Research ArticleMesenchymal StemStromal Cells Derived from InducedPluripotent Stem Cells Support CD34pos Hematopoietic StemCell Propagation and Suppress Inflammatory Reaction
1Department of Gastroenterology Hepatology and Endocrinology RG Translational Hepatology and Stem Cell Biology (OE 6817)Cluster-of-Excellence REBIRTH Hannover Medical School Carl-Neuberg-Street 30625 Hannover Germany2Department of Transfusion Medicine Cell Therapy and Hemostaseology Ludwig-Maximilian University HospitalMax-Lebsche-Platz 32 A 81377 Munich Germany3DRK Institute of Transfusion Medicine and Immune Hematology Frankfurt Germany4Cell and Developmental Biology Max Planck Institute for Molecular Biomedicine Munster Germany
Correspondence should be addressed to Reinhard Henschler reinhardhenschlermeduni-muenchendeand Tobias Cantz cantztobiasmh-hannoverde
Received 2 March 2015 Revised 14 May 2015 Accepted 25 May 2015
Academic Editor Igor Slukvin
Copyright copy 2015 Mohsen Moslem et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Mesenchymal stemstromal cells (MSCs) represent a promising cell source for research and therapeutic applications but theirrestricted ex vivo propagation capabilities limit putative applications Substantial self-renewing of stem cells can be achievedby reprogramming cells into induced pluripotent stem cells (iPSCs) that can be easily expanded as undifferentiated cells evenin mass culture Here we investigated a differentiation protocol enabling the generation and selection of human iPSC-derivedMSCs exhibiting relevant surface marker expression profiles (CD105 and CD73) and functional characteristics We generated suchiPSC-MSCs from fibroblasts and bone marrow MSCs utilizing two different reprogramming constructs All such iPSC-MSCsexhibited the characteristics of normal bone marrow-derived (BM) MSCs In direct comparison to BM-MSCs our iPSC-MSCsexhibited a similar surface marker expression profile but shorter doubling times without reaching senescence within 20 passagesConsidering functional capabilities iPSC-MSCs provided supportive feeder layer for CD34+ hematopoietic stem cellsrsquo self-renewaland colony forming capacities Furthermore iPSC-MSCs gained immunomodulatory function to suppress CD4+ cell proliferationreduce proinflammatory cytokines in mixed lymphocyte reaction and increase regulatory CD4+CD69+CD25+ T-lymphocytepopulation In conclusion we generated fully functional MSCs from various iPSC lines irrespective of their starting cell source orreprogramming factor composition andwe suggest that such iPSC-MSCs allow repetitive cell applications for advanced therapeuticapproaches
1 Introduction
Regarding clinical stem cell applications mesenchymal stemstromal cells (MSCs) have been introduced as a favorable celltype which can be maintained ex vivo and have the potentialto regenerate mesodermal tissues such as cartilage tendonbone and muscle in variety of skeletal diseases (for reviewsee [1]) FurthermoreMSCs can support hematopoiesis [2 3]and are able to modulate inflammatory reactions by dynamicinterplay with the innate and adaptive immune systems [4ndash6] However the limited proliferation capability of MSCs
during long-term culture leading to cellular senescence after8ndash10 passages challenges the generation of large-scale cellyields which would be essential for repetitive therapeuticapplications In principal such needs would be met bypluripotent stem cells exhibiting an unlimited proliferationcapacity and that can be generated from patientsrsquo samplesvia reprogramming of somatic cells into induced pluripotentstem cells (iPSCs) [7ndash10] Such human iPSCs are responsiveto differentiation stimuli during in vitro cultivation andin the recent past the generation of iPSC-derived MSCs(iPSC-MSCs) was described and it was demonstrated that
iPSC-MSCs displayed comparable antigen profile and dif-ferentiation capability to bone marrow MSCs (BM-MSCs)and exhibited considerable functional properties [11ndash16]Moreover there is convincing evidence that iPSC-MSCs withhigher expansion capacities can be transplanted in manydegenerative diseases resulting in similar outcomes as BM-MSCs [13 15 17] Increasing evidence however indicatesthat MSCs from different origins are heterogeneous popu-lations exhibiting variable gene expression patterns [18 19]presenting different surfacemarkers [20] or showing reducedproliferation potential and differentiation capacities [21ndash23]
Furthermore a successful approach of iPSC-based ther-apeutic cell applications in regenerative medicine dependson the ability to set up an efficient differentiation protocolresulting in a desired cell population with a high purityMost importantly harmful contaminations of undifferenti-ated pluripotent stem cells must be avoided to exclude therisk of teratoma formation Therefore the robust genera-tion of a homogenous iPSC-MSC population with cellularcharacteristics identical to bona fide MSCs and similar oreven enhanced functional capabilities such as proliferationhematopoietic support and anti-inflammatory responsesneed further attention Here we exploited the differentiationpotential of three iPSC lines generated from fibroblast orprimary MSCs with Yamanaka reprogramming factors [10]namely Oct4 Sox2 Klf4 and c-Myc (OSKM) or Thomsonfactors [7] namely Oct4 Sox2 Nanog and Lin28 (OSNL)Upon MSC differentiation we applied lentiviral selectionconstructs carrying CD105- and CD73-promoter drivenfluorescent reporter and NeomycinPuromycin-resistance-transgenes to enrich the bulk differentiation for fully differ-entiated MSCs Next we explored the antigen profile dif-ferentiation potential proliferation capacity hematopoieticsupport and immune-suppression potential in regulation oflymphocyte proliferation proinflammatory cytokine secre-tions and activation markers of such iPSC-MSCs in directcomparison to bone marrow MSCs (BM-MSCs) from threedifferent donors (LM02 LM05 and LM06)
2 Material and Methods
21 Human iPS Cell Culture Human fetal liver fibroblast(FLF) iPS cells were provided from in-house supplies usingtransduction via lentiviral reprogramming factors Oct4Sox2 Klf4 and c-Myc (OSKM) [24] and Oct4 Sox2 Nanogand Lin28 (OSNL) [25] Human iPSCs were cultured onirradiated mouse embryonic fibroblasts (MEF) in a humid-ified incubator at 37∘C and 5 CO
2in medium contain-
ing DMEMF-12 20 knockout serum replacement (LifeTechnologies) 20 ngmL human recombinant basic fibrob-last growth factor (bFGF provided from Leibniz UniversityHannover) 01mM 120573-mercaptoethanol (Life Technologies)1mM L-glutamine 1 nonessential amino acids and 1penicillinstreptomycin (all from Sigma-Aldrich)
Media were changed daily and cells were split weekly bydissociation with 200UmL of collagenase IV (Life Tech-nologies) and cells were plated on Matrigel-coated platesin medium supplemented with 40 ngmL bFGF for furtherdifferentiation
22 Derivation and Enrichment of Human MSC-Like CellsFor triggering iPSC differentiation toward MSC-like cellshuman iPSC colonies grown on Matrigel (Corning) weremaintained with MSC induction media consisting of DMEM(low-glucose Sigma-Aldrich) 10 defined fetal bovineserum (FBS STEMCELL Technologies) 1 nonessen-tial amino acids 1 penicillin-streptomycin and 2 ngmLhuman recombinant bFGF for 7 days Next cells were treatedwith collagenase IV for 3min at 37∘C dissociated by glassbeads and gentle pipetting and then passed through 40 mmcell strainers (Fisher Scientific) Single cells were seeded ontogelatin-coated plates at 1 times 104 cellscm2 in MSC media
To facilitate enrichment and screening of MSCs duringthe standard differentiation protocol of iPSCs into MSCsa combination of two positive markers namely CD73 andCD105 (which are consistently expressed in MSCs) was cho-sen to produce MSC-specific selection vectorsThe promoterregions for CD73 and CD105 were amplified and ligated intothe corresponding lentiviral backbone the CD105 promoterinto pRRL-Puro-IRES-GFP and the CD73 promoter intopRRL-neo-IRES-dTom (Lentiviral backbones provided in-house based on lentiviral constructs from Axel Scham-bach laboratory) Cells were selected by 500 120583gmL G-418and 4 120583gmL Puromycin-dihydrochloride (both from Sigma-Aldrich) in culture media for 2 weeks until the untransfectedcells were killed Functionality of the vectors confirmedfluorochrome expression in transduced cells by fluorescentmicroscope afterwards
Double-positive MSCs population transduced withpRRL-CD105-Puro-IRES-GFP and pRRLCD73-neo-IRES-dTom was purified with the FACSAria II cell sorter (BDBioscience) A total of 2 times 106 sorted cells were immediatelyplated back into gelatin-coated plates to facilitate adherenceAfter 24 hours fresh prewarmed MSCs medium was addedand cells were allowed to expand and reach nearly 100confluence Cells were counted in different time pointsBone marrow MSCs were isolated in Frankfurt universityhospital as previously described [26] Shortly 10ndash30mL ofbone marrow was aspirated from femoral cavity of patientswho needed hip joint replacement surgery after informedconsent in accordance with the Declaration of Helsinki Afterdensity gradient separation the light density mononuclearcell fraction was seeded on T25 (TPP) tissue culture flasks inpreviously mentioned MSC media
23 Lentiviral Vectors Production HEK 293T cells were usedfor virus production 3 times 106 cells were seeded one daybefore transfection in 10 cm dishes (TPP) in DMEM (highglucose Life Technologies) supplemented with 10 FBS and1penicillinstreptomycin and 1L-glutamineThenext daymedium was exchanged with 8mL DMEM supplementedwith 25 120583M Chloroquine (Sigma-Aldrich) Plasmids encod-ing for lentiviral gagpol (pCDNA3GPCCCC 10 120583g) RSV-Rev (pRSV-Rev 5120583g) VSV-G (pMD2G 2120583g) and packag-ing plasmid encoding for respective transgene into pRRL-Puro-IRES-GFP and pRRL-neo-IRES-dTom were mixed in400 120583L of ddH
2O and 100 120583L of 125M CaCl
2 The plasmids-
CaCl2mixture was added dropwise to 2xHBS and observed
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until precipitates became visible in phase-contrast micro-scope and then added toHEKcells 6 hours latermediumwasexchanged with 10mL DMEM high glucose supplementedwith 10 FBS and 1 penicillinstreptomycin and 1 L-glutamine 48 hours later supernatant was collected passedthrough 045 120583m filters and centrifuged at 14000timesg for8 h Virus pellet was resuspended in 200120583L PBS (Sigma-Aldrich) Viral titers were determined by transduction ofHEK 293T cells in serial dilutions and analysis of reportergene expression by flow cytometry Generally titers were inthe range of 1-2 times 107 viral particles per mL
24 Antigen Profiling by Flow Cytometry To assess the im-munophenotypic profile of BM-MSCs and iPSC-derivedMSCs single cell suspensions were prepared by trypsindigestion (Life Technologies) and washed with cold PBScontaining 1 bovine serum albumin BSA (MerckMillipore)Next 2 times 105 cells were incubated for 30 minutes with therespective APC-conjugated monoclonal antibodies CD73CD90 CD105 CD45 CD34 and CD19 (all from BD Bio-science listed in Table 1) and subsequently resuspended ina density of 2 times 105 cells per 200120583L in cold PBS contain-ing 1 BSA Nonspecific fluorescence was determined byincubation of cell aliquots with isotype-matched monoclonalantibodies
Samples were run on a FACS Calibur (BD Bioscience)cytometer using FACS Diva software For each analysis aminimum of 10000 cells was assayed Data was furtherprocessed using FlowJo Software (Tree Star)
25 Growth Kinetics Human BMSCs from 3 different donors(LM02 LM05 and LM06) and 3 iPSC-MSCs lineswere plated(2 times 104 cellswell) onto 12-well plates in triplicate Cellswere harvested after 72 hours in each passage (10 passagesfor BM-MSCs and 15 passages for iPSC-MSCs) Cumulativepopulation doublings were calculated using the formula 119909 =[log 10(NH) minus log 10(1198731)] log 10(2) [27] where 1198731 is theinoculum cell number and NH the cell harvest number Toyield the cumulated doubling level the population doublingfor each passage was calculated and then added to thepopulation doubling levels of the previous passages Thecultures were abandoned as soon as they showed a senescentphenotype when they ceased proliferation
26 In Vitro Adipogenic Chondrogenic and Osteogenic Dif-ferentiation Differentiation induction of iPSC-MSCs wascarried out for 21 days in different differentiation mediaTotally 104 cells were seeded per well in six-well plates(TPP) To induce osteogenic differentiation cells were cul-tured with MSC medium containing 1 120583M dexamethasone05 120583M ascorbic acid and 10mM b-glycerol phosphate (allfrom Sigma-Aldrich) For adipogenic induction cells werecultured in MSC medium supplemented with 50 120583gmLindomethacin (Sigma-Aldrich) 50120583gmL ascorbic acid and100 nM dexamethasone For chondrogenic differentiationiPSC-MSCs were centrifuged in 02mL of medium at500 g for 10min in 15 mL Falcon tubes to form a pelletThe pellets were cultured in MSC medium supplemented
Table 1 List of antibodies and ELISA kits used in this study
with 001120583M dexamethasone 397 120583gmL ascorbic acid-2-phosphate (Sigma-Aldrich) 1mM sodium pyruvate (Sigma-Aldrich) 10 ngmL transforming growth factor-1205731 (TGF-1205731Life Technologies) and 1 insulin-transferrin-selenium (LifeTechnologies) Osteogenesis was assessed by alizarin redstaining adipogenesis by oil red staining and chondrogen-esis by alcian blue staining (all from Sigma-Aldrich)
27 Real Time qRT PCR Total cellular RNA was isolatedusing TRIzol reagent (Life Technologies) Resultant RNAwas subjected to DNase treatment and cDNA Synthesis Kit(Life Technologies) with random hexamers Power SYBRGreen Master Mix based qRT PCR assays were performedon the StepOne Plus Cycler (Applied Biosystems) using thestandard settings We collected samples from at least threeindependent experiments Expression values of human per-oxisome proliferator-activated receptor 120574 (PPAR120574) PPAR120572lipoprotein lipase (LPL) collagen type II (COL2) aggre-can (ACAN) osteocalcin (OCN) and alkaline phosphatase(ALP) normalized to expression of GAPDH The primersequences are listed in Table 2
28 Coculture of CD34+ Progenitor Cells with iPSC-MSCsHuman iPSC-MSCs or BM-MSCs layers were grown until80 confluency in six-well plates and then treated withmitomycine-C (Sigma-Aldrich) to prevent cell overgrowthAfter 24 h medium was removed and purified CD34+cells (resuspended at 75 times 104 cells per well) were thenadded in 2mL of long-term culture medium containing 120572-minimal essentialmedium (Life Technologies) with 20FBS1 120583molL hydrocortisone (Sigma-Aldrich) and 01mM 120573-mercaptoethanol The cocultures were incubated for 20 daysmedium exchange twice per week Nonadherent viable cellswere counted at the indicated time points (day 10 and day20) CD-markers for hematopoietic differentiation were alsodetermined after 8 days for CD34 CD45 CD-11b and CD-14Experiments were repeated three times
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Table 2 List of primers used in this study qRT-PCR
29 In Vitro Progenitor Assays Effects of human iPSC-MSCsor BM-MSCs on progenitor cells were analyzed using acolony forming cell assay Human bone marrow CD34+ cells(2 times 106) were obtained from Lonza and were plated in 2mLof methylcellulose media (STEMCELL Technologies) with orwithout iPSC-MSCs and BMSCs Colonies of gt50 cells werescored after 4 and 8 days of incubation
210 Assessment of CD4+ T-Lymphocyte ProliferationResponseto iPSC-MSCs Standard 5-day MLR cultures were set upwith 5 times 104 Mitomycin Cndashtreated (Sigma-Aldrich) humanperipheral blood mononuclear cells (PBMCs) as stimula-tors and 2 times 105 human CD4+ T-cells (Lonza) in 96-wellround-bottom plates in 200120583L complete medium consistingRPMI 1640 (Life Technologies) supplemented with 01mM120573-mercaptoethanol 10 FBS GLUTAMAX I (Life Tech-nologies) 100UmL penicillin and 100 120583gmL streptomycinin the presence or absence of iPSC-MSCs and BM-MSCsFor analyzing expression of CD69+ and CD25+ regulatoryT-cell population 106 responder cells were mixed with 25times 105 stimulator PMNCs in presence or absence of 2 times105 iPSC-MSCs or BM-MSCs MLRs were performed ona layer of confluent Mitomycine C treated MSCs seededone day before Proliferation was determined with BrdUELISA assay (Roche) based on manufacturer instruction IL-2 and IFN-120574 concentration was determined in MSCMLRcoculture supernatants using a commercially available ELISA(BD Bioscience) according to manufacturerrsquos instructionsCD25 and CD69 (BD Bioscience) expression on CD4+ cellswere analyzed by flow cytometry
3 Results and Discussion
31 Generation of MSCs from Human iPSCs with Spindle-Shape Morphology The challenge of accessing an appro-priate and homogenous source for MSCs with sustainedgrowth kinetics immunosuppressive potentials and produc-tion of chemokines or growth factors supporting endogenousregeneration led to the question whether homogenouslydifferentiated MSCs could be derived from human inducedpluripotent stem cells (iPSCs) To address this question weused two different sources of somatic cells liver fibroblastand bone marrow-derived MSCs (BM-MSCs) and repro-grammed these cells into iPSCs Besides two sources of
somatic starting cells we also compared two slightly dif-ferent composition of reprograming factor cocktails Onefactor combination was comprised of Oct4 Sox2 Klf4 andc-Myc (OSKM) as it was originally described by ShinyaYamanaka and subsequently in multitudinous publications[8ndash10] and the other combination consisted of Oct4 Sox2Nanog and Lin28 (OSNL) as it was described by JamesThomson and some further groups [7 25] Taken together wegenerated fetal liver fibroblast-derived iPSCswithOSKMandOSNL (FLF iPSCs) and bone marrow MSC-derived iPSCswith OSKM (MSC-iPSCs) which were strongly expressingOCT4 SOX2 and SSEA-4 (Figure 1) All iPSC lines weredifferentiated based on a previously reported differentiationprotocol resulting in about 70 CD73+CD105+ cells [14]in which we have made some modifications to allow forantibiotic selection and fluorescent reporter-based purifi-cation (Figure 2(a)) As expected Epithelial-MesenchymalTransition occurred during differentiation giving rise toa heterogeneous population (Figure 2(b)) However withenrichment of early mesenchymal-like cells we observedintermediate and highly CD73-dTomCD105-GFP express-ing cells populations (Figure 2(c)) We sorted highly express-ing GFPdTom positive cells and obtained a much morehomogenous population (Figure 2(d)) Stimulated by the firstdescription of iPSC-derived MSCs by Lian et al in 2010[15] many other groups tried to do direct and spontaneouslydifferentiating iPSCs into MSCs by various means We con-sider the use of lentiviral reporter and selection constructsas important tools to monitor the purity of a cell populationduring differentiation processes and to ensure a high grade ofhomogeneity within the final cell population Such selectionconstructs were recently introduced in other lineagesrsquo differ-entiation protocols [28] Thus in the present study a similarvector architecture was applied to select for CD73+CD105+positive iPSC-MSCs Interestingly we obtained a high num-ber of CD73posCD105intermediate iPSC-MSCs (R1 633)and a smaller fraction of CD73posCD105high iPSC-MSCs(R2 643) and we concluded that sorting the less abun-dant CD73posCD105high population might provide the mosthomogenous cell population
32 Immunophenotype Proliferation and Differentiation Po-tential of iPSC-MSCs In order to characterize the iPSC-MSCs according to the International Society of Cell Therapy
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Figure 1 Generation and characterization iPS cells from human fetal liver fibroblasts (FLF) with Oct4 Sox2 Klf4 and c-Myc (OSKM) andOct4 Sox2 Nanog and Lin-28 (OSNL) and also from human bonemarrowMSCs with OSKMusing lentiviral vectors iPSCs stained positivefor humanOCT4 and SOX2DAPIwas used to stain the nuclei andmergedwith phase-contrast Expression of SSEA-4 is shown in histograms
(ISCT) criteria [29] cell surface marker expression was ana-lyzed by flow cytometry of all three iPSC-MSC lines and BM-MSCs at early passages (passages 3ndash6) All 3 differentiated andenriched iPSC-MSCs displayed a MSC-like antigen profilethat exhibited high CD105 CD73 and CD90 and absenceof CD34 CD45 and CD19 expression (Figure 3(a)) Thuswe were able to demonstrate that homogenous populationscan be isolated and purified from all three iPSC linesindependent to their somatic cell source (fibroblasts or bonemarrow MSCs) and method of reprogramming (OSKM or
OSNL factor cocktail) Strikingly the surface marker CD105and CD73 whose promoter motifs were utilized to expressthe fluorescent reporter transgenes and antibiotic selectioncassettes were readily detectable in almost 100 of purifiedcells indicating the high purity of our enriched iPSC-MSCsInterestingly CD90 was positive not only in all iPSC-MSCslines as well as the BM-MSCs but also in undifferenti-ated iPSCs (MSC-iPSCs) Furthermore the hematopoieticsurface markers were neither expressed in MSCs nor iniPSCs
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iPSCs in MSCsDifferentiation
(Epithelial Transduction MSC-likemedia morphology) and sorting cells
418 (500120583gmL) and puromycin (4120583gmL) and then sortedIRES-dTom and selected with G
lowastAfter plating cells were transduced with CD-105-Puro-IRES-GFP and CD-73-Neo-
(c) (d)
Figure 2 Derivation and enrichment of MSCs from human iPS cells (a) Schematic stepwise protocol for differentiation and selection ofMSC-like cells from human iPS cells (b) phase-contrast photos demonstrating Epithelial-Mesenchymal transition in cellular morphology(c) FACS dot blot showing intermediate (R1) and highly (P2) double-positive cells Highly positive CD-73 and CD-105 (R2) were sorted forupcoming experiments (d) iPS-MSCs after sorting showed more homogenous mesenchymal morphology expressing GFPdTom
Growth kinetics of iPSC-MSCs demonstrated a greaterproliferative capacity when compared with BM-MSCs withshorter doubling times (Figure 3(b)) In our experimentsthree independently derived BM-MSCs exhibited doublingtimes around 36 h in early passages that were prolongedabove 60 h around passage 8 and followed by a cessation ofproliferation with an apparent senescent phenotype aroundpassage 10 (Figure 3(b)) In contrast all three iPSC-MSCsexhibited significantly shorter doubling times (around 20 hin early passages) The prolonged doubling time of morethan 60 h did not occur before passage 15 and even after 20passages iPSC-MSCs did not show a senescent phenotypeThese results are in line with previously reported data fromSanchez et al who showed that human embryonic stem cell-derived CD73+ and CD90+ MSCs had higher proliferationrate than BM-MSCs (ESCs-MSCssim18 doubling compared toBM-MSCssim5 doubling in 30 days of culture) but were similar
