology vol 70 no 4 pp 622ndash630 2008
[38] H S Lee X F Yu J I Bang et al ldquoEnhanced histone acetylationin somatic cells induced by a histone deacetylase inhibitorimproved inter-generic cloned leopard cat blastocystsrdquo Theri-ogenology vol 74 no 8 pp 1439ndash1449 2010
[39] S-F Ning Q-Y Li M-M Liang et al ldquoMethylation charac-teristics and developmental potential of Guangxi Bamaminipig(Sus scrofa domestica) cloned embryos from donor cells treatedwith trichostatin A and 5-aza-21015840-deoxycytidinerdquo Zygote vol 21no 2 pp 178ndash186 2013
[40] B P Enright L-Y Sung C-C Chang X Yang and X C TianldquoMethylation and acetylation characteristics of cloned bovineembryos from donor cells treated with 5-aza-21015840-deoxycytidinerdquoBiology of Reproduction vol 72 no 4 pp 944ndash948 2005
[41] Y J Huan J Zhu B T Xie et al ldquoTreating cloned embryos butnot donor cells with 5-aza-21015840-deoxycytidine enhances thedevelopmental competence of porcine cloned embryosrdquo TheJournal of Reproduction andDevelopment vol 59 no 5 pp 442ndash449 2013
[42] B-G JeonG Coppola S D Perrault G-J RhoDH Betts andWAKing ldquoS-adenosylhomocysteine treatment of adult female
12 BioMed Research International
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
fibroblasts alters X-chromosome inactivation and improves invitro embryo development after somatic cell nuclear transferrdquoReproduction vol 135 no 6 pp 815ndash828 2008
[43] G Wee J-J Shim D-B Koo J-I Chae K-K Lee and Y-MHan ldquoEpigenetic alteration of the donor cells does not reca-pitulate the reprogramming of DNA methylation in clonedembryosrdquo Reproduction vol 134 no 6 pp 781ndash787 2007
[44] Y J Kim K S Ahn M Kim and H Shim ldquoComparison ofpotency between histone deacetylase inhibitors trichostatin Aand valproic acid on enhancing in vitro development of porcinesomatic cell nuclear transfer embryosrdquo In Vitro Cellular ampDevelopmental Biology Animal vol 47 no 4 pp 283ndash289 2011
[45] J R Sangalli M R Chiaratti T H De Bem et al ldquoDevelopmentto term of cloned cattle derived from donor cells treated withvalproic acidrdquo PLoS ONE vol 9 no 6 Article ID e101022 2014
[46] N vanThuan H-T Bui J-H Kim et al ldquoThe histone deacety-lase inhibitor scriptaid enhances nascent mRNA productionand rescues full-term development in cloned inbred micerdquoReproduction vol 138 no 2 pp 309ndash317 2009
[47] J Zhao J W Ross Y Hao et al ldquoSignificant improvementin cloning efficiency of an inbred miniature pig by histonedeacetylase inhibitor treatment after somatic cell nuclear trans-ferrdquo Biology of Reproduction vol 81 no 3 pp 525ndash530 2009
[48] W Xu Z Li B Yu et al ldquoEffects of DNMT1 and HDACinhibitors on gene-specific methylation reprogramming duringporcine somatic cell nuclear transferrdquo PLoS ONE vol 8 no 5Article ID e64705 2013
[49] B-Q Wen J Li J-J Li et al ldquoThe histone deacetylase inhibitorScriptaid improves in vitro developmental competence of ovinesomatic cell nuclear transferred embryosrdquo Theriogenology vol81 no 2 pp 332ndash339 2014
[50] L Liu Y Liu F Gao et al ldquoEmbryonic development andgene expression of porcine SCNT embryos treated with sodiumbutyraterdquo Journal of Experimental Zoology Part BMolecular andDevelopmental Evolution vol 318 no 3 pp 224ndash234 2012
[51] BM Kumar G-HMaeng Y-M Lee et al ldquoEpigeneticmodifi-cation of fetal fibroblasts improves developmental competencyand gene expression in porcine cloned embryosrdquo VeterinaryResearch Communications vol 37 no 1 pp 19ndash28 2013
[52] X Dai J Hao X J Hou et al ldquoSomatic nucleus reprogram-ming is significantly improved by m-carboxycinnamic acidbishydroxamide a histone deacetylase inhibitorrdquoThe Journal ofBiological Chemistry vol 285 no 40 pp 31002ndash31010 2010
[53] S-J Park H-J Park O-J Koo et al ldquoOxamflatin improvesdevelopmental competence of porcine somatic cell nucleartransfer embryosrdquo Cellular Reprogramming vol 14 no 5 pp398ndash406 2012
[54] L Shi and J Wu ldquoEpigenetic regulation in mammalian preim-plantation embryo developmentrdquo Reproductive Biology andEndocrinology vol 7 article 59 11 pages 2009
[55] Y-F Diao K-J Naruse R-X Han et al ldquoTreatment of fetalfibroblasts with DNA methylation inhibitors andor histonedeacetylase inhibitors improves the development of porcinenuclear transfer-derived embryosrdquo Animal Reproduction Sci-ence vol 141 no 3-4 pp 164ndash171 2013
[56] J OpielaM SamiecM Bochenek D Lipinski J Romanek andP Wilczek ldquoDNA aneuploidy in porcine bone marrow-derivedmesenchymal stem cells undergoing osteogenic and adipogenicin vitro differentiationrdquo Cellular Reprogramming vol 15 no 5pp 425ndash434 2013
[57] J Opiela Z Bartel J Romanek J Wieczorek and P WilczekldquoThe quality of porcinemesenchymal stem cells and their osteo-