to umbilical cord derived MSCs (sim15 doubling in 30 days)[12] The more robust proliferation potential of iPSC-MSCssuggests an important advantage over BM-MSCs wheneverrepetitive transplantations of the very sameMSCbatchwouldbe most preferential (for review of this impact on age-relateddisorders see [30]) Although the dosing of MSCs perfusionis currently controversially discussed for different disor-ders one can assume that an increasing demand of MSCstransplantation may arise in certain disorders For examplemusculoskeletal injuries with high occurrence in seniors [31]may urge for engineered MSCs with higher proliferationcapabilities but same functional abilities as BM-MSCs ThusiPSC-MSCs may serve as ldquooff the shelf transplantrdquo whichcan be provided by bloodstem cell banking institutions andused for several degenerative diseases Moreover the higherhomogeneity of such well-proliferating nonsenescent iPSC-MSCs populations suggest a higher safety and efficacy profile
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FLF-iPSC-MSCs (OSKM)FLF-iPSC-MSCs (OSNL)
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(b)
Figure 3 Antigen phenotype proliferation rate and functional characterization of hiPSC-MSCs (a) Immunophenotype of the three hiPS-MSCs lines generated Representative flow cytometry analysis of hBM-MSCs FLF-iPSC-MSCs (OSKM) FLF-iPSC-MSCs (OSNL) MSC-iPSC-MSCs (OSKM) and MSC-derived iPSC line MSC-related markers CD73 CD90 and CD105 and hematopoietic CD45 CD34 andCD19 were assessed (solid histogram) (b) In vitro cell growth measured as cumulative population of hiPSC-MSCs and BM-MSCs derivedfrom 3 different donors (LM02 LM05 and LM06)
andmay qualify such cells formore long-term treatment suchas inflammatory bowel diseases or during the prevention ofgraft versus host diseases or transplant rejection in futuretransplantation settings [12]
Addressing the functional capabilities of iPSC-derivedMSCs we applied differentiation protocols towards adipo-osteo- or chondrogenic lineages respectively and performedcytological staining andRT-PCR to investigate changes in cellmorphological and related marker gene expression Impor-tantly all 3 iPSC-MSCs could give rise to all of these threelineages However in comparison to BM-MSCs the qualityand morphology characteristics of the differentiated iPSC-MSCs exhibited slight differences All iPSC-MSCs were morereluctant to adipogenic differentiation and the respective totalnumbers of differentiated cells containing lipid droplets werelower than that of BM-MSCs (Figure 4) This observationwas confirmed with adipocytes specific mRNA level in whichexpression levels of PPAR-120572 and PPAR-120574 were significantlylower in iPSC-MSCs than BM-MSCs Also LPL expressionlevels were significantly (119901 le 005) lower in FLF-iPSC-MSCs (OKSM) and MSC-iPSC-MSCs (OKSM) compared toBM-MSCs On the other hand iPSC-MSCs had more affinity
to differentiate to osteogenic and chondrogenic lineagesGradually mineral nodules formation started 1 week earlierin iPSC-MSCs which were stained positive for alizarin red SThe expression of osteocalcin and alkaline phosphatase wascomparable to BM-MSCs Characterizing chondrogenesisthe respective pellets were stained more strongly with alcianblue and the respective gene expression profiles for collagenII showed higher expression in iPSC-MSCs compared toBM-MSCs However Aggrecan was similarly expressed inall three iPSC-MSCs and BM-MSCs It has been shown thatsomeMSCs for instance fromperiosteumand synoviumwereeasily capable of differentiating to bone and cartilage butonly a minor population amongst them could give rise toadipocytes [23 32] and we speculate that such a MSC-relatedphenotype is resembled by our iPSC-derived MSCs
33 Supportive Effects on Long-Term CD34+ Cells Mainte-nance Thebonemarrowniche plays a vital role in preservinghematopoietic progenitors to provide proper amounts ofblood cells throughout life [33 34]This activemicroenviron-ment is fostered by secreted factors of niche-accompanyingcells such asMSCs and sinusoidal endothelial cells to support
Figure 4 Adipogenic osteogenic and chondrogenic differentiation potential of hiPSC-MSCs and BM-MSC (LM06) Oil Red-O staining forlipid formation alizarin red staining of mineralized deposits and alcian blue staining for chondrocyte pellet formed by the three iPSC-MSC-like cell lines mRNA expression level of the relative expression of genes associated with adipogenesis PPAR120574 PPAR120572 and LPL osteogenesis(osteocalcin and alkaline phosphatase) and chondrogenesis (collagen type II and aggrecan) The data represent the mean expression valuesnormalized to the housekeeping gene GAPDH lowast significance difference with BM-MSCs 119901 le 005
the quiescent state of some of the hematopoietic progenitors[35 36] The supportive cellular microenvironment providedby MSCs regulates self-renewal versus differentiation ofhematopoietic stemprogenitor cells within the bone marrow[37 38] Moreover based on their secretion of cytokinessupportive for hematopoietic cell proliferation MSC areconsidered to serve as an excellent cell type for long-term
progenitor cell culture purposes [39] As further indicationfor the undisturbed functional capabilities of iPSC-MSCswe exploited a coculture system of iPSC-MSCs and CD34+hematopoietic stemprogenitor cells and investigated thetotal cell numbers the colony forming capacity and thehomogeneity of CD34+ cells After 10 days all three iPSC-MSC coculture assays contained significantly (119901 le 005)
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ells
()
CD-34CD-45
CD-11bCD-14
CD-34withoutstroma
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
lowast
lowastlowast
lowast
lowast
(c)
Figure 5 Coculture of CD34+ PBMCs with human iPSC-MSCs (a) Cell layers of iPSC-MSCs and BMSCs were established on 1 gelatinprecoated 24-well plates (80 confluent) CD34+ PBSCs were applied onto the stromal layers The cocultures were incubated for 20days Nonadherent viable cells were counted at the indicated time points (b) human CD34+ were plated with hiPSC-MSCs in 05mL ofmethylcellulose media containing human recombinant IL-3 SCF and EpoThe plates were incubated for 20 days following which progenitorswere scored (c) surface markers expression on CD34+ cells after coculture with mesenchymal stromal cells The results represent the mean(plusmnSD) of three replicates lowast significance difference with CD34 without stroma 119901 le 005
more nonadherent cells compared to CD34+ cells culturedwithout any stroma Comparing coculture of CD34+ cellswith iPSC-MSCs andBM-MSCs we observed a robust (but inour experiments not significant) increase in nonadherent cellnumbers for the iPSC-MSCs assays (Figure 5(a)) For OSKMfactors-derived iPSC-MSCs (Figure 5(a)) similar results wereobtained even at day 20 After replating CD34+ cells inMethoCult media for colony forming assays we observedsignificantly increased colonies in all iPSC-MSCs and BM-MSCs lines after 4 days of coculture comparing to singleCD34+ culture Furthermore MethoCult culture for 8 daysresulted in significantly (119901 le 005) higher colony numbersin iPSC-MSCs and in 2 lines of BM-MSCs (Figure 5(b))This data demonstrates a further important functional aspectand is supported by prior investigations that indicated thesupportive nature of MSCs on hematopoiesis by providinga suitable microenvironment for stemprogenitor cells ingrowing sites [40] With our data we also provided evidencethat significantly higher CD34+ cell number maintain theirstem cell status on the iPSC-MSCs and BM-MSCs rather than
conventional hematopoietic medium (Figure 5(c)) Theseenhanced proliferation and boosted colony forming abilitiescould be observed after 8 days of coculture in all iPSC-MSCs lines suggesting that these represent a reliable cellsource supporting the long-term culture of hematopoieticstemprogenitor cells Several publications are in favor of theeffects of different feeder layers and coatings for maintenanceand expansion of progenitors and somatic cells showing theimportance of mimicking in vivo conditions and providingsimilar microenvironment [41ndash43]
34 Immunosuppressive Effects of iPSC-MSCs In pioneer-ing studies mesenchymal stem cell based approaches wereapplied for suppressing immune reactions in autoimmunedisorders graft versus host disease (GVHD) or after solidorgan transplantation (for review see [44 45]) Duringallogeneic cell or organ transplantation cytotoxic and helperT-lymphocytes get activated and kill the targeted cells orpromote rejection of the transplanted organ [46] BecauseMSCs can secrete anti-inflammatory molecules to dampen
Stem Cells International 11
0
02
04
06
08
1
12
14
16
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
BrdU
mea
n ab
sorb
ance
in104
cells
lowastlowast
(a)
0
500
1000
1500
2000
2500
3000
3500
IFN
-120574(p
gm
L)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowast
lowastlowast
lowast
(b)
0
50
100
150
200
250
IL-2
(pg
mL)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
(c)
0
5
10
15
20
25
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowastlowastCD
4+C
D69
+(
)
(d)
0
5
10
15
20
25
lowast lowast
lowastlowast
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
CD4+
CD
25+
()
(e)
Figure 6 Status of activatedCD4+ T cells in the presence of hiPSC-MSCs (a) IFN-120574were determined at 48 hours by ELISAThe values are themeans plusmn SD from 3 independent experiments (b) concentrations of IL-2 and (c) proliferation in MLRMSC cocultures MLR cultures wereset up in presence or absence of hiPSC-MSCs BrdU incorporation was significantly lower in MSC-ips-MSCs and FLFiPSC-MSCs (OSNL)in comparison to absence of MSCs (d) Expression of the T-cell activation markers CD69 and (e) CD25 on CD4+ 5 days after stimulation ina 12-well dish in the presence or absence of hiPSC-MSCs lowast significance difference with MLR 119901 le 005
12 Stem Cells International
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
[1] M T Sutton and T L Bonfield ldquoStem cells innovations inclinical applicationsrdquo Stem Cells International vol 2014 ArticleID 516278 9 pages 2014
[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
iPSC-MSCs displayed comparable antigen profile and dif-ferentiation capability to bone marrow MSCs (BM-MSCs)and exhibited considerable functional properties [11ndash16]Moreover there is convincing evidence that iPSC-MSCs withhigher expansion capacities can be transplanted in manydegenerative diseases resulting in similar outcomes as BM-MSCs [13 15 17] Increasing evidence however indicatesthat MSCs from different origins are heterogeneous popu-lations exhibiting variable gene expression patterns [18 19]presenting different surfacemarkers [20] or showing reducedproliferation potential and differentiation capacities [21ndash23]
Furthermore a successful approach of iPSC-based ther-apeutic cell applications in regenerative medicine dependson the ability to set up an efficient differentiation protocolresulting in a desired cell population with a high purityMost importantly harmful contaminations of undifferenti-ated pluripotent stem cells must be avoided to exclude therisk of teratoma formation Therefore the robust genera-tion of a homogenous iPSC-MSC population with cellularcharacteristics identical to bona fide MSCs and similar oreven enhanced functional capabilities such as proliferationhematopoietic support and anti-inflammatory responsesneed further attention Here we exploited the differentiationpotential of three iPSC lines generated from fibroblast orprimary MSCs with Yamanaka reprogramming factors [10]namely Oct4 Sox2 Klf4 and c-Myc (OSKM) or Thomsonfactors [7] namely Oct4 Sox2 Nanog and Lin28 (OSNL)Upon MSC differentiation we applied lentiviral selectionconstructs carrying CD105- and CD73-promoter drivenfluorescent reporter and NeomycinPuromycin-resistance-transgenes to enrich the bulk differentiation for fully differ-entiated MSCs Next we explored the antigen profile dif-ferentiation potential proliferation capacity hematopoieticsupport and immune-suppression potential in regulation oflymphocyte proliferation proinflammatory cytokine secre-tions and activation markers of such iPSC-MSCs in directcomparison to bone marrow MSCs (BM-MSCs) from threedifferent donors (LM02 LM05 and LM06)
2 Material and Methods
21 Human iPS Cell Culture Human fetal liver fibroblast(FLF) iPS cells were provided from in-house supplies usingtransduction via lentiviral reprogramming factors Oct4Sox2 Klf4 and c-Myc (OSKM) [24] and Oct4 Sox2 Nanogand Lin28 (OSNL) [25] Human iPSCs were cultured onirradiated mouse embryonic fibroblasts (MEF) in a humid-ified incubator at 37∘C and 5 CO
2in medium contain-
ing DMEMF-12 20 knockout serum replacement (LifeTechnologies) 20 ngmL human recombinant basic fibrob-last growth factor (bFGF provided from Leibniz UniversityHannover) 01mM 120573-mercaptoethanol (Life Technologies)1mM L-glutamine 1 nonessential amino acids and 1penicillinstreptomycin (all from Sigma-Aldrich)
Media were changed daily and cells were split weekly bydissociation with 200UmL of collagenase IV (Life Tech-nologies) and cells were plated on Matrigel-coated platesin medium supplemented with 40 ngmL bFGF for furtherdifferentiation
22 Derivation and Enrichment of Human MSC-Like CellsFor triggering iPSC differentiation toward MSC-like cellshuman iPSC colonies grown on Matrigel (Corning) weremaintained with MSC induction media consisting of DMEM(low-glucose Sigma-Aldrich) 10 defined fetal bovineserum (FBS STEMCELL Technologies) 1 nonessen-tial amino acids 1 penicillin-streptomycin and 2 ngmLhuman recombinant bFGF for 7 days Next cells were treatedwith collagenase IV for 3min at 37∘C dissociated by glassbeads and gentle pipetting and then passed through 40 mmcell strainers (Fisher Scientific) Single cells were seeded ontogelatin-coated plates at 1 times 104 cellscm2 in MSC media
To facilitate enrichment and screening of MSCs duringthe standard differentiation protocol of iPSCs into MSCsa combination of two positive markers namely CD73 andCD105 (which are consistently expressed in MSCs) was cho-sen to produce MSC-specific selection vectorsThe promoterregions for CD73 and CD105 were amplified and ligated intothe corresponding lentiviral backbone the CD105 promoterinto pRRL-Puro-IRES-GFP and the CD73 promoter intopRRL-neo-IRES-dTom (Lentiviral backbones provided in-house based on lentiviral constructs from Axel Scham-bach laboratory) Cells were selected by 500 120583gmL G-418and 4 120583gmL Puromycin-dihydrochloride (both from Sigma-Aldrich) in culture media for 2 weeks until the untransfectedcells were killed Functionality of the vectors confirmedfluorochrome expression in transduced cells by fluorescentmicroscope afterwards
Double-positive MSCs population transduced withpRRL-CD105-Puro-IRES-GFP and pRRLCD73-neo-IRES-dTom was purified with the FACSAria II cell sorter (BDBioscience) A total of 2 times 106 sorted cells were immediatelyplated back into gelatin-coated plates to facilitate adherenceAfter 24 hours fresh prewarmed MSCs medium was addedand cells were allowed to expand and reach nearly 100confluence Cells were counted in different time pointsBone marrow MSCs were isolated in Frankfurt universityhospital as previously described [26] Shortly 10ndash30mL ofbone marrow was aspirated from femoral cavity of patientswho needed hip joint replacement surgery after informedconsent in accordance with the Declaration of Helsinki Afterdensity gradient separation the light density mononuclearcell fraction was seeded on T25 (TPP) tissue culture flasks inpreviously mentioned MSC media
23 Lentiviral Vectors Production HEK 293T cells were usedfor virus production 3 times 106 cells were seeded one daybefore transfection in 10 cm dishes (TPP) in DMEM (highglucose Life Technologies) supplemented with 10 FBS and1penicillinstreptomycin and 1L-glutamineThenext daymedium was exchanged with 8mL DMEM supplementedwith 25 120583M Chloroquine (Sigma-Aldrich) Plasmids encod-ing for lentiviral gagpol (pCDNA3GPCCCC 10 120583g) RSV-Rev (pRSV-Rev 5120583g) VSV-G (pMD2G 2120583g) and packag-ing plasmid encoding for respective transgene into pRRL-Puro-IRES-GFP and pRRL-neo-IRES-dTom were mixed in400 120583L of ddH
2O and 100 120583L of 125M CaCl
2 The plasmids-
CaCl2mixture was added dropwise to 2xHBS and observed
Stem Cells International 3
until precipitates became visible in phase-contrast micro-scope and then added toHEKcells 6 hours latermediumwasexchanged with 10mL DMEM high glucose supplementedwith 10 FBS and 1 penicillinstreptomycin and 1 L-glutamine 48 hours later supernatant was collected passedthrough 045 120583m filters and centrifuged at 14000timesg for8 h Virus pellet was resuspended in 200120583L PBS (Sigma-Aldrich) Viral titers were determined by transduction ofHEK 293T cells in serial dilutions and analysis of reportergene expression by flow cytometry Generally titers were inthe range of 1-2 times 107 viral particles per mL
24 Antigen Profiling by Flow Cytometry To assess the im-munophenotypic profile of BM-MSCs and iPSC-derivedMSCs single cell suspensions were prepared by trypsindigestion (Life Technologies) and washed with cold PBScontaining 1 bovine serum albumin BSA (MerckMillipore)Next 2 times 105 cells were incubated for 30 minutes with therespective APC-conjugated monoclonal antibodies CD73CD90 CD105 CD45 CD34 and CD19 (all from BD Bio-science listed in Table 1) and subsequently resuspended ina density of 2 times 105 cells per 200120583L in cold PBS contain-ing 1 BSA Nonspecific fluorescence was determined byincubation of cell aliquots with isotype-matched monoclonalantibodies
Samples were run on a FACS Calibur (BD Bioscience)cytometer using FACS Diva software For each analysis aminimum of 10000 cells was assayed Data was furtherprocessed using FlowJo Software (Tree Star)
25 Growth Kinetics Human BMSCs from 3 different donors(LM02 LM05 and LM06) and 3 iPSC-MSCs lineswere plated(2 times 104 cellswell) onto 12-well plates in triplicate Cellswere harvested after 72 hours in each passage (10 passagesfor BM-MSCs and 15 passages for iPSC-MSCs) Cumulativepopulation doublings were calculated using the formula 119909 =[log 10(NH) minus log 10(1198731)] log 10(2) [27] where 1198731 is theinoculum cell number and NH the cell harvest number Toyield the cumulated doubling level the population doublingfor each passage was calculated and then added to thepopulation doubling levels of the previous passages Thecultures were abandoned as soon as they showed a senescentphenotype when they ceased proliferation
26 In Vitro Adipogenic Chondrogenic and Osteogenic Dif-ferentiation Differentiation induction of iPSC-MSCs wascarried out for 21 days in different differentiation mediaTotally 104 cells were seeded per well in six-well plates(TPP) To induce osteogenic differentiation cells were cul-tured with MSC medium containing 1 120583M dexamethasone05 120583M ascorbic acid and 10mM b-glycerol phosphate (allfrom Sigma-Aldrich) For adipogenic induction cells werecultured in MSC medium supplemented with 50 120583gmLindomethacin (Sigma-Aldrich) 50120583gmL ascorbic acid and100 nM dexamethasone For chondrogenic differentiationiPSC-MSCs were centrifuged in 02mL of medium at500 g for 10min in 15 mL Falcon tubes to form a pelletThe pellets were cultured in MSC medium supplemented
Table 1 List of antibodies and ELISA kits used in this study
with 001120583M dexamethasone 397 120583gmL ascorbic acid-2-phosphate (Sigma-Aldrich) 1mM sodium pyruvate (Sigma-Aldrich) 10 ngmL transforming growth factor-1205731 (TGF-1205731Life Technologies) and 1 insulin-transferrin-selenium (LifeTechnologies) Osteogenesis was assessed by alizarin redstaining adipogenesis by oil red staining and chondrogen-esis by alcian blue staining (all from Sigma-Aldrich)
27 Real Time qRT PCR Total cellular RNA was isolatedusing TRIzol reagent (Life Technologies) Resultant RNAwas subjected to DNase treatment and cDNA Synthesis Kit(Life Technologies) with random hexamers Power SYBRGreen Master Mix based qRT PCR assays were performedon the StepOne Plus Cycler (Applied Biosystems) using thestandard settings We collected samples from at least threeindependent experiments Expression values of human per-oxisome proliferator-activated receptor 120574 (PPAR120574) PPAR120572lipoprotein lipase (LPL) collagen type II (COL2) aggre-can (ACAN) osteocalcin (OCN) and alkaline phosphatase(ALP) normalized to expression of GAPDH The primersequences are listed in Table 2
28 Coculture of CD34+ Progenitor Cells with iPSC-MSCsHuman iPSC-MSCs or BM-MSCs layers were grown until80 confluency in six-well plates and then treated withmitomycine-C (Sigma-Aldrich) to prevent cell overgrowthAfter 24 h medium was removed and purified CD34+cells (resuspended at 75 times 104 cells per well) were thenadded in 2mL of long-term culture medium containing 120572-minimal essentialmedium (Life Technologies) with 20FBS1 120583molL hydrocortisone (Sigma-Aldrich) and 01mM 120573-mercaptoethanol The cocultures were incubated for 20 daysmedium exchange twice per week Nonadherent viable cellswere counted at the indicated time points (day 10 and day20) CD-markers for hematopoietic differentiation were alsodetermined after 8 days for CD34 CD45 CD-11b and CD-14Experiments were repeated three times
4 Stem Cells International
Table 2 List of primers used in this study qRT-PCR
29 In Vitro Progenitor Assays Effects of human iPSC-MSCsor BM-MSCs on progenitor cells were analyzed using acolony forming cell assay Human bone marrow CD34+ cells(2 times 106) were obtained from Lonza and were plated in 2mLof methylcellulose media (STEMCELL Technologies) with orwithout iPSC-MSCs and BMSCs Colonies of gt50 cells werescored after 4 and 8 days of incubation
210 Assessment of CD4+ T-Lymphocyte ProliferationResponseto iPSC-MSCs Standard 5-day MLR cultures were set upwith 5 times 104 Mitomycin Cndashtreated (Sigma-Aldrich) humanperipheral blood mononuclear cells (PBMCs) as stimula-tors and 2 times 105 human CD4+ T-cells (Lonza) in 96-wellround-bottom plates in 200120583L complete medium consistingRPMI 1640 (Life Technologies) supplemented with 01mM120573-mercaptoethanol 10 FBS GLUTAMAX I (Life Tech-nologies) 100UmL penicillin and 100 120583gmL streptomycinin the presence or absence of iPSC-MSCs and BM-MSCsFor analyzing expression of CD69+ and CD25+ regulatoryT-cell population 106 responder cells were mixed with 25times 105 stimulator PMNCs in presence or absence of 2 times105 iPSC-MSCs or BM-MSCs MLRs were performed ona layer of confluent Mitomycine C treated MSCs seededone day before Proliferation was determined with BrdUELISA assay (Roche) based on manufacturer instruction IL-2 and IFN-120574 concentration was determined in MSCMLRcoculture supernatants using a commercially available ELISA(BD Bioscience) according to manufacturerrsquos instructionsCD25 and CD69 (BD Bioscience) expression on CD4+ cellswere analyzed by flow cytometry
3 Results and Discussion
31 Generation of MSCs from Human iPSCs with Spindle-Shape Morphology The challenge of accessing an appro-priate and homogenous source for MSCs with sustainedgrowth kinetics immunosuppressive potentials and produc-tion of chemokines or growth factors supporting endogenousregeneration led to the question whether homogenouslydifferentiated MSCs could be derived from human inducedpluripotent stem cells (iPSCs) To address this question weused two different sources of somatic cells liver fibroblastand bone marrow-derived MSCs (BM-MSCs) and repro-grammed these cells into iPSCs Besides two sources of
somatic starting cells we also compared two slightly dif-ferent composition of reprograming factor cocktails Onefactor combination was comprised of Oct4 Sox2 Klf4 andc-Myc (OSKM) as it was originally described by ShinyaYamanaka and subsequently in multitudinous publications[8ndash10] and the other combination consisted of Oct4 Sox2Nanog and Lin28 (OSNL) as it was described by JamesThomson and some further groups [7 25] Taken together wegenerated fetal liver fibroblast-derived iPSCswithOSKMandOSNL (FLF iPSCs) and bone marrow MSC-derived iPSCswith OSKM (MSC-iPSCs) which were strongly expressingOCT4 SOX2 and SSEA-4 (Figure 1) All iPSC lines weredifferentiated based on a previously reported differentiationprotocol resulting in about 70 CD73+CD105+ cells [14]in which we have made some modifications to allow forantibiotic selection and fluorescent reporter-based purifi-cation (Figure 2(a)) As expected Epithelial-MesenchymalTransition occurred during differentiation giving rise toa heterogeneous population (Figure 2(b)) However withenrichment of early mesenchymal-like cells we observedintermediate and highly CD73-dTomCD105-GFP express-ing cells populations (Figure 2(c)) We sorted highly express-ing GFPdTom positive cells and obtained a much morehomogenous population (Figure 2(d)) Stimulated by the firstdescription of iPSC-derived MSCs by Lian et al in 2010[15] many other groups tried to do direct and spontaneouslydifferentiating iPSCs into MSCs by various means We con-sider the use of lentiviral reporter and selection constructsas important tools to monitor the purity of a cell populationduring differentiation processes and to ensure a high grade ofhomogeneity within the final cell population Such selectionconstructs were recently introduced in other lineagesrsquo differ-entiation protocols [28] Thus in the present study a similarvector architecture was applied to select for CD73+CD105+positive iPSC-MSCs Interestingly we obtained a high num-ber of CD73posCD105intermediate iPSC-MSCs (R1 633)and a smaller fraction of CD73posCD105high iPSC-MSCs(R2 643) and we concluded that sorting the less abun-dant CD73posCD105high population might provide the mosthomogenous cell population
32 Immunophenotype Proliferation and Differentiation Po-tential of iPSC-MSCs In order to characterize the iPSC-MSCs according to the International Society of Cell Therapy
Stem Cells International 5
SSEA4 PE subset952
0
50
100
150 SSEA4 PE subset993
0
50
100
150SSEA4 PE subset997
0
100
200
300
FLF-iPSCs(OSKM)
FLF-iPSCs(OSNL)
MSC-iPSCs(OSKM)
DAPI
OCT-4
SOX-2
Merge
SSEA-4 SSEA-4 SSEA-4
Cou
nt
Cou
nt
Cou
nt
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
Figure 1 Generation and characterization iPS cells from human fetal liver fibroblasts (FLF) with Oct4 Sox2 Klf4 and c-Myc (OSKM) andOct4 Sox2 Nanog and Lin-28 (OSNL) and also from human bonemarrowMSCs with OSKMusing lentiviral vectors iPSCs stained positivefor humanOCT4 and SOX2DAPIwas used to stain the nuclei andmergedwith phase-contrast Expression of SSEA-4 is shown in histograms
(ISCT) criteria [29] cell surface marker expression was ana-lyzed by flow cytometry of all three iPSC-MSC lines and BM-MSCs at early passages (passages 3ndash6) All 3 differentiated andenriched iPSC-MSCs displayed a MSC-like antigen profilethat exhibited high CD105 CD73 and CD90 and absenceof CD34 CD45 and CD19 expression (Figure 3(a)) Thuswe were able to demonstrate that homogenous populationscan be isolated and purified from all three iPSC linesindependent to their somatic cell source (fibroblasts or bonemarrow MSCs) and method of reprogramming (OSKM or
OSNL factor cocktail) Strikingly the surface marker CD105and CD73 whose promoter motifs were utilized to expressthe fluorescent reporter transgenes and antibiotic selectioncassettes were readily detectable in almost 100 of purifiedcells indicating the high purity of our enriched iPSC-MSCsInterestingly CD90 was positive not only in all iPSC-MSCslines as well as the BM-MSCs but also in undifferenti-ated iPSCs (MSC-iPSCs) Furthermore the hematopoieticsurface markers were neither expressed in MSCs nor iniPSCs
6 Stem Cells International
iPSCs in MSCsDifferentiation
(Epithelial Transduction MSC-likemedia morphology) and sorting cells
418 (500120583gmL) and puromycin (4120583gmL) and then sortedIRES-dTom and selected with G
lowastAfter plating cells were transduced with CD-105-Puro-IRES-GFP and CD-73-Neo-
(c) (d)
Figure 2 Derivation and enrichment of MSCs from human iPS cells (a) Schematic stepwise protocol for differentiation and selection ofMSC-like cells from human iPS cells (b) phase-contrast photos demonstrating Epithelial-Mesenchymal transition in cellular morphology(c) FACS dot blot showing intermediate (R1) and highly (P2) double-positive cells Highly positive CD-73 and CD-105 (R2) were sorted forupcoming experiments (d) iPS-MSCs after sorting showed more homogenous mesenchymal morphology expressing GFPdTom
Growth kinetics of iPSC-MSCs demonstrated a greaterproliferative capacity when compared with BM-MSCs withshorter doubling times (Figure 3(b)) In our experimentsthree independently derived BM-MSCs exhibited doublingtimes around 36 h in early passages that were prolongedabove 60 h around passage 8 and followed by a cessation ofproliferation with an apparent senescent phenotype aroundpassage 10 (Figure 3(b)) In contrast all three iPSC-MSCsexhibited significantly shorter doubling times (around 20 hin early passages) The prolonged doubling time of morethan 60 h did not occur before passage 15 and even after 20passages iPSC-MSCs did not show a senescent phenotypeThese results are in line with previously reported data fromSanchez et al who showed that human embryonic stem cell-derived CD73+ and CD90+ MSCs had higher proliferationrate than BM-MSCs (ESCs-MSCssim18 doubling compared toBM-MSCssim5 doubling in 30 days of culture) but were similar
to umbilical cord derived MSCs (sim15 doubling in 30 days)[12] The more robust proliferation potential of iPSC-MSCssuggests an important advantage over BM-MSCs wheneverrepetitive transplantations of the very sameMSCbatchwouldbe most preferential (for review of this impact on age-relateddisorders see [30]) Although the dosing of MSCs perfusionis currently controversially discussed for different disor-ders one can assume that an increasing demand of MSCstransplantation may arise in certain disorders For examplemusculoskeletal injuries with high occurrence in seniors [31]may urge for engineered MSCs with higher proliferationcapabilities but same functional abilities as BM-MSCs ThusiPSC-MSCs may serve as ldquooff the shelf transplantrdquo whichcan be provided by bloodstem cell banking institutions andused for several degenerative diseases Moreover the higherhomogeneity of such well-proliferating nonsenescent iPSC-MSCs populations suggest a higher safety and efficacy profile
Stem Cells International 7
0
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10
20
30
05
10152025
MSC-iPSCs(OSKM)
hBM-MSCs(LM06)
FLF-iPSC-MSCs (OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs (OSKM)
CD73
CD10
5CD
90CD
45CD
34CD
19
10010
110
210
310
410
510
010
110
210
310
410
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010
110
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10010
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10010
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410
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10010
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410
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10010
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410
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410
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10010
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110
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410
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410
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100 10
110
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410
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10010
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210
310
410
5
10010
110
210
310
410
510
010
110
210
310
410
5
10010
110
210
310
410
5
100 10
110
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310
410
510
0 10110
210
310
410
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0 10110
210
310
410
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10010
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410
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10010
110
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410
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10010
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(a)
Figure 3 Continued
8 Stem Cells International
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10
BM-MSC LM02BM-MSC LM05
BM-MSC LM06
Passages
Dou
blin
g tim
e (ho
urs)
Dou
blin
g tim
e (ho
urs)
Passages
01020304050607080
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
FLF-iPSC-MSCs (OSKM)FLF-iPSC-MSCs (OSNL)
MSC-iPS-MSCs (OSKM)
(b)
Figure 3 Antigen phenotype proliferation rate and functional characterization of hiPSC-MSCs (a) Immunophenotype of the three hiPS-MSCs lines generated Representative flow cytometry analysis of hBM-MSCs FLF-iPSC-MSCs (OSKM) FLF-iPSC-MSCs (OSNL) MSC-iPSC-MSCs (OSKM) and MSC-derived iPSC line MSC-related markers CD73 CD90 and CD105 and hematopoietic CD45 CD34 andCD19 were assessed (solid histogram) (b) In vitro cell growth measured as cumulative population of hiPSC-MSCs and BM-MSCs derivedfrom 3 different donors (LM02 LM05 and LM06)
andmay qualify such cells formore long-term treatment suchas inflammatory bowel diseases or during the prevention ofgraft versus host diseases or transplant rejection in futuretransplantation settings [12]
Addressing the functional capabilities of iPSC-derivedMSCs we applied differentiation protocols towards adipo-osteo- or chondrogenic lineages respectively and performedcytological staining andRT-PCR to investigate changes in cellmorphological and related marker gene expression Impor-tantly all 3 iPSC-MSCs could give rise to all of these threelineages However in comparison to BM-MSCs the qualityand morphology characteristics of the differentiated iPSC-MSCs exhibited slight differences All iPSC-MSCs were morereluctant to adipogenic differentiation and the respective totalnumbers of differentiated cells containing lipid droplets werelower than that of BM-MSCs (Figure 4) This observationwas confirmed with adipocytes specific mRNA level in whichexpression levels of PPAR-120572 and PPAR-120574 were significantlylower in iPSC-MSCs than BM-MSCs Also LPL expressionlevels were significantly (119901 le 005) lower in FLF-iPSC-MSCs (OKSM) and MSC-iPSC-MSCs (OKSM) compared toBM-MSCs On the other hand iPSC-MSCs had more affinity
to differentiate to osteogenic and chondrogenic lineagesGradually mineral nodules formation started 1 week earlierin iPSC-MSCs which were stained positive for alizarin red SThe expression of osteocalcin and alkaline phosphatase wascomparable to BM-MSCs Characterizing chondrogenesisthe respective pellets were stained more strongly with alcianblue and the respective gene expression profiles for collagenII showed higher expression in iPSC-MSCs compared toBM-MSCs However Aggrecan was similarly expressed inall three iPSC-MSCs and BM-MSCs It has been shown thatsomeMSCs for instance fromperiosteumand synoviumwereeasily capable of differentiating to bone and cartilage butonly a minor population amongst them could give rise toadipocytes [23 32] and we speculate that such a MSC-relatedphenotype is resembled by our iPSC-derived MSCs
33 Supportive Effects on Long-Term CD34+ Cells Mainte-nance Thebonemarrowniche plays a vital role in preservinghematopoietic progenitors to provide proper amounts ofblood cells throughout life [33 34]This activemicroenviron-ment is fostered by secreted factors of niche-accompanyingcells such asMSCs and sinusoidal endothelial cells to support
Figure 4 Adipogenic osteogenic and chondrogenic differentiation potential of hiPSC-MSCs and BM-MSC (LM06) Oil Red-O staining forlipid formation alizarin red staining of mineralized deposits and alcian blue staining for chondrocyte pellet formed by the three iPSC-MSC-like cell lines mRNA expression level of the relative expression of genes associated with adipogenesis PPAR120574 PPAR120572 and LPL osteogenesis(osteocalcin and alkaline phosphatase) and chondrogenesis (collagen type II and aggrecan) The data represent the mean expression valuesnormalized to the housekeeping gene GAPDH lowast significance difference with BM-MSCs 119901 le 005
the quiescent state of some of the hematopoietic progenitors[35 36] The supportive cellular microenvironment providedby MSCs regulates self-renewal versus differentiation ofhematopoietic stemprogenitor cells within the bone marrow[37 38] Moreover based on their secretion of cytokinessupportive for hematopoietic cell proliferation MSC areconsidered to serve as an excellent cell type for long-term
progenitor cell culture purposes [39] As further indicationfor the undisturbed functional capabilities of iPSC-MSCswe exploited a coculture system of iPSC-MSCs and CD34+hematopoietic stemprogenitor cells and investigated thetotal cell numbers the colony forming capacity and thehomogeneity of CD34+ cells After 10 days all three iPSC-MSC coculture assays contained significantly (119901 le 005)
10 Stem Cells International
0
100
200
300
400
500
600
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
Day 0Day 10
Day 20
Tota
l non
adhe
rent
via
ble c
ellstimes104
lowast lowastlowast lowast lowast
lowastlowast
(a)
0
20
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180
Tota
l col
ony
form
ing
units
wel
l
Day 4Day 8
lowast lowastlowast lowast
lowast
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
(b)
0102030405060708090
100
Posit
ive c
ells
()
CD-34CD-45
CD-11bCD-14
CD-34withoutstroma
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
lowast
lowastlowast
lowast
lowast
(c)
Figure 5 Coculture of CD34+ PBMCs with human iPSC-MSCs (a) Cell layers of iPSC-MSCs and BMSCs were established on 1 gelatinprecoated 24-well plates (80 confluent) CD34+ PBSCs were applied onto the stromal layers The cocultures were incubated for 20days Nonadherent viable cells were counted at the indicated time points (b) human CD34+ were plated with hiPSC-MSCs in 05mL ofmethylcellulose media containing human recombinant IL-3 SCF and EpoThe plates were incubated for 20 days following which progenitorswere scored (c) surface markers expression on CD34+ cells after coculture with mesenchymal stromal cells The results represent the mean(plusmnSD) of three replicates lowast significance difference with CD34 without stroma 119901 le 005
more nonadherent cells compared to CD34+ cells culturedwithout any stroma Comparing coculture of CD34+ cellswith iPSC-MSCs andBM-MSCs we observed a robust (but inour experiments not significant) increase in nonadherent cellnumbers for the iPSC-MSCs assays (Figure 5(a)) For OSKMfactors-derived iPSC-MSCs (Figure 5(a)) similar results wereobtained even at day 20 After replating CD34+ cells inMethoCult media for colony forming assays we observedsignificantly increased colonies in all iPSC-MSCs and BM-MSCs lines after 4 days of coculture comparing to singleCD34+ culture Furthermore MethoCult culture for 8 daysresulted in significantly (119901 le 005) higher colony numbersin iPSC-MSCs and in 2 lines of BM-MSCs (Figure 5(b))This data demonstrates a further important functional aspectand is supported by prior investigations that indicated thesupportive nature of MSCs on hematopoiesis by providinga suitable microenvironment for stemprogenitor cells ingrowing sites [40] With our data we also provided evidencethat significantly higher CD34+ cell number maintain theirstem cell status on the iPSC-MSCs and BM-MSCs rather than
conventional hematopoietic medium (Figure 5(c)) Theseenhanced proliferation and boosted colony forming abilitiescould be observed after 8 days of coculture in all iPSC-MSCs lines suggesting that these represent a reliable cellsource supporting the long-term culture of hematopoieticstemprogenitor cells Several publications are in favor of theeffects of different feeder layers and coatings for maintenanceand expansion of progenitors and somatic cells showing theimportance of mimicking in vivo conditions and providingsimilar microenvironment [41ndash43]
34 Immunosuppressive Effects of iPSC-MSCs In pioneer-ing studies mesenchymal stem cell based approaches wereapplied for suppressing immune reactions in autoimmunedisorders graft versus host disease (GVHD) or after solidorgan transplantation (for review see [44 45]) Duringallogeneic cell or organ transplantation cytotoxic and helperT-lymphocytes get activated and kill the targeted cells orpromote rejection of the transplanted organ [46] BecauseMSCs can secrete anti-inflammatory molecules to dampen
Stem Cells International 11
0
02
04
06
08
1
12
14
16
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
BrdU
mea
n ab
sorb
ance
in104
cells
lowastlowast
(a)
0
500
1000
1500
2000
2500
3000
3500
IFN
-120574(p
gm
L)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowast
lowastlowast
lowast
(b)
0
50
100
150
200
250
IL-2
(pg
mL)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
(c)
0
5
10
15
20
25
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowastlowastCD
4+C
D69
+(
)
(d)
0
5
10
15
20
25
lowast lowast
lowastlowast
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
CD4+
CD
25+
()
(e)
Figure 6 Status of activatedCD4+ T cells in the presence of hiPSC-MSCs (a) IFN-120574were determined at 48 hours by ELISAThe values are themeans plusmn SD from 3 independent experiments (b) concentrations of IL-2 and (c) proliferation in MLRMSC cocultures MLR cultures wereset up in presence or absence of hiPSC-MSCs BrdU incorporation was significantly lower in MSC-ips-MSCs and FLFiPSC-MSCs (OSNL)in comparison to absence of MSCs (d) Expression of the T-cell activation markers CD69 and (e) CD25 on CD4+ 5 days after stimulation ina 12-well dish in the presence or absence of hiPSC-MSCs lowast significance difference with MLR 119901 le 005
12 Stem Cells International
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
[1] M T Sutton and T L Bonfield ldquoStem cells innovations inclinical applicationsrdquo Stem Cells International vol 2014 ArticleID 516278 9 pages 2014
[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
until precipitates became visible in phase-contrast micro-scope and then added toHEKcells 6 hours latermediumwasexchanged with 10mL DMEM high glucose supplementedwith 10 FBS and 1 penicillinstreptomycin and 1 L-glutamine 48 hours later supernatant was collected passedthrough 045 120583m filters and centrifuged at 14000timesg for8 h Virus pellet was resuspended in 200120583L PBS (Sigma-Aldrich) Viral titers were determined by transduction ofHEK 293T cells in serial dilutions and analysis of reportergene expression by flow cytometry Generally titers were inthe range of 1-2 times 107 viral particles per mL
24 Antigen Profiling by Flow Cytometry To assess the im-munophenotypic profile of BM-MSCs and iPSC-derivedMSCs single cell suspensions were prepared by trypsindigestion (Life Technologies) and washed with cold PBScontaining 1 bovine serum albumin BSA (MerckMillipore)Next 2 times 105 cells were incubated for 30 minutes with therespective APC-conjugated monoclonal antibodies CD73CD90 CD105 CD45 CD34 and CD19 (all from BD Bio-science listed in Table 1) and subsequently resuspended ina density of 2 times 105 cells per 200120583L in cold PBS contain-ing 1 BSA Nonspecific fluorescence was determined byincubation of cell aliquots with isotype-matched monoclonalantibodies
Samples were run on a FACS Calibur (BD Bioscience)cytometer using FACS Diva software For each analysis aminimum of 10000 cells was assayed Data was furtherprocessed using FlowJo Software (Tree Star)
25 Growth Kinetics Human BMSCs from 3 different donors(LM02 LM05 and LM06) and 3 iPSC-MSCs lineswere plated(2 times 104 cellswell) onto 12-well plates in triplicate Cellswere harvested after 72 hours in each passage (10 passagesfor BM-MSCs and 15 passages for iPSC-MSCs) Cumulativepopulation doublings were calculated using the formula 119909 =[log 10(NH) minus log 10(1198731)] log 10(2) [27] where 1198731 is theinoculum cell number and NH the cell harvest number Toyield the cumulated doubling level the population doublingfor each passage was calculated and then added to thepopulation doubling levels of the previous passages Thecultures were abandoned as soon as they showed a senescentphenotype when they ceased proliferation
26 In Vitro Adipogenic Chondrogenic and Osteogenic Dif-ferentiation Differentiation induction of iPSC-MSCs wascarried out for 21 days in different differentiation mediaTotally 104 cells were seeded per well in six-well plates(TPP) To induce osteogenic differentiation cells were cul-tured with MSC medium containing 1 120583M dexamethasone05 120583M ascorbic acid and 10mM b-glycerol phosphate (allfrom Sigma-Aldrich) For adipogenic induction cells werecultured in MSC medium supplemented with 50 120583gmLindomethacin (Sigma-Aldrich) 50120583gmL ascorbic acid and100 nM dexamethasone For chondrogenic differentiationiPSC-MSCs were centrifuged in 02mL of medium at500 g for 10min in 15 mL Falcon tubes to form a pelletThe pellets were cultured in MSC medium supplemented