and adipogenic cell derivativesmdashthe level of proapoptotic badprotein expressionrdquo Annals of Animal Science vol 13 no 4 pp753ndash763 2013
[58] J OpielaM Samiec D LipinskiM Bochenek and J RomanekldquoThe effect of trichostatinA-dependent epigenomicmodulationon the phenotype multipotency extent and suitability of adultbone marrow-derived mesenchymal stem cells for efficientsomatic cell cloning in pigsrdquo Stem Cells International In press
[59] M Samiec M Skrzyszowska and D Lipinski ldquoPseudophysio-logical transcomplementary activation of reconstructed oocytesas a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetalfibroblast cellsrdquo Polish Journal of Veterinary Sciences vol 15 no3 pp 509ndash516 2012
[60] M Samiec M Skrzyszowska andM Bochenek ldquoIn vitro devel-opment of porcine nuclear-transferred embryos derived fromfibroblast cells analysed cytometrically for apoptosis incidenceand accuracy of cell cycle synchronization at the G0G1 stagesrdquoAnnals of Animal Science vol 13 no 4 pp 735ndash752 2013
[61] J Opiela E Latasiewicz and Z Smorag ldquoOptimal concentra-tion of hyaluronan and plant protein in different culture systemsfor in vitro maturation of bovine oocytesrdquo Indian Journal ofExperimental Biology vol 50 no 12 pp 839ndash846 2012
[62] J Opiela J Romanek D Lipinski and Z Smorąg ldquoEffectof hyaluronan on developmental competence and quality ofoocytes and obtained blastocysts from in vitro maturationof bovine oocytesrdquo BioMed Research International vol 2014Article ID 519189 8 pages 2014
[63] L Armstrong M LakoW Dean andM Stojkovic ldquoEpigeneticmodification is central to genome reprogramming in somaticcell nuclear transferrdquo Stem Cells vol 24 no 4 pp 805ndash8142006
[64] G N Corry B Tanasijevic E R Barry W Krueger and T PRasmussen ldquoEpigenetic regulatory mechanisms during preim-plantation developmentrdquo Birth Defects Research Part C EmbryoToday Reviews vol 87 no 4 pp 297ndash313 2009
[65] M Samiec ldquoThe effect of mitochondrial genome on archi-tectural remodeling and epigenetic reprogramming of donorcell nuclei in mammalian nuclear transfer-derived embryosrdquoJournal of Animal and Feed Sciences vol 14 no 3 pp 393ndash4222005
[66] M Samiec and M Skrzyszowska ldquoTransgenic mammalianspecies generated by somatic cell cloning in biomedicinebiopharmaceutical industry and human nutritiondieteticsmdashrecent achievementsrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 317ndash328 2011
[67] M Samiec and M Skrzyszowska ldquoThe possibilities of practicalapplication of transgenic mammalian species generated bysomatic cell cloning in pharmacology veterinary medicine andxenotransplantologyrdquo Polish Journal of Veterinary Sciences vol14 no 2 pp 329ndash340 2011
[68] N Kirchhof J W Carnwath E Lemme K Anastassiadis HScholer and H Niemann ldquoExpression pattern of Oct-4 in pre-implantation embryos of different speciesrdquo Biology of Reproduc-tion vol 63 no 6 pp 1698ndash1705 2000
[69] SMasui Y Nakatake Y Toyooka et al ldquoPluripotency governedby Sox2 via regulation ofOct34 expression inmouse embryonicstem cellsrdquo Nature Cell Biology vol 9 no 6 pp 625ndash635 2007
[70] W A Kues M Nowak-Imialek S Haridoss and H NiemannldquoStrategies for the derivation of pluripotent cells from farm ani-malsrdquo Reproduction in Domestic Animals vol 45 supplement 3pp 25ndash31 2010
BioMed Research International 13
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
[71] KMitsui Y TokuzawaH Itoh et al ldquoThehomeoproteinNanogis required for maintenance of pluripotency in mouse epiblastand ES cellsrdquo Cell vol 113 no 5 pp 631ndash642 2003
[72] S-Y Hatano M Tada H Kimura et al ldquoPluripotential com-petence of cells associated with Nanog activityrdquo Mechanisms ofDevelopment vol 122 no 1 pp 67ndash79 2005
[73] Y Wang J Su L Wang et al ldquoThe effects of 5-aza-21015840-deoxy-cytidine and trichostatin A on gene expression and DNAmeth-ylation status in cloned bovine blastocystsrdquoCellular Reprogram-ming vol 13 no 4 pp 297ndash306 2011
![)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/bmri/2014/101286.pdf · the mitochondrial FAD transporter [ ]. is deleted strain showed a small-colony phenotype, on](https://static.documents.pub/doc/80x56/5e946a1c5bc80640124d9250/joebxj1vcmjtijohpsqpsbujpo-the-mitochondrial-fad-transporter-is-deleted-strain.jpg)
![)JOEBXJ1VCMJTIJOH$PSQPSBUJPO 7FUFSJOBSZ.FEJDJOF ...downloads.hindawi.com/journals/vmi/2014/892421.pdf · neonatal deaths, and loss of animals [ , , ]. In contrast to M. agalactiae](https://static.documents.pub/doc/80x56/5ea3bf5453c3a36c7d50daa4/joebxj1vcmjtijohpsqpsbujpo-downloadshindawicomjournalsvmi2014892421pdf.jpg)