Table 1 List of antibodies and ELISA kits used in this study
with 001120583M dexamethasone 397 120583gmL ascorbic acid-2-phosphate (Sigma-Aldrich) 1mM sodium pyruvate (Sigma-Aldrich) 10 ngmL transforming growth factor-1205731 (TGF-1205731Life Technologies) and 1 insulin-transferrin-selenium (LifeTechnologies) Osteogenesis was assessed by alizarin redstaining adipogenesis by oil red staining and chondrogen-esis by alcian blue staining (all from Sigma-Aldrich)
27 Real Time qRT PCR Total cellular RNA was isolatedusing TRIzol reagent (Life Technologies) Resultant RNAwas subjected to DNase treatment and cDNA Synthesis Kit(Life Technologies) with random hexamers Power SYBRGreen Master Mix based qRT PCR assays were performedon the StepOne Plus Cycler (Applied Biosystems) using thestandard settings We collected samples from at least threeindependent experiments Expression values of human per-oxisome proliferator-activated receptor 120574 (PPAR120574) PPAR120572lipoprotein lipase (LPL) collagen type II (COL2) aggre-can (ACAN) osteocalcin (OCN) and alkaline phosphatase(ALP) normalized to expression of GAPDH The primersequences are listed in Table 2
28 Coculture of CD34+ Progenitor Cells with iPSC-MSCsHuman iPSC-MSCs or BM-MSCs layers were grown until80 confluency in six-well plates and then treated withmitomycine-C (Sigma-Aldrich) to prevent cell overgrowthAfter 24 h medium was removed and purified CD34+cells (resuspended at 75 times 104 cells per well) were thenadded in 2mL of long-term culture medium containing 120572-minimal essentialmedium (Life Technologies) with 20FBS1 120583molL hydrocortisone (Sigma-Aldrich) and 01mM 120573-mercaptoethanol The cocultures were incubated for 20 daysmedium exchange twice per week Nonadherent viable cellswere counted at the indicated time points (day 10 and day20) CD-markers for hematopoietic differentiation were alsodetermined after 8 days for CD34 CD45 CD-11b and CD-14Experiments were repeated three times
4 Stem Cells International
Table 2 List of primers used in this study qRT-PCR
29 In Vitro Progenitor Assays Effects of human iPSC-MSCsor BM-MSCs on progenitor cells were analyzed using acolony forming cell assay Human bone marrow CD34+ cells(2 times 106) were obtained from Lonza and were plated in 2mLof methylcellulose media (STEMCELL Technologies) with orwithout iPSC-MSCs and BMSCs Colonies of gt50 cells werescored after 4 and 8 days of incubation
210 Assessment of CD4+ T-Lymphocyte ProliferationResponseto iPSC-MSCs Standard 5-day MLR cultures were set upwith 5 times 104 Mitomycin Cndashtreated (Sigma-Aldrich) humanperipheral blood mononuclear cells (PBMCs) as stimula-tors and 2 times 105 human CD4+ T-cells (Lonza) in 96-wellround-bottom plates in 200120583L complete medium consistingRPMI 1640 (Life Technologies) supplemented with 01mM120573-mercaptoethanol 10 FBS GLUTAMAX I (Life Tech-nologies) 100UmL penicillin and 100 120583gmL streptomycinin the presence or absence of iPSC-MSCs and BM-MSCsFor analyzing expression of CD69+ and CD25+ regulatoryT-cell population 106 responder cells were mixed with 25times 105 stimulator PMNCs in presence or absence of 2 times105 iPSC-MSCs or BM-MSCs MLRs were performed ona layer of confluent Mitomycine C treated MSCs seededone day before Proliferation was determined with BrdUELISA assay (Roche) based on manufacturer instruction IL-2 and IFN-120574 concentration was determined in MSCMLRcoculture supernatants using a commercially available ELISA(BD Bioscience) according to manufacturerrsquos instructionsCD25 and CD69 (BD Bioscience) expression on CD4+ cellswere analyzed by flow cytometry
3 Results and Discussion
31 Generation of MSCs from Human iPSCs with Spindle-Shape Morphology The challenge of accessing an appro-priate and homogenous source for MSCs with sustainedgrowth kinetics immunosuppressive potentials and produc-tion of chemokines or growth factors supporting endogenousregeneration led to the question whether homogenouslydifferentiated MSCs could be derived from human inducedpluripotent stem cells (iPSCs) To address this question weused two different sources of somatic cells liver fibroblastand bone marrow-derived MSCs (BM-MSCs) and repro-grammed these cells into iPSCs Besides two sources of
somatic starting cells we also compared two slightly dif-ferent composition of reprograming factor cocktails Onefactor combination was comprised of Oct4 Sox2 Klf4 andc-Myc (OSKM) as it was originally described by ShinyaYamanaka and subsequently in multitudinous publications[8ndash10] and the other combination consisted of Oct4 Sox2Nanog and Lin28 (OSNL) as it was described by JamesThomson and some further groups [7 25] Taken together wegenerated fetal liver fibroblast-derived iPSCswithOSKMandOSNL (FLF iPSCs) and bone marrow MSC-derived iPSCswith OSKM (MSC-iPSCs) which were strongly expressingOCT4 SOX2 and SSEA-4 (Figure 1) All iPSC lines weredifferentiated based on a previously reported differentiationprotocol resulting in about 70 CD73+CD105+ cells [14]in which we have made some modifications to allow forantibiotic selection and fluorescent reporter-based purifi-cation (Figure 2(a)) As expected Epithelial-MesenchymalTransition occurred during differentiation giving rise toa heterogeneous population (Figure 2(b)) However withenrichment of early mesenchymal-like cells we observedintermediate and highly CD73-dTomCD105-GFP express-ing cells populations (Figure 2(c)) We sorted highly express-ing GFPdTom positive cells and obtained a much morehomogenous population (Figure 2(d)) Stimulated by the firstdescription of iPSC-derived MSCs by Lian et al in 2010[15] many other groups tried to do direct and spontaneouslydifferentiating iPSCs into MSCs by various means We con-sider the use of lentiviral reporter and selection constructsas important tools to monitor the purity of a cell populationduring differentiation processes and to ensure a high grade ofhomogeneity within the final cell population Such selectionconstructs were recently introduced in other lineagesrsquo differ-entiation protocols [28] Thus in the present study a similarvector architecture was applied to select for CD73+CD105+positive iPSC-MSCs Interestingly we obtained a high num-ber of CD73posCD105intermediate iPSC-MSCs (R1 633)and a smaller fraction of CD73posCD105high iPSC-MSCs(R2 643) and we concluded that sorting the less abun-dant CD73posCD105high population might provide the mosthomogenous cell population
32 Immunophenotype Proliferation and Differentiation Po-tential of iPSC-MSCs In order to characterize the iPSC-MSCs according to the International Society of Cell Therapy
Stem Cells International 5
SSEA4 PE subset952
0
50
100
150 SSEA4 PE subset993
0
50
100
150SSEA4 PE subset997
0
100
200
300
FLF-iPSCs(OSKM)
FLF-iPSCs(OSNL)
MSC-iPSCs(OSKM)
DAPI
OCT-4
SOX-2
Merge
SSEA-4 SSEA-4 SSEA-4
Cou
nt
Cou
nt
Cou
nt
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
Figure 1 Generation and characterization iPS cells from human fetal liver fibroblasts (FLF) with Oct4 Sox2 Klf4 and c-Myc (OSKM) andOct4 Sox2 Nanog and Lin-28 (OSNL) and also from human bonemarrowMSCs with OSKMusing lentiviral vectors iPSCs stained positivefor humanOCT4 and SOX2DAPIwas used to stain the nuclei andmergedwith phase-contrast Expression of SSEA-4 is shown in histograms
(ISCT) criteria [29] cell surface marker expression was ana-lyzed by flow cytometry of all three iPSC-MSC lines and BM-MSCs at early passages (passages 3ndash6) All 3 differentiated andenriched iPSC-MSCs displayed a MSC-like antigen profilethat exhibited high CD105 CD73 and CD90 and absenceof CD34 CD45 and CD19 expression (Figure 3(a)) Thuswe were able to demonstrate that homogenous populationscan be isolated and purified from all three iPSC linesindependent to their somatic cell source (fibroblasts or bonemarrow MSCs) and method of reprogramming (OSKM or
OSNL factor cocktail) Strikingly the surface marker CD105and CD73 whose promoter motifs were utilized to expressthe fluorescent reporter transgenes and antibiotic selectioncassettes were readily detectable in almost 100 of purifiedcells indicating the high purity of our enriched iPSC-MSCsInterestingly CD90 was positive not only in all iPSC-MSCslines as well as the BM-MSCs but also in undifferenti-ated iPSCs (MSC-iPSCs) Furthermore the hematopoieticsurface markers were neither expressed in MSCs nor iniPSCs
6 Stem Cells International
iPSCs in MSCsDifferentiation
(Epithelial Transduction MSC-likemedia morphology) and sorting cells
418 (500120583gmL) and puromycin (4120583gmL) and then sortedIRES-dTom and selected with G
lowastAfter plating cells were transduced with CD-105-Puro-IRES-GFP and CD-73-Neo-
(c) (d)
Figure 2 Derivation and enrichment of MSCs from human iPS cells (a) Schematic stepwise protocol for differentiation and selection ofMSC-like cells from human iPS cells (b) phase-contrast photos demonstrating Epithelial-Mesenchymal transition in cellular morphology(c) FACS dot blot showing intermediate (R1) and highly (P2) double-positive cells Highly positive CD-73 and CD-105 (R2) were sorted forupcoming experiments (d) iPS-MSCs after sorting showed more homogenous mesenchymal morphology expressing GFPdTom
Growth kinetics of iPSC-MSCs demonstrated a greaterproliferative capacity when compared with BM-MSCs withshorter doubling times (Figure 3(b)) In our experimentsthree independently derived BM-MSCs exhibited doublingtimes around 36 h in early passages that were prolongedabove 60 h around passage 8 and followed by a cessation ofproliferation with an apparent senescent phenotype aroundpassage 10 (Figure 3(b)) In contrast all three iPSC-MSCsexhibited significantly shorter doubling times (around 20 hin early passages) The prolonged doubling time of morethan 60 h did not occur before passage 15 and even after 20passages iPSC-MSCs did not show a senescent phenotypeThese results are in line with previously reported data fromSanchez et al who showed that human embryonic stem cell-derived CD73+ and CD90+ MSCs had higher proliferationrate than BM-MSCs (ESCs-MSCssim18 doubling compared toBM-MSCssim5 doubling in 30 days of culture) but were similar
to umbilical cord derived MSCs (sim15 doubling in 30 days)[12] The more robust proliferation potential of iPSC-MSCssuggests an important advantage over BM-MSCs wheneverrepetitive transplantations of the very sameMSCbatchwouldbe most preferential (for review of this impact on age-relateddisorders see [30]) Although the dosing of MSCs perfusionis currently controversially discussed for different disor-ders one can assume that an increasing demand of MSCstransplantation may arise in certain disorders For examplemusculoskeletal injuries with high occurrence in seniors [31]may urge for engineered MSCs with higher proliferationcapabilities but same functional abilities as BM-MSCs ThusiPSC-MSCs may serve as ldquooff the shelf transplantrdquo whichcan be provided by bloodstem cell banking institutions andused for several degenerative diseases Moreover the higherhomogeneity of such well-proliferating nonsenescent iPSC-MSCs populations suggest a higher safety and efficacy profile
Stem Cells International 7
0
50
100
150
0
30
60
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120
0
30
60
90
120
0
30
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90
120
0
30
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120
0
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0
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150
0
50
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200
0
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150
0
50
100
150
0
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120
0
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0
100
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050
100150200250
0
30
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120
0
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0
30
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120
0
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0
30
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0
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0
20
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80
0
50
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150
0
20
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80
0
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0
30
60
90
120
0
30
60
90
120
0
50
100
150
200
0
10
20
30
05
10152025
MSC-iPSCs(OSKM)
hBM-MSCs(LM06)
FLF-iPSC-MSCs (OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs (OSKM)
CD73
CD10
5CD
90CD
45CD
34CD
19
10010
110
210
310
410
510
010
110
210
310
410
5 10010
110
210
310
410
510
010
110
210
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410
5 10010
110
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410
5
10010
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410
5
10010
110
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410
5
100 10
110
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310
410
5
10010
110
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410
5
10010
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410
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10010
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410
5 10010
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5 10010
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5
10010
110
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510010
110
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410
510010
110
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510010
110
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5
100 10
110
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5
10010
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5
10010
110
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510
010
110
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410
5
10010
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410
5
100 10
110
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410
510
0 10110
210
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510
0 10110
210
310
410
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10010
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5 10010
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5
10010
110
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310
410
5 10010
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5
10010
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410
5
(a)
Figure 3 Continued
8 Stem Cells International
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10
BM-MSC LM02BM-MSC LM05
BM-MSC LM06
Passages
Dou
blin
g tim
e (ho
urs)
Dou
blin
g tim
e (ho
urs)
Passages
01020304050607080
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
FLF-iPSC-MSCs (OSKM)FLF-iPSC-MSCs (OSNL)
MSC-iPS-MSCs (OSKM)
(b)
Figure 3 Antigen phenotype proliferation rate and functional characterization of hiPSC-MSCs (a) Immunophenotype of the three hiPS-MSCs lines generated Representative flow cytometry analysis of hBM-MSCs FLF-iPSC-MSCs (OSKM) FLF-iPSC-MSCs (OSNL) MSC-iPSC-MSCs (OSKM) and MSC-derived iPSC line MSC-related markers CD73 CD90 and CD105 and hematopoietic CD45 CD34 andCD19 were assessed (solid histogram) (b) In vitro cell growth measured as cumulative population of hiPSC-MSCs and BM-MSCs derivedfrom 3 different donors (LM02 LM05 and LM06)
andmay qualify such cells formore long-term treatment suchas inflammatory bowel diseases or during the prevention ofgraft versus host diseases or transplant rejection in futuretransplantation settings [12]
Addressing the functional capabilities of iPSC-derivedMSCs we applied differentiation protocols towards adipo-osteo- or chondrogenic lineages respectively and performedcytological staining andRT-PCR to investigate changes in cellmorphological and related marker gene expression Impor-tantly all 3 iPSC-MSCs could give rise to all of these threelineages However in comparison to BM-MSCs the qualityand morphology characteristics of the differentiated iPSC-MSCs exhibited slight differences All iPSC-MSCs were morereluctant to adipogenic differentiation and the respective totalnumbers of differentiated cells containing lipid droplets werelower than that of BM-MSCs (Figure 4) This observationwas confirmed with adipocytes specific mRNA level in whichexpression levels of PPAR-120572 and PPAR-120574 were significantlylower in iPSC-MSCs than BM-MSCs Also LPL expressionlevels were significantly (119901 le 005) lower in FLF-iPSC-MSCs (OKSM) and MSC-iPSC-MSCs (OKSM) compared toBM-MSCs On the other hand iPSC-MSCs had more affinity
to differentiate to osteogenic and chondrogenic lineagesGradually mineral nodules formation started 1 week earlierin iPSC-MSCs which were stained positive for alizarin red SThe expression of osteocalcin and alkaline phosphatase wascomparable to BM-MSCs Characterizing chondrogenesisthe respective pellets were stained more strongly with alcianblue and the respective gene expression profiles for collagenII showed higher expression in iPSC-MSCs compared toBM-MSCs However Aggrecan was similarly expressed inall three iPSC-MSCs and BM-MSCs It has been shown thatsomeMSCs for instance fromperiosteumand synoviumwereeasily capable of differentiating to bone and cartilage butonly a minor population amongst them could give rise toadipocytes [23 32] and we speculate that such a MSC-relatedphenotype is resembled by our iPSC-derived MSCs
33 Supportive Effects on Long-Term CD34+ Cells Mainte-nance Thebonemarrowniche plays a vital role in preservinghematopoietic progenitors to provide proper amounts ofblood cells throughout life [33 34]This activemicroenviron-ment is fostered by secreted factors of niche-accompanyingcells such asMSCs and sinusoidal endothelial cells to support
Figure 4 Adipogenic osteogenic and chondrogenic differentiation potential of hiPSC-MSCs and BM-MSC (LM06) Oil Red-O staining forlipid formation alizarin red staining of mineralized deposits and alcian blue staining for chondrocyte pellet formed by the three iPSC-MSC-like cell lines mRNA expression level of the relative expression of genes associated with adipogenesis PPAR120574 PPAR120572 and LPL osteogenesis(osteocalcin and alkaline phosphatase) and chondrogenesis (collagen type II and aggrecan) The data represent the mean expression valuesnormalized to the housekeeping gene GAPDH lowast significance difference with BM-MSCs 119901 le 005
the quiescent state of some of the hematopoietic progenitors[35 36] The supportive cellular microenvironment providedby MSCs regulates self-renewal versus differentiation ofhematopoietic stemprogenitor cells within the bone marrow[37 38] Moreover based on their secretion of cytokinessupportive for hematopoietic cell proliferation MSC areconsidered to serve as an excellent cell type for long-term
progenitor cell culture purposes [39] As further indicationfor the undisturbed functional capabilities of iPSC-MSCswe exploited a coculture system of iPSC-MSCs and CD34+hematopoietic stemprogenitor cells and investigated thetotal cell numbers the colony forming capacity and thehomogeneity of CD34+ cells After 10 days all three iPSC-MSC coculture assays contained significantly (119901 le 005)
10 Stem Cells International
0
100
200
300
400
500
600
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
Day 0Day 10
Day 20
Tota
l non
adhe
rent
via
ble c
ellstimes104
lowast lowastlowast lowast lowast
lowastlowast
(a)
0
20
40
60
80
100
120
140
160
180
Tota
l col
ony
form
ing
units
wel
l
Day 4Day 8
lowast lowastlowast lowast
lowast
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
(b)
0102030405060708090
100
Posit
ive c
ells
()
CD-34CD-45
CD-11bCD-14
CD-34withoutstroma
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
lowast
lowastlowast
lowast
lowast
(c)
Figure 5 Coculture of CD34+ PBMCs with human iPSC-MSCs (a) Cell layers of iPSC-MSCs and BMSCs were established on 1 gelatinprecoated 24-well plates (80 confluent) CD34+ PBSCs were applied onto the stromal layers The cocultures were incubated for 20days Nonadherent viable cells were counted at the indicated time points (b) human CD34+ were plated with hiPSC-MSCs in 05mL ofmethylcellulose media containing human recombinant IL-3 SCF and EpoThe plates were incubated for 20 days following which progenitorswere scored (c) surface markers expression on CD34+ cells after coculture with mesenchymal stromal cells The results represent the mean(plusmnSD) of three replicates lowast significance difference with CD34 without stroma 119901 le 005
more nonadherent cells compared to CD34+ cells culturedwithout any stroma Comparing coculture of CD34+ cellswith iPSC-MSCs andBM-MSCs we observed a robust (but inour experiments not significant) increase in nonadherent cellnumbers for the iPSC-MSCs assays (Figure 5(a)) For OSKMfactors-derived iPSC-MSCs (Figure 5(a)) similar results wereobtained even at day 20 After replating CD34+ cells inMethoCult media for colony forming assays we observedsignificantly increased colonies in all iPSC-MSCs and BM-MSCs lines after 4 days of coculture comparing to singleCD34+ culture Furthermore MethoCult culture for 8 daysresulted in significantly (119901 le 005) higher colony numbersin iPSC-MSCs and in 2 lines of BM-MSCs (Figure 5(b))This data demonstrates a further important functional aspectand is supported by prior investigations that indicated thesupportive nature of MSCs on hematopoiesis by providinga suitable microenvironment for stemprogenitor cells ingrowing sites [40] With our data we also provided evidencethat significantly higher CD34+ cell number maintain theirstem cell status on the iPSC-MSCs and BM-MSCs rather than
conventional hematopoietic medium (Figure 5(c)) Theseenhanced proliferation and boosted colony forming abilitiescould be observed after 8 days of coculture in all iPSC-MSCs lines suggesting that these represent a reliable cellsource supporting the long-term culture of hematopoieticstemprogenitor cells Several publications are in favor of theeffects of different feeder layers and coatings for maintenanceand expansion of progenitors and somatic cells showing theimportance of mimicking in vivo conditions and providingsimilar microenvironment [41ndash43]
34 Immunosuppressive Effects of iPSC-MSCs In pioneer-ing studies mesenchymal stem cell based approaches wereapplied for suppressing immune reactions in autoimmunedisorders graft versus host disease (GVHD) or after solidorgan transplantation (for review see [44 45]) Duringallogeneic cell or organ transplantation cytotoxic and helperT-lymphocytes get activated and kill the targeted cells orpromote rejection of the transplanted organ [46] BecauseMSCs can secrete anti-inflammatory molecules to dampen
Stem Cells International 11
0
02
04
06
08
1
12
14
16
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
BrdU
mea
n ab
sorb
ance
in104
cells
lowastlowast
(a)
0
500
1000
1500
2000
2500
3000
3500
IFN
-120574(p
gm
L)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowast
lowastlowast
lowast
(b)
0
50
100
150
200
250
IL-2
(pg
mL)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
(c)
0
5
10
15
20
25
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowastlowastCD
4+C
D69
+(
)
(d)
0
5
10
15
20
25
lowast lowast
lowastlowast
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
CD4+
CD
25+
()
(e)
Figure 6 Status of activatedCD4+ T cells in the presence of hiPSC-MSCs (a) IFN-120574were determined at 48 hours by ELISAThe values are themeans plusmn SD from 3 independent experiments (b) concentrations of IL-2 and (c) proliferation in MLRMSC cocultures MLR cultures wereset up in presence or absence of hiPSC-MSCs BrdU incorporation was significantly lower in MSC-ips-MSCs and FLFiPSC-MSCs (OSNL)in comparison to absence of MSCs (d) Expression of the T-cell activation markers CD69 and (e) CD25 on CD4+ 5 days after stimulation ina 12-well dish in the presence or absence of hiPSC-MSCs lowast significance difference with MLR 119901 le 005
12 Stem Cells International
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
[1] M T Sutton and T L Bonfield ldquoStem cells innovations inclinical applicationsrdquo Stem Cells International vol 2014 ArticleID 516278 9 pages 2014
[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
29 In Vitro Progenitor Assays Effects of human iPSC-MSCsor BM-MSCs on progenitor cells were analyzed using acolony forming cell assay Human bone marrow CD34+ cells(2 times 106) were obtained from Lonza and were plated in 2mLof methylcellulose media (STEMCELL Technologies) with orwithout iPSC-MSCs and BMSCs Colonies of gt50 cells werescored after 4 and 8 days of incubation
210 Assessment of CD4+ T-Lymphocyte ProliferationResponseto iPSC-MSCs Standard 5-day MLR cultures were set upwith 5 times 104 Mitomycin Cndashtreated (Sigma-Aldrich) humanperipheral blood mononuclear cells (PBMCs) as stimula-tors and 2 times 105 human CD4+ T-cells (Lonza) in 96-wellround-bottom plates in 200120583L complete medium consistingRPMI 1640 (Life Technologies) supplemented with 01mM120573-mercaptoethanol 10 FBS GLUTAMAX I (Life Tech-nologies) 100UmL penicillin and 100 120583gmL streptomycinin the presence or absence of iPSC-MSCs and BM-MSCsFor analyzing expression of CD69+ and CD25+ regulatoryT-cell population 106 responder cells were mixed with 25times 105 stimulator PMNCs in presence or absence of 2 times105 iPSC-MSCs or BM-MSCs MLRs were performed ona layer of confluent Mitomycine C treated MSCs seededone day before Proliferation was determined with BrdUELISA assay (Roche) based on manufacturer instruction IL-2 and IFN-120574 concentration was determined in MSCMLRcoculture supernatants using a commercially available ELISA(BD Bioscience) according to manufacturerrsquos instructionsCD25 and CD69 (BD Bioscience) expression on CD4+ cellswere analyzed by flow cytometry
3 Results and Discussion
31 Generation of MSCs from Human iPSCs with Spindle-Shape Morphology The challenge of accessing an appro-priate and homogenous source for MSCs with sustainedgrowth kinetics immunosuppressive potentials and produc-tion of chemokines or growth factors supporting endogenousregeneration led to the question whether homogenouslydifferentiated MSCs could be derived from human inducedpluripotent stem cells (iPSCs) To address this question weused two different sources of somatic cells liver fibroblastand bone marrow-derived MSCs (BM-MSCs) and repro-grammed these cells into iPSCs Besides two sources of
somatic starting cells we also compared two slightly dif-ferent composition of reprograming factor cocktails Onefactor combination was comprised of Oct4 Sox2 Klf4 andc-Myc (OSKM) as it was originally described by ShinyaYamanaka and subsequently in multitudinous publications[8ndash10] and the other combination consisted of Oct4 Sox2Nanog and Lin28 (OSNL) as it was described by JamesThomson and some further groups [7 25] Taken together wegenerated fetal liver fibroblast-derived iPSCswithOSKMandOSNL (FLF iPSCs) and bone marrow MSC-derived iPSCswith OSKM (MSC-iPSCs) which were strongly expressingOCT4 SOX2 and SSEA-4 (Figure 1) All iPSC lines weredifferentiated based on a previously reported differentiationprotocol resulting in about 70 CD73+CD105+ cells [14]in which we have made some modifications to allow forantibiotic selection and fluorescent reporter-based purifi-cation (Figure 2(a)) As expected Epithelial-MesenchymalTransition occurred during differentiation giving rise toa heterogeneous population (Figure 2(b)) However withenrichment of early mesenchymal-like cells we observedintermediate and highly CD73-dTomCD105-GFP express-ing cells populations (Figure 2(c)) We sorted highly express-ing GFPdTom positive cells and obtained a much morehomogenous population (Figure 2(d)) Stimulated by the firstdescription of iPSC-derived MSCs by Lian et al in 2010[15] many other groups tried to do direct and spontaneouslydifferentiating iPSCs into MSCs by various means We con-sider the use of lentiviral reporter and selection constructsas important tools to monitor the purity of a cell populationduring differentiation processes and to ensure a high grade ofhomogeneity within the final cell population Such selectionconstructs were recently introduced in other lineagesrsquo differ-entiation protocols [28] Thus in the present study a similarvector architecture was applied to select for CD73+CD105+positive iPSC-MSCs Interestingly we obtained a high num-ber of CD73posCD105intermediate iPSC-MSCs (R1 633)and a smaller fraction of CD73posCD105high iPSC-MSCs(R2 643) and we concluded that sorting the less abun-dant CD73posCD105high population might provide the mosthomogenous cell population
32 Immunophenotype Proliferation and Differentiation Po-tential of iPSC-MSCs In order to characterize the iPSC-MSCs according to the International Society of Cell Therapy
Stem Cells International 5
SSEA4 PE subset952
0
50
100
150 SSEA4 PE subset993
0
50
100
150SSEA4 PE subset997
0
100
200
300
FLF-iPSCs(OSKM)
FLF-iPSCs(OSNL)
MSC-iPSCs(OSKM)
DAPI
OCT-4
SOX-2
Merge
SSEA-4 SSEA-4 SSEA-4
Cou
nt
Cou
nt
Cou
nt
100
101
102
103
104
100
101
102
103
104
100
101
102
103
104
Figure 1 Generation and characterization iPS cells from human fetal liver fibroblasts (FLF) with Oct4 Sox2 Klf4 and c-Myc (OSKM) andOct4 Sox2 Nanog and Lin-28 (OSNL) and also from human bonemarrowMSCs with OSKMusing lentiviral vectors iPSCs stained positivefor humanOCT4 and SOX2DAPIwas used to stain the nuclei andmergedwith phase-contrast Expression of SSEA-4 is shown in histograms
(ISCT) criteria [29] cell surface marker expression was ana-lyzed by flow cytometry of all three iPSC-MSC lines and BM-MSCs at early passages (passages 3ndash6) All 3 differentiated andenriched iPSC-MSCs displayed a MSC-like antigen profilethat exhibited high CD105 CD73 and CD90 and absenceof CD34 CD45 and CD19 expression (Figure 3(a)) Thuswe were able to demonstrate that homogenous populationscan be isolated and purified from all three iPSC linesindependent to their somatic cell source (fibroblasts or bonemarrow MSCs) and method of reprogramming (OSKM or
OSNL factor cocktail) Strikingly the surface marker CD105and CD73 whose promoter motifs were utilized to expressthe fluorescent reporter transgenes and antibiotic selectioncassettes were readily detectable in almost 100 of purifiedcells indicating the high purity of our enriched iPSC-MSCsInterestingly CD90 was positive not only in all iPSC-MSCslines as well as the BM-MSCs but also in undifferenti-ated iPSCs (MSC-iPSCs) Furthermore the hematopoieticsurface markers were neither expressed in MSCs nor iniPSCs
6 Stem Cells International
iPSCs in MSCsDifferentiation
(Epithelial Transduction MSC-likemedia morphology) and sorting cells
418 (500120583gmL) and puromycin (4120583gmL) and then sortedIRES-dTom and selected with G
lowastAfter plating cells were transduced with CD-105-Puro-IRES-GFP and CD-73-Neo-
(c) (d)
Figure 2 Derivation and enrichment of MSCs from human iPS cells (a) Schematic stepwise protocol for differentiation and selection ofMSC-like cells from human iPS cells (b) phase-contrast photos demonstrating Epithelial-Mesenchymal transition in cellular morphology(c) FACS dot blot showing intermediate (R1) and highly (P2) double-positive cells Highly positive CD-73 and CD-105 (R2) were sorted forupcoming experiments (d) iPS-MSCs after sorting showed more homogenous mesenchymal morphology expressing GFPdTom
Growth kinetics of iPSC-MSCs demonstrated a greaterproliferative capacity when compared with BM-MSCs withshorter doubling times (Figure 3(b)) In our experimentsthree independently derived BM-MSCs exhibited doublingtimes around 36 h in early passages that were prolongedabove 60 h around passage 8 and followed by a cessation ofproliferation with an apparent senescent phenotype aroundpassage 10 (Figure 3(b)) In contrast all three iPSC-MSCsexhibited significantly shorter doubling times (around 20 hin early passages) The prolonged doubling time of morethan 60 h did not occur before passage 15 and even after 20passages iPSC-MSCs did not show a senescent phenotypeThese results are in line with previously reported data fromSanchez et al who showed that human embryonic stem cell-derived CD73+ and CD90+ MSCs had higher proliferationrate than BM-MSCs (ESCs-MSCssim18 doubling compared toBM-MSCssim5 doubling in 30 days of culture) but were similar
to umbilical cord derived MSCs (sim15 doubling in 30 days)[12] The more robust proliferation potential of iPSC-MSCssuggests an important advantage over BM-MSCs wheneverrepetitive transplantations of the very sameMSCbatchwouldbe most preferential (for review of this impact on age-relateddisorders see [30]) Although the dosing of MSCs perfusionis currently controversially discussed for different disor-ders one can assume that an increasing demand of MSCstransplantation may arise in certain disorders For examplemusculoskeletal injuries with high occurrence in seniors [31]may urge for engineered MSCs with higher proliferationcapabilities but same functional abilities as BM-MSCs ThusiPSC-MSCs may serve as ldquooff the shelf transplantrdquo whichcan be provided by bloodstem cell banking institutions andused for several degenerative diseases Moreover the higherhomogeneity of such well-proliferating nonsenescent iPSC-MSCs populations suggest a higher safety and efficacy profile
Stem Cells International 7
0
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30
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120
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050
100150200250
0
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150
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0
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60
90
120
0
30
60
90
120
0
50
100
150
200
0
10
20
30
05
10152025
MSC-iPSCs(OSKM)
hBM-MSCs(LM06)
FLF-iPSC-MSCs (OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs (OSKM)
CD73
CD10
5CD
90CD
45CD
34CD
19
10010
110
210
310
410
510
010
110
210
310
410
5 10010
110
210
310
410
510
010
110
210
310
410
5 10010
110
210
310
410
5
10010
110
210
310
410
5
10010
110
210
310
410
5
100 10
110
210
310
410
5
10010
110
210
310
410
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10010
110
210
310
410
5
10010
110
210
310
410
5 10010
110
210
310
410
5 10010
110
210
310
410
5
10010
110
210
310
410
510010
110
210
310
410
510010
110
210
310
410
510010
110
210
310
410
5
100 10
110
210
310
410
5
10010
110
210
310
410
5
10010
110
210
310
410
510
010
110
210
310
410
5
10010
110
210
310
410
5
100 10
110
210
310
410
510
0 10110
210
310
410
510
0 10110
210
310
410
5
10010
110
210
310
410
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210
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410
5
10010
110
210
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410
5 10010
110
210
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10010
110
210
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410
5
(a)
Figure 3 Continued
8 Stem Cells International
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10
BM-MSC LM02BM-MSC LM05
BM-MSC LM06
Passages
Dou
blin
g tim
e (ho
urs)
Dou
blin
g tim
e (ho
urs)
Passages
01020304050607080
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
FLF-iPSC-MSCs (OSKM)FLF-iPSC-MSCs (OSNL)
MSC-iPS-MSCs (OSKM)
(b)
Figure 3 Antigen phenotype proliferation rate and functional characterization of hiPSC-MSCs (a) Immunophenotype of the three hiPS-MSCs lines generated Representative flow cytometry analysis of hBM-MSCs FLF-iPSC-MSCs (OSKM) FLF-iPSC-MSCs (OSNL) MSC-iPSC-MSCs (OSKM) and MSC-derived iPSC line MSC-related markers CD73 CD90 and CD105 and hematopoietic CD45 CD34 andCD19 were assessed (solid histogram) (b) In vitro cell growth measured as cumulative population of hiPSC-MSCs and BM-MSCs derivedfrom 3 different donors (LM02 LM05 and LM06)
andmay qualify such cells formore long-term treatment suchas inflammatory bowel diseases or during the prevention ofgraft versus host diseases or transplant rejection in futuretransplantation settings [12]
Addressing the functional capabilities of iPSC-derivedMSCs we applied differentiation protocols towards adipo-osteo- or chondrogenic lineages respectively and performedcytological staining andRT-PCR to investigate changes in cellmorphological and related marker gene expression Impor-tantly all 3 iPSC-MSCs could give rise to all of these threelineages However in comparison to BM-MSCs the qualityand morphology characteristics of the differentiated iPSC-MSCs exhibited slight differences All iPSC-MSCs were morereluctant to adipogenic differentiation and the respective totalnumbers of differentiated cells containing lipid droplets werelower than that of BM-MSCs (Figure 4) This observationwas confirmed with adipocytes specific mRNA level in whichexpression levels of PPAR-120572 and PPAR-120574 were significantlylower in iPSC-MSCs than BM-MSCs Also LPL expressionlevels were significantly (119901 le 005) lower in FLF-iPSC-MSCs (OKSM) and MSC-iPSC-MSCs (OKSM) compared toBM-MSCs On the other hand iPSC-MSCs had more affinity
to differentiate to osteogenic and chondrogenic lineagesGradually mineral nodules formation started 1 week earlierin iPSC-MSCs which were stained positive for alizarin red SThe expression of osteocalcin and alkaline phosphatase wascomparable to BM-MSCs Characterizing chondrogenesisthe respective pellets were stained more strongly with alcianblue and the respective gene expression profiles for collagenII showed higher expression in iPSC-MSCs compared toBM-MSCs However Aggrecan was similarly expressed inall three iPSC-MSCs and BM-MSCs It has been shown thatsomeMSCs for instance fromperiosteumand synoviumwereeasily capable of differentiating to bone and cartilage butonly a minor population amongst them could give rise toadipocytes [23 32] and we speculate that such a MSC-relatedphenotype is resembled by our iPSC-derived MSCs
33 Supportive Effects on Long-Term CD34+ Cells Mainte-nance Thebonemarrowniche plays a vital role in preservinghematopoietic progenitors to provide proper amounts ofblood cells throughout life [33 34]This activemicroenviron-ment is fostered by secreted factors of niche-accompanyingcells such asMSCs and sinusoidal endothelial cells to support
Figure 4 Adipogenic osteogenic and chondrogenic differentiation potential of hiPSC-MSCs and BM-MSC (LM06) Oil Red-O staining forlipid formation alizarin red staining of mineralized deposits and alcian blue staining for chondrocyte pellet formed by the three iPSC-MSC-like cell lines mRNA expression level of the relative expression of genes associated with adipogenesis PPAR120574 PPAR120572 and LPL osteogenesis(osteocalcin and alkaline phosphatase) and chondrogenesis (collagen type II and aggrecan) The data represent the mean expression valuesnormalized to the housekeeping gene GAPDH lowast significance difference with BM-MSCs 119901 le 005
the quiescent state of some of the hematopoietic progenitors[35 36] The supportive cellular microenvironment providedby MSCs regulates self-renewal versus differentiation ofhematopoietic stemprogenitor cells within the bone marrow[37 38] Moreover based on their secretion of cytokinessupportive for hematopoietic cell proliferation MSC areconsidered to serve as an excellent cell type for long-term
progenitor cell culture purposes [39] As further indicationfor the undisturbed functional capabilities of iPSC-MSCswe exploited a coculture system of iPSC-MSCs and CD34+hematopoietic stemprogenitor cells and investigated thetotal cell numbers the colony forming capacity and thehomogeneity of CD34+ cells After 10 days all three iPSC-MSC coculture assays contained significantly (119901 le 005)
10 Stem Cells International
0
100
200
300
400
500
600
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
Day 0Day 10
Day 20
Tota
l non
adhe
rent
via
ble c
ellstimes104
lowast lowastlowast lowast lowast
lowastlowast
(a)
0
20
40
60
80
100
120
140
160
180
Tota
l col
ony
form
ing
units
wel
l
Day 4Day 8
lowast lowastlowast lowast
lowast
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
(b)
0102030405060708090
100
Posit
ive c
ells
()
CD-34CD-45
CD-11bCD-14
CD-34withoutstroma
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
lowast
lowastlowast
lowast
lowast
(c)
Figure 5 Coculture of CD34+ PBMCs with human iPSC-MSCs (a) Cell layers of iPSC-MSCs and BMSCs were established on 1 gelatinprecoated 24-well plates (80 confluent) CD34+ PBSCs were applied onto the stromal layers The cocultures were incubated for 20days Nonadherent viable cells were counted at the indicated time points (b) human CD34+ were plated with hiPSC-MSCs in 05mL ofmethylcellulose media containing human recombinant IL-3 SCF and EpoThe plates were incubated for 20 days following which progenitorswere scored (c) surface markers expression on CD34+ cells after coculture with mesenchymal stromal cells The results represent the mean(plusmnSD) of three replicates lowast significance difference with CD34 without stroma 119901 le 005
more nonadherent cells compared to CD34+ cells culturedwithout any stroma Comparing coculture of CD34+ cellswith iPSC-MSCs andBM-MSCs we observed a robust (but inour experiments not significant) increase in nonadherent cellnumbers for the iPSC-MSCs assays (Figure 5(a)) For OSKMfactors-derived iPSC-MSCs (Figure 5(a)) similar results wereobtained even at day 20 After replating CD34+ cells inMethoCult media for colony forming assays we observedsignificantly increased colonies in all iPSC-MSCs and BM-MSCs lines after 4 days of coculture comparing to singleCD34+ culture Furthermore MethoCult culture for 8 daysresulted in significantly (119901 le 005) higher colony numbersin iPSC-MSCs and in 2 lines of BM-MSCs (Figure 5(b))This data demonstrates a further important functional aspectand is supported by prior investigations that indicated thesupportive nature of MSCs on hematopoiesis by providinga suitable microenvironment for stemprogenitor cells ingrowing sites [40] With our data we also provided evidencethat significantly higher CD34+ cell number maintain theirstem cell status on the iPSC-MSCs and BM-MSCs rather than
conventional hematopoietic medium (Figure 5(c)) Theseenhanced proliferation and boosted colony forming abilitiescould be observed after 8 days of coculture in all iPSC-MSCs lines suggesting that these represent a reliable cellsource supporting the long-term culture of hematopoieticstemprogenitor cells Several publications are in favor of theeffects of different feeder layers and coatings for maintenanceand expansion of progenitors and somatic cells showing theimportance of mimicking in vivo conditions and providingsimilar microenvironment [41ndash43]
34 Immunosuppressive Effects of iPSC-MSCs In pioneer-ing studies mesenchymal stem cell based approaches wereapplied for suppressing immune reactions in autoimmunedisorders graft versus host disease (GVHD) or after solidorgan transplantation (for review see [44 45]) Duringallogeneic cell or organ transplantation cytotoxic and helperT-lymphocytes get activated and kill the targeted cells orpromote rejection of the transplanted organ [46] BecauseMSCs can secrete anti-inflammatory molecules to dampen
Stem Cells International 11
0
02
04
06
08
1
12
14
16
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
BrdU
mea
n ab
sorb
ance
in104
cells
lowastlowast
(a)
0
500
1000
1500
2000
2500
3000
3500
IFN
-120574(p
gm
L)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowast
lowastlowast
lowast
(b)
0
50
100
150
200
250
IL-2
(pg
mL)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
(c)
0
5
10
15
20
25
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowastlowastCD
4+C
D69
+(
)
(d)
0
5
10
15
20
25
lowast lowast
lowastlowast
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
CD4+
CD
25+
()
(e)
Figure 6 Status of activatedCD4+ T cells in the presence of hiPSC-MSCs (a) IFN-120574were determined at 48 hours by ELISAThe values are themeans plusmn SD from 3 independent experiments (b) concentrations of IL-2 and (c) proliferation in MLRMSC cocultures MLR cultures wereset up in presence or absence of hiPSC-MSCs BrdU incorporation was significantly lower in MSC-ips-MSCs and FLFiPSC-MSCs (OSNL)in comparison to absence of MSCs (d) Expression of the T-cell activation markers CD69 and (e) CD25 on CD4+ 5 days after stimulation ina 12-well dish in the presence or absence of hiPSC-MSCs lowast significance difference with MLR 119901 le 005
12 Stem Cells International
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
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[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
Figure 1 Generation and characterization iPS cells from human fetal liver fibroblasts (FLF) with Oct4 Sox2 Klf4 and c-Myc (OSKM) andOct4 Sox2 Nanog and Lin-28 (OSNL) and also from human bonemarrowMSCs with OSKMusing lentiviral vectors iPSCs stained positivefor humanOCT4 and SOX2DAPIwas used to stain the nuclei andmergedwith phase-contrast Expression of SSEA-4 is shown in histograms
(ISCT) criteria [29] cell surface marker expression was ana-lyzed by flow cytometry of all three iPSC-MSC lines and BM-MSCs at early passages (passages 3ndash6) All 3 differentiated andenriched iPSC-MSCs displayed a MSC-like antigen profilethat exhibited high CD105 CD73 and CD90 and absenceof CD34 CD45 and CD19 expression (Figure 3(a)) Thuswe were able to demonstrate that homogenous populationscan be isolated and purified from all three iPSC linesindependent to their somatic cell source (fibroblasts or bonemarrow MSCs) and method of reprogramming (OSKM or
OSNL factor cocktail) Strikingly the surface marker CD105and CD73 whose promoter motifs were utilized to expressthe fluorescent reporter transgenes and antibiotic selectioncassettes were readily detectable in almost 100 of purifiedcells indicating the high purity of our enriched iPSC-MSCsInterestingly CD90 was positive not only in all iPSC-MSCslines as well as the BM-MSCs but also in undifferenti-ated iPSCs (MSC-iPSCs) Furthermore the hematopoieticsurface markers were neither expressed in MSCs nor iniPSCs
6 Stem Cells International
iPSCs in MSCsDifferentiation
(Epithelial Transduction MSC-likemedia morphology) and sorting cells
418 (500120583gmL) and puromycin (4120583gmL) and then sortedIRES-dTom and selected with G
lowastAfter plating cells were transduced with CD-105-Puro-IRES-GFP and CD-73-Neo-
(c) (d)
Figure 2 Derivation and enrichment of MSCs from human iPS cells (a) Schematic stepwise protocol for differentiation and selection ofMSC-like cells from human iPS cells (b) phase-contrast photos demonstrating Epithelial-Mesenchymal transition in cellular morphology(c) FACS dot blot showing intermediate (R1) and highly (P2) double-positive cells Highly positive CD-73 and CD-105 (R2) were sorted forupcoming experiments (d) iPS-MSCs after sorting showed more homogenous mesenchymal morphology expressing GFPdTom
Growth kinetics of iPSC-MSCs demonstrated a greaterproliferative capacity when compared with BM-MSCs withshorter doubling times (Figure 3(b)) In our experimentsthree independently derived BM-MSCs exhibited doublingtimes around 36 h in early passages that were prolongedabove 60 h around passage 8 and followed by a cessation ofproliferation with an apparent senescent phenotype aroundpassage 10 (Figure 3(b)) In contrast all three iPSC-MSCsexhibited significantly shorter doubling times (around 20 hin early passages) The prolonged doubling time of morethan 60 h did not occur before passage 15 and even after 20passages iPSC-MSCs did not show a senescent phenotypeThese results are in line with previously reported data fromSanchez et al who showed that human embryonic stem cell-derived CD73+ and CD90+ MSCs had higher proliferationrate than BM-MSCs (ESCs-MSCssim18 doubling compared toBM-MSCssim5 doubling in 30 days of culture) but were similar
to umbilical cord derived MSCs (sim15 doubling in 30 days)[12] The more robust proliferation potential of iPSC-MSCssuggests an important advantage over BM-MSCs wheneverrepetitive transplantations of the very sameMSCbatchwouldbe most preferential (for review of this impact on age-relateddisorders see [30]) Although the dosing of MSCs perfusionis currently controversially discussed for different disor-ders one can assume that an increasing demand of MSCstransplantation may arise in certain disorders For examplemusculoskeletal injuries with high occurrence in seniors [31]may urge for engineered MSCs with higher proliferationcapabilities but same functional abilities as BM-MSCs ThusiPSC-MSCs may serve as ldquooff the shelf transplantrdquo whichcan be provided by bloodstem cell banking institutions andused for several degenerative diseases Moreover the higherhomogeneity of such well-proliferating nonsenescent iPSC-MSCs populations suggest a higher safety and efficacy profile
Stem Cells International 7
0
50
100
150
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
50
100
150
0
100
200
300
0
50
100
150
0
50
100
150
200
0
50
100
150
0
50
100
150
0
30
60
90
120
0
100
200
300
0
100
200
300
050
100150200250
0
30
60
90
120
0
50
100
150
0
30
60
90
120
0
50
100
150
0
30
60
90
120
0
50
100
150
200
0
20
40
60
80
0
50
100
150
0
20
40
60
80
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
50
100
150
200
0
10
20
30
05
10152025
MSC-iPSCs(OSKM)
hBM-MSCs(LM06)
FLF-iPSC-MSCs (OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs (OSKM)
CD73
CD10
5CD
90CD
45CD
34CD
19
10010
110
210
310
410
510
010
110
210
310
410
5 10010
110
210
310
410
510
010
110
210
310
410
5 10010
110
210
310
410
5
10010
110
210
310
410
5
10010
110
210
310
410
5
100 10
110
210
310
410
5
10010
110
210
310
410
5
10010
110
210
310
410
5
10010
110
210
310
410
5 10010
110
210
310
410
5 10010
110
210
310
410
5
10010
110
210
310
410
510010
110
210
310
410
510010
110
210
310
410
510010
110
210
310
410
5
100 10
110
210
310
410
5
10010
110
210
310
410
5
10010
110
210
310
410
510
010
110
210
310
410
5
10010
110
210
310
410
5
100 10
110
210
310
410
510
0 10110
210
310
410
510
0 10110
210
310
410
5
10010
110
210
310
410
5 10010
110
210
310
410
5
10010
110
210
310
410
5 10010
110
210
310
410
5
10010
110
210
310
410
5
(a)
Figure 3 Continued
8 Stem Cells International
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10
BM-MSC LM02BM-MSC LM05
BM-MSC LM06
Passages
Dou
blin
g tim
e (ho
urs)
Dou
blin
g tim
e (ho
urs)
Passages
01020304050607080
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
FLF-iPSC-MSCs (OSKM)FLF-iPSC-MSCs (OSNL)
MSC-iPS-MSCs (OSKM)
(b)
Figure 3 Antigen phenotype proliferation rate and functional characterization of hiPSC-MSCs (a) Immunophenotype of the three hiPS-MSCs lines generated Representative flow cytometry analysis of hBM-MSCs FLF-iPSC-MSCs (OSKM) FLF-iPSC-MSCs (OSNL) MSC-iPSC-MSCs (OSKM) and MSC-derived iPSC line MSC-related markers CD73 CD90 and CD105 and hematopoietic CD45 CD34 andCD19 were assessed (solid histogram) (b) In vitro cell growth measured as cumulative population of hiPSC-MSCs and BM-MSCs derivedfrom 3 different donors (LM02 LM05 and LM06)
andmay qualify such cells formore long-term treatment suchas inflammatory bowel diseases or during the prevention ofgraft versus host diseases or transplant rejection in futuretransplantation settings [12]
Addressing the functional capabilities of iPSC-derivedMSCs we applied differentiation protocols towards adipo-osteo- or chondrogenic lineages respectively and performedcytological staining andRT-PCR to investigate changes in cellmorphological and related marker gene expression Impor-tantly all 3 iPSC-MSCs could give rise to all of these threelineages However in comparison to BM-MSCs the qualityand morphology characteristics of the differentiated iPSC-MSCs exhibited slight differences All iPSC-MSCs were morereluctant to adipogenic differentiation and the respective totalnumbers of differentiated cells containing lipid droplets werelower than that of BM-MSCs (Figure 4) This observationwas confirmed with adipocytes specific mRNA level in whichexpression levels of PPAR-120572 and PPAR-120574 were significantlylower in iPSC-MSCs than BM-MSCs Also LPL expressionlevels were significantly (119901 le 005) lower in FLF-iPSC-MSCs (OKSM) and MSC-iPSC-MSCs (OKSM) compared toBM-MSCs On the other hand iPSC-MSCs had more affinity
to differentiate to osteogenic and chondrogenic lineagesGradually mineral nodules formation started 1 week earlierin iPSC-MSCs which were stained positive for alizarin red SThe expression of osteocalcin and alkaline phosphatase wascomparable to BM-MSCs Characterizing chondrogenesisthe respective pellets were stained more strongly with alcianblue and the respective gene expression profiles for collagenII showed higher expression in iPSC-MSCs compared toBM-MSCs However Aggrecan was similarly expressed inall three iPSC-MSCs and BM-MSCs It has been shown thatsomeMSCs for instance fromperiosteumand synoviumwereeasily capable of differentiating to bone and cartilage butonly a minor population amongst them could give rise toadipocytes [23 32] and we speculate that such a MSC-relatedphenotype is resembled by our iPSC-derived MSCs
33 Supportive Effects on Long-Term CD34+ Cells Mainte-nance Thebonemarrowniche plays a vital role in preservinghematopoietic progenitors to provide proper amounts ofblood cells throughout life [33 34]This activemicroenviron-ment is fostered by secreted factors of niche-accompanyingcells such asMSCs and sinusoidal endothelial cells to support
Figure 4 Adipogenic osteogenic and chondrogenic differentiation potential of hiPSC-MSCs and BM-MSC (LM06) Oil Red-O staining forlipid formation alizarin red staining of mineralized deposits and alcian blue staining for chondrocyte pellet formed by the three iPSC-MSC-like cell lines mRNA expression level of the relative expression of genes associated with adipogenesis PPAR120574 PPAR120572 and LPL osteogenesis(osteocalcin and alkaline phosphatase) and chondrogenesis (collagen type II and aggrecan) The data represent the mean expression valuesnormalized to the housekeeping gene GAPDH lowast significance difference with BM-MSCs 119901 le 005
the quiescent state of some of the hematopoietic progenitors[35 36] The supportive cellular microenvironment providedby MSCs regulates self-renewal versus differentiation ofhematopoietic stemprogenitor cells within the bone marrow[37 38] Moreover based on their secretion of cytokinessupportive for hematopoietic cell proliferation MSC areconsidered to serve as an excellent cell type for long-term
progenitor cell culture purposes [39] As further indicationfor the undisturbed functional capabilities of iPSC-MSCswe exploited a coculture system of iPSC-MSCs and CD34+hematopoietic stemprogenitor cells and investigated thetotal cell numbers the colony forming capacity and thehomogeneity of CD34+ cells After 10 days all three iPSC-MSC coculture assays contained significantly (119901 le 005)
10 Stem Cells International
0
100
200
300
400
500
600
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
Day 0Day 10
Day 20
Tota
l non
adhe
rent
via
ble c
ellstimes104
lowast lowastlowast lowast lowast
lowastlowast
(a)
0
20
40
60
80
100
120
140
160
180
Tota
l col
ony
form
ing
units
wel
l
Day 4Day 8
lowast lowastlowast lowast
lowast
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
(b)
0102030405060708090
100
Posit
ive c
ells
()
CD-34CD-45
CD-11bCD-14
CD-34withoutstroma
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
lowast
lowastlowast
lowast
lowast
(c)
Figure 5 Coculture of CD34+ PBMCs with human iPSC-MSCs (a) Cell layers of iPSC-MSCs and BMSCs were established on 1 gelatinprecoated 24-well plates (80 confluent) CD34+ PBSCs were applied onto the stromal layers The cocultures were incubated for 20days Nonadherent viable cells were counted at the indicated time points (b) human CD34+ were plated with hiPSC-MSCs in 05mL ofmethylcellulose media containing human recombinant IL-3 SCF and EpoThe plates were incubated for 20 days following which progenitorswere scored (c) surface markers expression on CD34+ cells after coculture with mesenchymal stromal cells The results represent the mean(plusmnSD) of three replicates lowast significance difference with CD34 without stroma 119901 le 005
more nonadherent cells compared to CD34+ cells culturedwithout any stroma Comparing coculture of CD34+ cellswith iPSC-MSCs andBM-MSCs we observed a robust (but inour experiments not significant) increase in nonadherent cellnumbers for the iPSC-MSCs assays (Figure 5(a)) For OSKMfactors-derived iPSC-MSCs (Figure 5(a)) similar results wereobtained even at day 20 After replating CD34+ cells inMethoCult media for colony forming assays we observedsignificantly increased colonies in all iPSC-MSCs and BM-MSCs lines after 4 days of coculture comparing to singleCD34+ culture Furthermore MethoCult culture for 8 daysresulted in significantly (119901 le 005) higher colony numbersin iPSC-MSCs and in 2 lines of BM-MSCs (Figure 5(b))This data demonstrates a further important functional aspectand is supported by prior investigations that indicated thesupportive nature of MSCs on hematopoiesis by providinga suitable microenvironment for stemprogenitor cells ingrowing sites [40] With our data we also provided evidencethat significantly higher CD34+ cell number maintain theirstem cell status on the iPSC-MSCs and BM-MSCs rather than
conventional hematopoietic medium (Figure 5(c)) Theseenhanced proliferation and boosted colony forming abilitiescould be observed after 8 days of coculture in all iPSC-MSCs lines suggesting that these represent a reliable cellsource supporting the long-term culture of hematopoieticstemprogenitor cells Several publications are in favor of theeffects of different feeder layers and coatings for maintenanceand expansion of progenitors and somatic cells showing theimportance of mimicking in vivo conditions and providingsimilar microenvironment [41ndash43]
34 Immunosuppressive Effects of iPSC-MSCs In pioneer-ing studies mesenchymal stem cell based approaches wereapplied for suppressing immune reactions in autoimmunedisorders graft versus host disease (GVHD) or after solidorgan transplantation (for review see [44 45]) Duringallogeneic cell or organ transplantation cytotoxic and helperT-lymphocytes get activated and kill the targeted cells orpromote rejection of the transplanted organ [46] BecauseMSCs can secrete anti-inflammatory molecules to dampen
Stem Cells International 11
0
02
04
06
08
1
12
14
16
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
BrdU
mea
n ab
sorb
ance
in104
cells
lowastlowast
(a)
0
500
1000
1500
2000
2500
3000
3500
IFN
-120574(p
gm
L)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowast
lowastlowast
lowast
(b)
0
50
100
150
200
250
IL-2
(pg
mL)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
(c)
0
5
10
15
20
25
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowastlowastCD
4+C
D69
+(
)
(d)
0
5
10
15
20
25
lowast lowast
lowastlowast
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
CD4+
CD
25+
()
(e)
Figure 6 Status of activatedCD4+ T cells in the presence of hiPSC-MSCs (a) IFN-120574were determined at 48 hours by ELISAThe values are themeans plusmn SD from 3 independent experiments (b) concentrations of IL-2 and (c) proliferation in MLRMSC cocultures MLR cultures wereset up in presence or absence of hiPSC-MSCs BrdU incorporation was significantly lower in MSC-ips-MSCs and FLFiPSC-MSCs (OSNL)in comparison to absence of MSCs (d) Expression of the T-cell activation markers CD69 and (e) CD25 on CD4+ 5 days after stimulation ina 12-well dish in the presence or absence of hiPSC-MSCs lowast significance difference with MLR 119901 le 005
12 Stem Cells International
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
[1] M T Sutton and T L Bonfield ldquoStem cells innovations inclinical applicationsrdquo Stem Cells International vol 2014 ArticleID 516278 9 pages 2014
[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
418 (500120583gmL) and puromycin (4120583gmL) and then sortedIRES-dTom and selected with G
lowastAfter plating cells were transduced with CD-105-Puro-IRES-GFP and CD-73-Neo-
(c) (d)
Figure 2 Derivation and enrichment of MSCs from human iPS cells (a) Schematic stepwise protocol for differentiation and selection ofMSC-like cells from human iPS cells (b) phase-contrast photos demonstrating Epithelial-Mesenchymal transition in cellular morphology(c) FACS dot blot showing intermediate (R1) and highly (P2) double-positive cells Highly positive CD-73 and CD-105 (R2) were sorted forupcoming experiments (d) iPS-MSCs after sorting showed more homogenous mesenchymal morphology expressing GFPdTom
Growth kinetics of iPSC-MSCs demonstrated a greaterproliferative capacity when compared with BM-MSCs withshorter doubling times (Figure 3(b)) In our experimentsthree independently derived BM-MSCs exhibited doublingtimes around 36 h in early passages that were prolongedabove 60 h around passage 8 and followed by a cessation ofproliferation with an apparent senescent phenotype aroundpassage 10 (Figure 3(b)) In contrast all three iPSC-MSCsexhibited significantly shorter doubling times (around 20 hin early passages) The prolonged doubling time of morethan 60 h did not occur before passage 15 and even after 20passages iPSC-MSCs did not show a senescent phenotypeThese results are in line with previously reported data fromSanchez et al who showed that human embryonic stem cell-derived CD73+ and CD90+ MSCs had higher proliferationrate than BM-MSCs (ESCs-MSCssim18 doubling compared toBM-MSCssim5 doubling in 30 days of culture) but were similar
to umbilical cord derived MSCs (sim15 doubling in 30 days)[12] The more robust proliferation potential of iPSC-MSCssuggests an important advantage over BM-MSCs wheneverrepetitive transplantations of the very sameMSCbatchwouldbe most preferential (for review of this impact on age-relateddisorders see [30]) Although the dosing of MSCs perfusionis currently controversially discussed for different disor-ders one can assume that an increasing demand of MSCstransplantation may arise in certain disorders For examplemusculoskeletal injuries with high occurrence in seniors [31]may urge for engineered MSCs with higher proliferationcapabilities but same functional abilities as BM-MSCs ThusiPSC-MSCs may serve as ldquooff the shelf transplantrdquo whichcan be provided by bloodstem cell banking institutions andused for several degenerative diseases Moreover the higherhomogeneity of such well-proliferating nonsenescent iPSC-MSCs populations suggest a higher safety and efficacy profile
Stem Cells International 7
0
50
100
150
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
50
100
150
0
100
200
300
0
50
100
150
0
50
100
150
200
0
50
100
150
0
50
100
150
0
30
60
90
120
0
100
200
300
0
100
200
300
050
100150200250
0
30
60
90
120
0
50
100
150
0
30
60
90
120
0
50
100
150
0
30
60
90
120
0
50
100
150
200
0
20
40
60
80
0
50
100
150
0
20
40
60
80
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
50
100
150
200
0
10
20
30
05
10152025
MSC-iPSCs(OSKM)
hBM-MSCs(LM06)
FLF-iPSC-MSCs (OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs (OSKM)
CD73
CD10
5CD
90CD
45CD
34CD
19
10010
110
210
310
410
510
010
110
210
310
410
5 10010
110
210
310
410
510
010
110
210
310
410
5 10010
110
210
310
410
5
10010
110
210
310
410
5
10010
110
210
310
410
5
100 10
110
210
310
410
5
10010
110
210
310
410
5
10010
110
210
310
410
5
10010
110
210
310
410
5 10010
110
210
310
410
5 10010
110
210
310
410
5
10010
110
210
310
410
510010
110
210
310
410
510010
110
210
310
410
510010
110
210
310
410
5
100 10
110
210
310
410
5
10010
110
210
310
410
5
10010
110
210
310
410
510
010
110
210
310
410
5
10010
110
210
310
410
5
100 10
110
210
310
410
510
0 10110
210
310
410
510
0 10110
210
310
410
5
10010
110
210
310
410
5 10010
110
210
310
410
5
10010
110
210
310
410
5 10010
110
210
310
410
5
10010
110
210
310
410
5
(a)
Figure 3 Continued
8 Stem Cells International
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10
BM-MSC LM02BM-MSC LM05
BM-MSC LM06
Passages
Dou
blin
g tim
e (ho
urs)
Dou
blin
g tim
e (ho
urs)
Passages
01020304050607080
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
FLF-iPSC-MSCs (OSKM)FLF-iPSC-MSCs (OSNL)
MSC-iPS-MSCs (OSKM)
(b)
Figure 3 Antigen phenotype proliferation rate and functional characterization of hiPSC-MSCs (a) Immunophenotype of the three hiPS-MSCs lines generated Representative flow cytometry analysis of hBM-MSCs FLF-iPSC-MSCs (OSKM) FLF-iPSC-MSCs (OSNL) MSC-iPSC-MSCs (OSKM) and MSC-derived iPSC line MSC-related markers CD73 CD90 and CD105 and hematopoietic CD45 CD34 andCD19 were assessed (solid histogram) (b) In vitro cell growth measured as cumulative population of hiPSC-MSCs and BM-MSCs derivedfrom 3 different donors (LM02 LM05 and LM06)
andmay qualify such cells formore long-term treatment suchas inflammatory bowel diseases or during the prevention ofgraft versus host diseases or transplant rejection in futuretransplantation settings [12]
Addressing the functional capabilities of iPSC-derivedMSCs we applied differentiation protocols towards adipo-osteo- or chondrogenic lineages respectively and performedcytological staining andRT-PCR to investigate changes in cellmorphological and related marker gene expression Impor-tantly all 3 iPSC-MSCs could give rise to all of these threelineages However in comparison to BM-MSCs the qualityand morphology characteristics of the differentiated iPSC-MSCs exhibited slight differences All iPSC-MSCs were morereluctant to adipogenic differentiation and the respective totalnumbers of differentiated cells containing lipid droplets werelower than that of BM-MSCs (Figure 4) This observationwas confirmed with adipocytes specific mRNA level in whichexpression levels of PPAR-120572 and PPAR-120574 were significantlylower in iPSC-MSCs than BM-MSCs Also LPL expressionlevels were significantly (119901 le 005) lower in FLF-iPSC-MSCs (OKSM) and MSC-iPSC-MSCs (OKSM) compared toBM-MSCs On the other hand iPSC-MSCs had more affinity
to differentiate to osteogenic and chondrogenic lineagesGradually mineral nodules formation started 1 week earlierin iPSC-MSCs which were stained positive for alizarin red SThe expression of osteocalcin and alkaline phosphatase wascomparable to BM-MSCs Characterizing chondrogenesisthe respective pellets were stained more strongly with alcianblue and the respective gene expression profiles for collagenII showed higher expression in iPSC-MSCs compared toBM-MSCs However Aggrecan was similarly expressed inall three iPSC-MSCs and BM-MSCs It has been shown thatsomeMSCs for instance fromperiosteumand synoviumwereeasily capable of differentiating to bone and cartilage butonly a minor population amongst them could give rise toadipocytes [23 32] and we speculate that such a MSC-relatedphenotype is resembled by our iPSC-derived MSCs
33 Supportive Effects on Long-Term CD34+ Cells Mainte-nance Thebonemarrowniche plays a vital role in preservinghematopoietic progenitors to provide proper amounts ofblood cells throughout life [33 34]This activemicroenviron-ment is fostered by secreted factors of niche-accompanyingcells such asMSCs and sinusoidal endothelial cells to support
Figure 4 Adipogenic osteogenic and chondrogenic differentiation potential of hiPSC-MSCs and BM-MSC (LM06) Oil Red-O staining forlipid formation alizarin red staining of mineralized deposits and alcian blue staining for chondrocyte pellet formed by the three iPSC-MSC-like cell lines mRNA expression level of the relative expression of genes associated with adipogenesis PPAR120574 PPAR120572 and LPL osteogenesis(osteocalcin and alkaline phosphatase) and chondrogenesis (collagen type II and aggrecan) The data represent the mean expression valuesnormalized to the housekeeping gene GAPDH lowast significance difference with BM-MSCs 119901 le 005
the quiescent state of some of the hematopoietic progenitors[35 36] The supportive cellular microenvironment providedby MSCs regulates self-renewal versus differentiation ofhematopoietic stemprogenitor cells within the bone marrow[37 38] Moreover based on their secretion of cytokinessupportive for hematopoietic cell proliferation MSC areconsidered to serve as an excellent cell type for long-term
progenitor cell culture purposes [39] As further indicationfor the undisturbed functional capabilities of iPSC-MSCswe exploited a coculture system of iPSC-MSCs and CD34+hematopoietic stemprogenitor cells and investigated thetotal cell numbers the colony forming capacity and thehomogeneity of CD34+ cells After 10 days all three iPSC-MSC coculture assays contained significantly (119901 le 005)
10 Stem Cells International
0
100
200
300
400
500
600
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
Day 0Day 10
Day 20
Tota
l non
adhe
rent
via
ble c
ellstimes104
lowast lowastlowast lowast lowast
lowastlowast
(a)
0
20
40
60
80
100
120
140
160
180
Tota
l col
ony
form
ing
units
wel
l
Day 4Day 8
lowast lowastlowast lowast
lowast
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
(b)
0102030405060708090
100
Posit
ive c
ells
()
CD-34CD-45
CD-11bCD-14
CD-34withoutstroma
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
lowast
lowastlowast
lowast
lowast
(c)
Figure 5 Coculture of CD34+ PBMCs with human iPSC-MSCs (a) Cell layers of iPSC-MSCs and BMSCs were established on 1 gelatinprecoated 24-well plates (80 confluent) CD34+ PBSCs were applied onto the stromal layers The cocultures were incubated for 20days Nonadherent viable cells were counted at the indicated time points (b) human CD34+ were plated with hiPSC-MSCs in 05mL ofmethylcellulose media containing human recombinant IL-3 SCF and EpoThe plates were incubated for 20 days following which progenitorswere scored (c) surface markers expression on CD34+ cells after coculture with mesenchymal stromal cells The results represent the mean(plusmnSD) of three replicates lowast significance difference with CD34 without stroma 119901 le 005
more nonadherent cells compared to CD34+ cells culturedwithout any stroma Comparing coculture of CD34+ cellswith iPSC-MSCs andBM-MSCs we observed a robust (but inour experiments not significant) increase in nonadherent cellnumbers for the iPSC-MSCs assays (Figure 5(a)) For OSKMfactors-derived iPSC-MSCs (Figure 5(a)) similar results wereobtained even at day 20 After replating CD34+ cells inMethoCult media for colony forming assays we observedsignificantly increased colonies in all iPSC-MSCs and BM-MSCs lines after 4 days of coculture comparing to singleCD34+ culture Furthermore MethoCult culture for 8 daysresulted in significantly (119901 le 005) higher colony numbersin iPSC-MSCs and in 2 lines of BM-MSCs (Figure 5(b))This data demonstrates a further important functional aspectand is supported by prior investigations that indicated thesupportive nature of MSCs on hematopoiesis by providinga suitable microenvironment for stemprogenitor cells ingrowing sites [40] With our data we also provided evidencethat significantly higher CD34+ cell number maintain theirstem cell status on the iPSC-MSCs and BM-MSCs rather than
conventional hematopoietic medium (Figure 5(c)) Theseenhanced proliferation and boosted colony forming abilitiescould be observed after 8 days of coculture in all iPSC-MSCs lines suggesting that these represent a reliable cellsource supporting the long-term culture of hematopoieticstemprogenitor cells Several publications are in favor of theeffects of different feeder layers and coatings for maintenanceand expansion of progenitors and somatic cells showing theimportance of mimicking in vivo conditions and providingsimilar microenvironment [41ndash43]
34 Immunosuppressive Effects of iPSC-MSCs In pioneer-ing studies mesenchymal stem cell based approaches wereapplied for suppressing immune reactions in autoimmunedisorders graft versus host disease (GVHD) or after solidorgan transplantation (for review see [44 45]) Duringallogeneic cell or organ transplantation cytotoxic and helperT-lymphocytes get activated and kill the targeted cells orpromote rejection of the transplanted organ [46] BecauseMSCs can secrete anti-inflammatory molecules to dampen
Stem Cells International 11
0
02
04
06
08
1
12
14
16
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
BrdU
mea
n ab
sorb
ance
in104
cells
lowastlowast
(a)
0
500
1000
1500
2000
2500
3000
3500
IFN
-120574(p
gm
L)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowast
lowastlowast
lowast
(b)
0
50
100
150
200
250
IL-2
(pg
mL)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
(c)
0
5
10
15
20
25
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowastlowastCD
4+C
D69
+(
)
(d)
0
5
10
15
20
25
lowast lowast
lowastlowast
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
CD4+
CD
25+
()
(e)
Figure 6 Status of activatedCD4+ T cells in the presence of hiPSC-MSCs (a) IFN-120574were determined at 48 hours by ELISAThe values are themeans plusmn SD from 3 independent experiments (b) concentrations of IL-2 and (c) proliferation in MLRMSC cocultures MLR cultures wereset up in presence or absence of hiPSC-MSCs BrdU incorporation was significantly lower in MSC-ips-MSCs and FLFiPSC-MSCs (OSNL)in comparison to absence of MSCs (d) Expression of the T-cell activation markers CD69 and (e) CD25 on CD4+ 5 days after stimulation ina 12-well dish in the presence or absence of hiPSC-MSCs lowast significance difference with MLR 119901 le 005
12 Stem Cells International
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
[1] M T Sutton and T L Bonfield ldquoStem cells innovations inclinical applicationsrdquo Stem Cells International vol 2014 ArticleID 516278 9 pages 2014
[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
Figure 3 Antigen phenotype proliferation rate and functional characterization of hiPSC-MSCs (a) Immunophenotype of the three hiPS-MSCs lines generated Representative flow cytometry analysis of hBM-MSCs FLF-iPSC-MSCs (OSKM) FLF-iPSC-MSCs (OSNL) MSC-iPSC-MSCs (OSKM) and MSC-derived iPSC line MSC-related markers CD73 CD90 and CD105 and hematopoietic CD45 CD34 andCD19 were assessed (solid histogram) (b) In vitro cell growth measured as cumulative population of hiPSC-MSCs and BM-MSCs derivedfrom 3 different donors (LM02 LM05 and LM06)
andmay qualify such cells formore long-term treatment suchas inflammatory bowel diseases or during the prevention ofgraft versus host diseases or transplant rejection in futuretransplantation settings [12]
Addressing the functional capabilities of iPSC-derivedMSCs we applied differentiation protocols towards adipo-osteo- or chondrogenic lineages respectively and performedcytological staining andRT-PCR to investigate changes in cellmorphological and related marker gene expression Impor-tantly all 3 iPSC-MSCs could give rise to all of these threelineages However in comparison to BM-MSCs the qualityand morphology characteristics of the differentiated iPSC-MSCs exhibited slight differences All iPSC-MSCs were morereluctant to adipogenic differentiation and the respective totalnumbers of differentiated cells containing lipid droplets werelower than that of BM-MSCs (Figure 4) This observationwas confirmed with adipocytes specific mRNA level in whichexpression levels of PPAR-120572 and PPAR-120574 were significantlylower in iPSC-MSCs than BM-MSCs Also LPL expressionlevels were significantly (119901 le 005) lower in FLF-iPSC-MSCs (OKSM) and MSC-iPSC-MSCs (OKSM) compared toBM-MSCs On the other hand iPSC-MSCs had more affinity
to differentiate to osteogenic and chondrogenic lineagesGradually mineral nodules formation started 1 week earlierin iPSC-MSCs which were stained positive for alizarin red SThe expression of osteocalcin and alkaline phosphatase wascomparable to BM-MSCs Characterizing chondrogenesisthe respective pellets were stained more strongly with alcianblue and the respective gene expression profiles for collagenII showed higher expression in iPSC-MSCs compared toBM-MSCs However Aggrecan was similarly expressed inall three iPSC-MSCs and BM-MSCs It has been shown thatsomeMSCs for instance fromperiosteumand synoviumwereeasily capable of differentiating to bone and cartilage butonly a minor population amongst them could give rise toadipocytes [23 32] and we speculate that such a MSC-relatedphenotype is resembled by our iPSC-derived MSCs
33 Supportive Effects on Long-Term CD34+ Cells Mainte-nance Thebonemarrowniche plays a vital role in preservinghematopoietic progenitors to provide proper amounts ofblood cells throughout life [33 34]This activemicroenviron-ment is fostered by secreted factors of niche-accompanyingcells such asMSCs and sinusoidal endothelial cells to support
Figure 4 Adipogenic osteogenic and chondrogenic differentiation potential of hiPSC-MSCs and BM-MSC (LM06) Oil Red-O staining forlipid formation alizarin red staining of mineralized deposits and alcian blue staining for chondrocyte pellet formed by the three iPSC-MSC-like cell lines mRNA expression level of the relative expression of genes associated with adipogenesis PPAR120574 PPAR120572 and LPL osteogenesis(osteocalcin and alkaline phosphatase) and chondrogenesis (collagen type II and aggrecan) The data represent the mean expression valuesnormalized to the housekeeping gene GAPDH lowast significance difference with BM-MSCs 119901 le 005
the quiescent state of some of the hematopoietic progenitors[35 36] The supportive cellular microenvironment providedby MSCs regulates self-renewal versus differentiation ofhematopoietic stemprogenitor cells within the bone marrow[37 38] Moreover based on their secretion of cytokinessupportive for hematopoietic cell proliferation MSC areconsidered to serve as an excellent cell type for long-term
progenitor cell culture purposes [39] As further indicationfor the undisturbed functional capabilities of iPSC-MSCswe exploited a coculture system of iPSC-MSCs and CD34+hematopoietic stemprogenitor cells and investigated thetotal cell numbers the colony forming capacity and thehomogeneity of CD34+ cells After 10 days all three iPSC-MSC coculture assays contained significantly (119901 le 005)
10 Stem Cells International
0
100
200
300
400
500
600
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
Day 0Day 10
Day 20
Tota
l non
adhe
rent
via
ble c
ellstimes104
lowast lowastlowast lowast lowast
lowastlowast
(a)
0
20
40
60
80
100
120
140
160
180
Tota
l col
ony
form
ing
units
wel
l
Day 4Day 8
lowast lowastlowast lowast
lowast
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
(b)
0102030405060708090
100
Posit
ive c
ells
()
CD-34CD-45
CD-11bCD-14
CD-34withoutstroma
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
lowast
lowastlowast
lowast
lowast
(c)
Figure 5 Coculture of CD34+ PBMCs with human iPSC-MSCs (a) Cell layers of iPSC-MSCs and BMSCs were established on 1 gelatinprecoated 24-well plates (80 confluent) CD34+ PBSCs were applied onto the stromal layers The cocultures were incubated for 20days Nonadherent viable cells were counted at the indicated time points (b) human CD34+ were plated with hiPSC-MSCs in 05mL ofmethylcellulose media containing human recombinant IL-3 SCF and EpoThe plates were incubated for 20 days following which progenitorswere scored (c) surface markers expression on CD34+ cells after coculture with mesenchymal stromal cells The results represent the mean(plusmnSD) of three replicates lowast significance difference with CD34 without stroma 119901 le 005
more nonadherent cells compared to CD34+ cells culturedwithout any stroma Comparing coculture of CD34+ cellswith iPSC-MSCs andBM-MSCs we observed a robust (but inour experiments not significant) increase in nonadherent cellnumbers for the iPSC-MSCs assays (Figure 5(a)) For OSKMfactors-derived iPSC-MSCs (Figure 5(a)) similar results wereobtained even at day 20 After replating CD34+ cells inMethoCult media for colony forming assays we observedsignificantly increased colonies in all iPSC-MSCs and BM-MSCs lines after 4 days of coculture comparing to singleCD34+ culture Furthermore MethoCult culture for 8 daysresulted in significantly (119901 le 005) higher colony numbersin iPSC-MSCs and in 2 lines of BM-MSCs (Figure 5(b))This data demonstrates a further important functional aspectand is supported by prior investigations that indicated thesupportive nature of MSCs on hematopoiesis by providinga suitable microenvironment for stemprogenitor cells ingrowing sites [40] With our data we also provided evidencethat significantly higher CD34+ cell number maintain theirstem cell status on the iPSC-MSCs and BM-MSCs rather than
conventional hematopoietic medium (Figure 5(c)) Theseenhanced proliferation and boosted colony forming abilitiescould be observed after 8 days of coculture in all iPSC-MSCs lines suggesting that these represent a reliable cellsource supporting the long-term culture of hematopoieticstemprogenitor cells Several publications are in favor of theeffects of different feeder layers and coatings for maintenanceand expansion of progenitors and somatic cells showing theimportance of mimicking in vivo conditions and providingsimilar microenvironment [41ndash43]
34 Immunosuppressive Effects of iPSC-MSCs In pioneer-ing studies mesenchymal stem cell based approaches wereapplied for suppressing immune reactions in autoimmunedisorders graft versus host disease (GVHD) or after solidorgan transplantation (for review see [44 45]) Duringallogeneic cell or organ transplantation cytotoxic and helperT-lymphocytes get activated and kill the targeted cells orpromote rejection of the transplanted organ [46] BecauseMSCs can secrete anti-inflammatory molecules to dampen
Stem Cells International 11
0
02
04
06
08
1
12
14
16
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
BrdU
mea
n ab
sorb
ance
in104
cells
lowastlowast
(a)
0
500
1000
1500
2000
2500
3000
3500
IFN
-120574(p
gm
L)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowast
lowastlowast
lowast
(b)
0
50
100
150
200
250
IL-2
(pg
mL)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
(c)
0
5
10
15
20
25
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowastlowastCD
4+C
D69
+(
)
(d)
0
5
10
15
20
25
lowast lowast
lowastlowast
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
CD4+
CD
25+
()
(e)
Figure 6 Status of activatedCD4+ T cells in the presence of hiPSC-MSCs (a) IFN-120574were determined at 48 hours by ELISAThe values are themeans plusmn SD from 3 independent experiments (b) concentrations of IL-2 and (c) proliferation in MLRMSC cocultures MLR cultures wereset up in presence or absence of hiPSC-MSCs BrdU incorporation was significantly lower in MSC-ips-MSCs and FLFiPSC-MSCs (OSNL)in comparison to absence of MSCs (d) Expression of the T-cell activation markers CD69 and (e) CD25 on CD4+ 5 days after stimulation ina 12-well dish in the presence or absence of hiPSC-MSCs lowast significance difference with MLR 119901 le 005
12 Stem Cells International
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
[1] M T Sutton and T L Bonfield ldquoStem cells innovations inclinical applicationsrdquo Stem Cells International vol 2014 ArticleID 516278 9 pages 2014
[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
Figure 3 Antigen phenotype proliferation rate and functional characterization of hiPSC-MSCs (a) Immunophenotype of the three hiPS-MSCs lines generated Representative flow cytometry analysis of hBM-MSCs FLF-iPSC-MSCs (OSKM) FLF-iPSC-MSCs (OSNL) MSC-iPSC-MSCs (OSKM) and MSC-derived iPSC line MSC-related markers CD73 CD90 and CD105 and hematopoietic CD45 CD34 andCD19 were assessed (solid histogram) (b) In vitro cell growth measured as cumulative population of hiPSC-MSCs and BM-MSCs derivedfrom 3 different donors (LM02 LM05 and LM06)
andmay qualify such cells formore long-term treatment suchas inflammatory bowel diseases or during the prevention ofgraft versus host diseases or transplant rejection in futuretransplantation settings [12]
Addressing the functional capabilities of iPSC-derivedMSCs we applied differentiation protocols towards adipo-osteo- or chondrogenic lineages respectively and performedcytological staining andRT-PCR to investigate changes in cellmorphological and related marker gene expression Impor-tantly all 3 iPSC-MSCs could give rise to all of these threelineages However in comparison to BM-MSCs the qualityand morphology characteristics of the differentiated iPSC-MSCs exhibited slight differences All iPSC-MSCs were morereluctant to adipogenic differentiation and the respective totalnumbers of differentiated cells containing lipid droplets werelower than that of BM-MSCs (Figure 4) This observationwas confirmed with adipocytes specific mRNA level in whichexpression levels of PPAR-120572 and PPAR-120574 were significantlylower in iPSC-MSCs than BM-MSCs Also LPL expressionlevels were significantly (119901 le 005) lower in FLF-iPSC-MSCs (OKSM) and MSC-iPSC-MSCs (OKSM) compared toBM-MSCs On the other hand iPSC-MSCs had more affinity
to differentiate to osteogenic and chondrogenic lineagesGradually mineral nodules formation started 1 week earlierin iPSC-MSCs which were stained positive for alizarin red SThe expression of osteocalcin and alkaline phosphatase wascomparable to BM-MSCs Characterizing chondrogenesisthe respective pellets were stained more strongly with alcianblue and the respective gene expression profiles for collagenII showed higher expression in iPSC-MSCs compared toBM-MSCs However Aggrecan was similarly expressed inall three iPSC-MSCs and BM-MSCs It has been shown thatsomeMSCs for instance fromperiosteumand synoviumwereeasily capable of differentiating to bone and cartilage butonly a minor population amongst them could give rise toadipocytes [23 32] and we speculate that such a MSC-relatedphenotype is resembled by our iPSC-derived MSCs
33 Supportive Effects on Long-Term CD34+ Cells Mainte-nance Thebonemarrowniche plays a vital role in preservinghematopoietic progenitors to provide proper amounts ofblood cells throughout life [33 34]This activemicroenviron-ment is fostered by secreted factors of niche-accompanyingcells such asMSCs and sinusoidal endothelial cells to support
Figure 4 Adipogenic osteogenic and chondrogenic differentiation potential of hiPSC-MSCs and BM-MSC (LM06) Oil Red-O staining forlipid formation alizarin red staining of mineralized deposits and alcian blue staining for chondrocyte pellet formed by the three iPSC-MSC-like cell lines mRNA expression level of the relative expression of genes associated with adipogenesis PPAR120574 PPAR120572 and LPL osteogenesis(osteocalcin and alkaline phosphatase) and chondrogenesis (collagen type II and aggrecan) The data represent the mean expression valuesnormalized to the housekeeping gene GAPDH lowast significance difference with BM-MSCs 119901 le 005
the quiescent state of some of the hematopoietic progenitors[35 36] The supportive cellular microenvironment providedby MSCs regulates self-renewal versus differentiation ofhematopoietic stemprogenitor cells within the bone marrow[37 38] Moreover based on their secretion of cytokinessupportive for hematopoietic cell proliferation MSC areconsidered to serve as an excellent cell type for long-term
progenitor cell culture purposes [39] As further indicationfor the undisturbed functional capabilities of iPSC-MSCswe exploited a coculture system of iPSC-MSCs and CD34+hematopoietic stemprogenitor cells and investigated thetotal cell numbers the colony forming capacity and thehomogeneity of CD34+ cells After 10 days all three iPSC-MSC coculture assays contained significantly (119901 le 005)
10 Stem Cells International
0
100
200
300
400
500
600
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
Day 0Day 10
Day 20
Tota
l non
adhe
rent
via
ble c
ellstimes104
lowast lowastlowast lowast lowast
lowastlowast
(a)
0
20
40
60
80
100
120
140
160
180
Tota
l col
ony
form
ing
units
wel
l
Day 4Day 8
lowast lowastlowast lowast
lowast
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
(b)
0102030405060708090
100
Posit
ive c
ells
()
CD-34CD-45
CD-11bCD-14
CD-34withoutstroma
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
lowast
lowastlowast
lowast
lowast
(c)
Figure 5 Coculture of CD34+ PBMCs with human iPSC-MSCs (a) Cell layers of iPSC-MSCs and BMSCs were established on 1 gelatinprecoated 24-well plates (80 confluent) CD34+ PBSCs were applied onto the stromal layers The cocultures were incubated for 20days Nonadherent viable cells were counted at the indicated time points (b) human CD34+ were plated with hiPSC-MSCs in 05mL ofmethylcellulose media containing human recombinant IL-3 SCF and EpoThe plates were incubated for 20 days following which progenitorswere scored (c) surface markers expression on CD34+ cells after coculture with mesenchymal stromal cells The results represent the mean(plusmnSD) of three replicates lowast significance difference with CD34 without stroma 119901 le 005
more nonadherent cells compared to CD34+ cells culturedwithout any stroma Comparing coculture of CD34+ cellswith iPSC-MSCs andBM-MSCs we observed a robust (but inour experiments not significant) increase in nonadherent cellnumbers for the iPSC-MSCs assays (Figure 5(a)) For OSKMfactors-derived iPSC-MSCs (Figure 5(a)) similar results wereobtained even at day 20 After replating CD34+ cells inMethoCult media for colony forming assays we observedsignificantly increased colonies in all iPSC-MSCs and BM-MSCs lines after 4 days of coculture comparing to singleCD34+ culture Furthermore MethoCult culture for 8 daysresulted in significantly (119901 le 005) higher colony numbersin iPSC-MSCs and in 2 lines of BM-MSCs (Figure 5(b))This data demonstrates a further important functional aspectand is supported by prior investigations that indicated thesupportive nature of MSCs on hematopoiesis by providinga suitable microenvironment for stemprogenitor cells ingrowing sites [40] With our data we also provided evidencethat significantly higher CD34+ cell number maintain theirstem cell status on the iPSC-MSCs and BM-MSCs rather than
conventional hematopoietic medium (Figure 5(c)) Theseenhanced proliferation and boosted colony forming abilitiescould be observed after 8 days of coculture in all iPSC-MSCs lines suggesting that these represent a reliable cellsource supporting the long-term culture of hematopoieticstemprogenitor cells Several publications are in favor of theeffects of different feeder layers and coatings for maintenanceand expansion of progenitors and somatic cells showing theimportance of mimicking in vivo conditions and providingsimilar microenvironment [41ndash43]
34 Immunosuppressive Effects of iPSC-MSCs In pioneer-ing studies mesenchymal stem cell based approaches wereapplied for suppressing immune reactions in autoimmunedisorders graft versus host disease (GVHD) or after solidorgan transplantation (for review see [44 45]) Duringallogeneic cell or organ transplantation cytotoxic and helperT-lymphocytes get activated and kill the targeted cells orpromote rejection of the transplanted organ [46] BecauseMSCs can secrete anti-inflammatory molecules to dampen
Stem Cells International 11
0
02
04
06
08
1
12
14
16
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
BrdU
mea
n ab
sorb
ance
in104
cells
lowastlowast
(a)
0
500
1000
1500
2000
2500
3000
3500
IFN
-120574(p
gm
L)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowast
lowastlowast
lowast
(b)
0
50
100
150
200
250
IL-2
(pg
mL)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
(c)
0
5
10
15
20
25
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowastlowastCD
4+C
D69
+(
)
(d)
0
5
10
15
20
25
lowast lowast
lowastlowast
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
CD4+
CD
25+
()
(e)
Figure 6 Status of activatedCD4+ T cells in the presence of hiPSC-MSCs (a) IFN-120574were determined at 48 hours by ELISAThe values are themeans plusmn SD from 3 independent experiments (b) concentrations of IL-2 and (c) proliferation in MLRMSC cocultures MLR cultures wereset up in presence or absence of hiPSC-MSCs BrdU incorporation was significantly lower in MSC-ips-MSCs and FLFiPSC-MSCs (OSNL)in comparison to absence of MSCs (d) Expression of the T-cell activation markers CD69 and (e) CD25 on CD4+ 5 days after stimulation ina 12-well dish in the presence or absence of hiPSC-MSCs lowast significance difference with MLR 119901 le 005
12 Stem Cells International
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
[1] M T Sutton and T L Bonfield ldquoStem cells innovations inclinical applicationsrdquo Stem Cells International vol 2014 ArticleID 516278 9 pages 2014
[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
Figure 4 Adipogenic osteogenic and chondrogenic differentiation potential of hiPSC-MSCs and BM-MSC (LM06) Oil Red-O staining forlipid formation alizarin red staining of mineralized deposits and alcian blue staining for chondrocyte pellet formed by the three iPSC-MSC-like cell lines mRNA expression level of the relative expression of genes associated with adipogenesis PPAR120574 PPAR120572 and LPL osteogenesis(osteocalcin and alkaline phosphatase) and chondrogenesis (collagen type II and aggrecan) The data represent the mean expression valuesnormalized to the housekeeping gene GAPDH lowast significance difference with BM-MSCs 119901 le 005
the quiescent state of some of the hematopoietic progenitors[35 36] The supportive cellular microenvironment providedby MSCs regulates self-renewal versus differentiation ofhematopoietic stemprogenitor cells within the bone marrow[37 38] Moreover based on their secretion of cytokinessupportive for hematopoietic cell proliferation MSC areconsidered to serve as an excellent cell type for long-term
progenitor cell culture purposes [39] As further indicationfor the undisturbed functional capabilities of iPSC-MSCswe exploited a coculture system of iPSC-MSCs and CD34+hematopoietic stemprogenitor cells and investigated thetotal cell numbers the colony forming capacity and thehomogeneity of CD34+ cells After 10 days all three iPSC-MSC coculture assays contained significantly (119901 le 005)
10 Stem Cells International
0
100
200
300
400
500
600
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
Day 0Day 10
Day 20
Tota
l non
adhe
rent
via
ble c
ellstimes104
lowast lowastlowast lowast lowast
lowastlowast
(a)
0
20
40
60
80
100
120
140
160
180
Tota
l col
ony
form
ing
units
wel
l
Day 4Day 8
lowast lowastlowast lowast
lowast
CD-34withoutstroma
BM-MSCLM02
BM-MSCLM05
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
(b)
0102030405060708090
100
Posit
ive c
ells
()
CD-34CD-45
CD-11bCD-14
CD-34withoutstroma
BM-MSCLM06
FLF-iPSC-MSCs
(OSKM)
FLF-iPSC-MSCs(OSNL)
MSC-iPSC-MSCs
(OSKM)
lowast
lowastlowast
lowast
lowast
(c)
Figure 5 Coculture of CD34+ PBMCs with human iPSC-MSCs (a) Cell layers of iPSC-MSCs and BMSCs were established on 1 gelatinprecoated 24-well plates (80 confluent) CD34+ PBSCs were applied onto the stromal layers The cocultures were incubated for 20days Nonadherent viable cells were counted at the indicated time points (b) human CD34+ were plated with hiPSC-MSCs in 05mL ofmethylcellulose media containing human recombinant IL-3 SCF and EpoThe plates were incubated for 20 days following which progenitorswere scored (c) surface markers expression on CD34+ cells after coculture with mesenchymal stromal cells The results represent the mean(plusmnSD) of three replicates lowast significance difference with CD34 without stroma 119901 le 005
more nonadherent cells compared to CD34+ cells culturedwithout any stroma Comparing coculture of CD34+ cellswith iPSC-MSCs andBM-MSCs we observed a robust (but inour experiments not significant) increase in nonadherent cellnumbers for the iPSC-MSCs assays (Figure 5(a)) For OSKMfactors-derived iPSC-MSCs (Figure 5(a)) similar results wereobtained even at day 20 After replating CD34+ cells inMethoCult media for colony forming assays we observedsignificantly increased colonies in all iPSC-MSCs and BM-MSCs lines after 4 days of coculture comparing to singleCD34+ culture Furthermore MethoCult culture for 8 daysresulted in significantly (119901 le 005) higher colony numbersin iPSC-MSCs and in 2 lines of BM-MSCs (Figure 5(b))This data demonstrates a further important functional aspectand is supported by prior investigations that indicated thesupportive nature of MSCs on hematopoiesis by providinga suitable microenvironment for stemprogenitor cells ingrowing sites [40] With our data we also provided evidencethat significantly higher CD34+ cell number maintain theirstem cell status on the iPSC-MSCs and BM-MSCs rather than
conventional hematopoietic medium (Figure 5(c)) Theseenhanced proliferation and boosted colony forming abilitiescould be observed after 8 days of coculture in all iPSC-MSCs lines suggesting that these represent a reliable cellsource supporting the long-term culture of hematopoieticstemprogenitor cells Several publications are in favor of theeffects of different feeder layers and coatings for maintenanceand expansion of progenitors and somatic cells showing theimportance of mimicking in vivo conditions and providingsimilar microenvironment [41ndash43]
34 Immunosuppressive Effects of iPSC-MSCs In pioneer-ing studies mesenchymal stem cell based approaches wereapplied for suppressing immune reactions in autoimmunedisorders graft versus host disease (GVHD) or after solidorgan transplantation (for review see [44 45]) Duringallogeneic cell or organ transplantation cytotoxic and helperT-lymphocytes get activated and kill the targeted cells orpromote rejection of the transplanted organ [46] BecauseMSCs can secrete anti-inflammatory molecules to dampen
Stem Cells International 11
0
02
04
06
08
1
12
14
16
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
BrdU
mea
n ab
sorb
ance
in104
cells
lowastlowast
(a)
0
500
1000
1500
2000
2500
3000
3500
IFN
-120574(p
gm
L)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowast
lowastlowast
lowast
(b)
0
50
100
150
200
250
IL-2
(pg
mL)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
(c)
0
5
10
15
20
25
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowastlowastCD
4+C
D69
+(
)
(d)
0
5
10
15
20
25
lowast lowast
lowastlowast
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
CD4+
CD
25+
()
(e)
Figure 6 Status of activatedCD4+ T cells in the presence of hiPSC-MSCs (a) IFN-120574were determined at 48 hours by ELISAThe values are themeans plusmn SD from 3 independent experiments (b) concentrations of IL-2 and (c) proliferation in MLRMSC cocultures MLR cultures wereset up in presence or absence of hiPSC-MSCs BrdU incorporation was significantly lower in MSC-ips-MSCs and FLFiPSC-MSCs (OSNL)in comparison to absence of MSCs (d) Expression of the T-cell activation markers CD69 and (e) CD25 on CD4+ 5 days after stimulation ina 12-well dish in the presence or absence of hiPSC-MSCs lowast significance difference with MLR 119901 le 005
12 Stem Cells International
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
[1] M T Sutton and T L Bonfield ldquoStem cells innovations inclinical applicationsrdquo Stem Cells International vol 2014 ArticleID 516278 9 pages 2014
[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
Figure 5 Coculture of CD34+ PBMCs with human iPSC-MSCs (a) Cell layers of iPSC-MSCs and BMSCs were established on 1 gelatinprecoated 24-well plates (80 confluent) CD34+ PBSCs were applied onto the stromal layers The cocultures were incubated for 20days Nonadherent viable cells were counted at the indicated time points (b) human CD34+ were plated with hiPSC-MSCs in 05mL ofmethylcellulose media containing human recombinant IL-3 SCF and EpoThe plates were incubated for 20 days following which progenitorswere scored (c) surface markers expression on CD34+ cells after coculture with mesenchymal stromal cells The results represent the mean(plusmnSD) of three replicates lowast significance difference with CD34 without stroma 119901 le 005
more nonadherent cells compared to CD34+ cells culturedwithout any stroma Comparing coculture of CD34+ cellswith iPSC-MSCs andBM-MSCs we observed a robust (but inour experiments not significant) increase in nonadherent cellnumbers for the iPSC-MSCs assays (Figure 5(a)) For OSKMfactors-derived iPSC-MSCs (Figure 5(a)) similar results wereobtained even at day 20 After replating CD34+ cells inMethoCult media for colony forming assays we observedsignificantly increased colonies in all iPSC-MSCs and BM-MSCs lines after 4 days of coculture comparing to singleCD34+ culture Furthermore MethoCult culture for 8 daysresulted in significantly (119901 le 005) higher colony numbersin iPSC-MSCs and in 2 lines of BM-MSCs (Figure 5(b))This data demonstrates a further important functional aspectand is supported by prior investigations that indicated thesupportive nature of MSCs on hematopoiesis by providinga suitable microenvironment for stemprogenitor cells ingrowing sites [40] With our data we also provided evidencethat significantly higher CD34+ cell number maintain theirstem cell status on the iPSC-MSCs and BM-MSCs rather than
conventional hematopoietic medium (Figure 5(c)) Theseenhanced proliferation and boosted colony forming abilitiescould be observed after 8 days of coculture in all iPSC-MSCs lines suggesting that these represent a reliable cellsource supporting the long-term culture of hematopoieticstemprogenitor cells Several publications are in favor of theeffects of different feeder layers and coatings for maintenanceand expansion of progenitors and somatic cells showing theimportance of mimicking in vivo conditions and providingsimilar microenvironment [41ndash43]
34 Immunosuppressive Effects of iPSC-MSCs In pioneer-ing studies mesenchymal stem cell based approaches wereapplied for suppressing immune reactions in autoimmunedisorders graft versus host disease (GVHD) or after solidorgan transplantation (for review see [44 45]) Duringallogeneic cell or organ transplantation cytotoxic and helperT-lymphocytes get activated and kill the targeted cells orpromote rejection of the transplanted organ [46] BecauseMSCs can secrete anti-inflammatory molecules to dampen
Stem Cells International 11
0
02
04
06
08
1
12
14
16
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
BrdU
mea
n ab
sorb
ance
in104
cells
lowastlowast
(a)
0
500
1000
1500
2000
2500
3000
3500
IFN
-120574(p
gm
L)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowast
lowastlowast
lowast
(b)
0
50
100
150
200
250
IL-2
(pg
mL)
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
(c)
0
5
10
15
20
25
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
lowastlowastCD
4+C
D69
+(
)
(d)
0
5
10
15
20
25
lowast lowast
lowastlowast
MLR BM-MSC(LM06) + MLR
FLF-iPSC-MSCs
(OSKM) + MLR
FLF-iPSC-MSCs
(OSNL) + MLR
MSC-iPSC-MSCs
(OSKM) + MLR
CD4+
CD
25+
()
(e)
Figure 6 Status of activatedCD4+ T cells in the presence of hiPSC-MSCs (a) IFN-120574were determined at 48 hours by ELISAThe values are themeans plusmn SD from 3 independent experiments (b) concentrations of IL-2 and (c) proliferation in MLRMSC cocultures MLR cultures wereset up in presence or absence of hiPSC-MSCs BrdU incorporation was significantly lower in MSC-ips-MSCs and FLFiPSC-MSCs (OSNL)in comparison to absence of MSCs (d) Expression of the T-cell activation markers CD69 and (e) CD25 on CD4+ 5 days after stimulation ina 12-well dish in the presence or absence of hiPSC-MSCs lowast significance difference with MLR 119901 le 005
12 Stem Cells International
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
[1] M T Sutton and T L Bonfield ldquoStem cells innovations inclinical applicationsrdquo Stem Cells International vol 2014 ArticleID 516278 9 pages 2014
[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
Figure 6 Status of activatedCD4+ T cells in the presence of hiPSC-MSCs (a) IFN-120574were determined at 48 hours by ELISAThe values are themeans plusmn SD from 3 independent experiments (b) concentrations of IL-2 and (c) proliferation in MLRMSC cocultures MLR cultures wereset up in presence or absence of hiPSC-MSCs BrdU incorporation was significantly lower in MSC-ips-MSCs and FLFiPSC-MSCs (OSNL)in comparison to absence of MSCs (d) Expression of the T-cell activation markers CD69 and (e) CD25 on CD4+ 5 days after stimulation ina 12-well dish in the presence or absence of hiPSC-MSCs lowast significance difference with MLR 119901 le 005
12 Stem Cells International
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
[1] M T Sutton and T L Bonfield ldquoStem cells innovations inclinical applicationsrdquo Stem Cells International vol 2014 ArticleID 516278 9 pages 2014
[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
inflammatory reaction [47] one can speculate that iPSC-derived MSCs could also provide a valuable cell source forimmunomodulatory therapies (for review see [11]) In orderto investigate the immunomodulatory properties of iPSC-MSCs we have used Mixed Lymphocyte Reaction (MLR)to mimic inflammatory reaction by mixing CD4+ lympho-cytes with healthy donor peripheral blood mononuclearcells (PMNCs) on iPSC-MSCs and BM-MSCs feeder layersrespectively First we checked the CD4+ lymphocyte prolif-eration in MLR assay by BrdU incorporation Human FLF-iPSC-MSCs (OSNL) and hMSC-iPSC-MSCs (OSKM) couldsignificantly (119901 le 005) dampen lymphocyte proliferationand we observed a similar decrease in FLF-iPSC-MSCs(OSKM) and BM-MSCs (Figure 6(a)) MSCs are known toexhibit regulatory properties on different kinds of immunecells including T-lymphocytes but so far it has been insuf-ficiently considered to what extent iPSC-MSCs display thismodulating activity Previously immune regulatory effects ofiPSC-MSCs on Natural Killer (NK) cells have been studiedby Giuliani et al where it was indicated that the NK-cellcytolytic machinery was disrupted by inhibiting NK-cellproliferation and IL-2 activation via expression of differentactivation markers and ERK12 signaling [48] There is alsoplenty of evidence that lymphocyte can be suppressed byMSCs secreting anti-inflammatory cytokines in response toproinflammatory stimuli mediated through IL-2 and IFN-120574 [49 50] Therefore we investigated the amount of IFN-120574 (Figure 6(b)) and IL-2 (Figure 6(c)) in the supernatantof MLR assays from the iPSC-MSC coculture experimentsWhile we could observe a significant decrease of IFN-120574 levelsin the iPSC-MSC and BM-MSCs coculture experimentswe could only detect a minor reduction of IL-2 levels inthe control BM-MSC coculture experiment as well as inthe iPSC-MSC experiments Nevertheless these results aresupporting previous findings that MSCs can dampen inflam-matory response via suppressing T-cell proliferation [51] anddecreasing proinflammatory cytokines due to nitric oxideproduction that inhibits Stat-5 phosphorylation in memoryand cytotoxic T-cells [4] Previously it has been reported thatMLR coculture with MSCs significantly increases regulatorymarkers (CD69 and CD25) expressing population in CD4+cells [5 52ndash54] Our results indicated that MSC-iPSC-MSCsand BM-MSCs significantly increased the early T-cell acti-vation marker CD69+ population compared to MLR alone(Figure 6(d)) Even if the increase in CD69+ population inFLF-iPSC-MSCs did not reach the level of significance wespeculate that these cells have an immunomodulatory impactas well Moreover all iPSC-MSCs as feeder layer have theability to significantly increase CD25+ population comparedto MLR alone (Figure 6(e)) which is in line with previouspublications [52 55]
4 Conclusion
Here we describe a lentiviral selection cassette mediatedallowing the enrichment of highly functional human iPSC-derived MSCs from different somatic starting cells SuchiPSC-MSCs exhibited higher proliferation capabilities andsimilar surface marker compared to bona fide MSCs derived
from bone marrow Moreover we were able to demonstratethat iPSC-MSCs support the long-term culture of CD34+hematopoietic stemprogenitor cells with undisturbed colonyforming abilities Finally human iPSC-MSCs also exhib-ited immunomodulatory function with lowering CD4+ T-lymphocyte population decreasing IFN-120574 secretion andincreasing regulatory T-cell population Thus iPSC-MSCsmight be considered as relevant cell resource for futuretransplantation studies in preclinical models of GVHD anddegenerative autoimmune diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors would like to thank Matthias Ballmaier andthe flow cytometry unit of Hannover Medical School fortheir technical assistance The authors are grateful to UrsulaRinas (Leibniz University Hannover) for support with bFGFand Axel Schambach (Hannover Medical School) for sup-port with lentiviral vectors as well as Reto Eggenschwiler(Hannover Medical School) for help with iPSC cultureand cell characterization The authors also thank RaymundBuhmann and Christian Wichmann (Ludwig MaximillianrsquosUniversity Munich) for their support with designing andinterpretation of results for immunomodulatory effects ofiPSC-MSCs on CD4+ T-Lymphocytes Parts of the studywere funded through the REBIRTH cluster of excellenceDFG (EXC 622) and the LOEWE Center for Cell and GeneTherapy Frankfurt
References
[1] M T Sutton and T L Bonfield ldquoStem cells innovations inclinical applicationsrdquo Stem Cells International vol 2014 ArticleID 516278 9 pages 2014
[2] A Ludwig R Saffrich V Eckstein et al ldquoFunctional potentialsof human hematopoietic progenitor cells are maintained bymesenchymal stromal cells and not impaired by plerixaforrdquoCytotherapy vol 16 no 1 pp 111ndash121 2014
[3] A Oodi M Noruzinia M H Roudkenar et al ldquoExpressionof P16 cell cycle inhibitor in human cord blood CD34+expanded cells following co-culture with bone marrow-derivedmesenchymal stem cellsrdquo Hematology vol 17 no 6 pp 334ndash340 2012
[4] K Sato K Ozaki I Oh et al ldquoNitric oxide plays a critical role insuppression of T-cell proliferation by mesenchymal stem cellsrdquoBlood vol 109 no 1 pp 228ndash234 2007
[5] K Le Blanc I Rasmusson C Gotherstrom et al ldquoMesenchy-mal stem cells inhibit the expression of CD25 (interleukin-2receptor) and CD38 on phytohaemagglutinin-activated lym-phocytesrdquo Scandinavian Journal of Immunology vol 60 no 3pp 307ndash315 2004
[6] G Ren L Zhang X Zhao et al ldquoMesenchymal stem cell-mediated immunosuppression occurs via concerted action ofchemokines and nitric oxiderdquo Cell Stem Cell vol 2 no 2 pp141ndash150 2008
Stem Cells International 13
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
[7] J Yu M A Vodyanik K Smuga-Otto et al ldquoInduced pluripo-tent stem cell lines derived from human somatic cellsrdquo Sciencevol 318 no 5858 pp 1917ndash1920 2007
[8] B Groszlig M Sgodda M Rasche et al ldquoImproved genera-tion of patient-specific induced pluripotent stem cells usinga chemically-defined and matrigel-based approachrdquo CurrentMolecular Medicine vol 13 no 5 pp 765ndash776 2013
[9] H Zaehres G Kogler M J Arauzo-Bravo et al ldquoInduction ofpluripotency in human cord blood unrestricted somatic stemcellsrdquo Experimental Hematology vol 38 no 9 pp 809ndash8182010
[10] K Takahashi K Tanabe M Ohnuki et al ldquoInduction ofpluripotent stem cells from adult human fibroblasts by definedfactorsrdquo Cell vol 131 no 5 pp 861ndash872 2007
[11] I Eberle M Moslem R Henschler and T Cantz ldquoEngineeredMSCs from patient-specific iPS cellsrdquo in Mesenchymal StemCellsmdashBasics and Clinical Application II vol 130 of Advancesin Biochemical EngineeringBiotechnology pp 1ndash17 SpringerBerlin Germany 2013
[12] L Sanchez I Gutierrez-Aranda G Ligero et al ldquoEnrichment ofhuman ESC-derived multipotent mesenchymal stem cells withimmunosuppressive and anti-inflammatory properties capableto protect against experimental inflammatory bowel diseaserdquoStem Cells vol 29 no 2 pp 251ndash262 2011
[13] Y-Q Sun M-X Deng J He et al ldquoHuman pluripotent stemcell-derived mesenchymal stem cells prevent allergic airwayinflammation inmicerdquo StemCells vol 30 no 12 pp 2692ndash26992012
[14] M Moslem M R Valojerdi B Pournasr A Muhammadnejadand H Baharvand ldquoTherapeutic potential of human inducedpluripotent stem cell-derived mesenchymal stem cells in micewith lethal fulminant hepatic failurerdquo Cell Transplantation vol22 no 10 pp 1785ndash1799 2013
[15] Q Lian Y Zhang J Zhang et al ldquoFunctional mesenchymalstem cells derived from human induced pluripotent stem cellsattenuate limb ischemia in micerdquo Circulation vol 121 no 9 pp1113ndash1123 2010
[16] Y Jung G Bauer and J A Nolta ldquoConcise review inducedpluripotent stem cell-derived mesenchymal stem cells progresstoward safe clinical productsrdquo Stem Cells vol 30 no 1 pp 42ndash47 2012
[17] K Hynes D Menicanin J Han et al ldquoMesenchymal stem cellsfrom iPS cells facilitate periodontal regenerationrdquo Journal ofDental Research vol 92 no 9 pp 833ndash839 2013
[18] W Wagner F Wein A Seckinger et al ldquoComparative charac-teristics of mesenchymal stem cells from human bone marrowadipose tissue and umbilical cord bloodrdquo Experimental Hema-tology vol 33 no 11 pp 1402ndash1416 2005
[19] R Torensma H-J Prins E Schrama et al ldquoThe impact of cellsource culture methodology culture location and individualdonors on gene expression profiles of bonemarrow-derived andadipose-derived stromal cellsrdquo StemCells andDevelopment vol22 no 7 pp 1086ndash1096 2013
[20] G Pachon-Pena G Yu A Tucker et al ldquoStromal stem cellsfrom adipose tissue and bone marrow of age-matched femaledonors display distinct immunophenotypic profilesrdquo Journal ofCellular Physiology vol 226 no 3 pp 843ndash851 2011
[21] B Shen A Wei S Whittaker et al ldquoThe role of BMP-7 in chondrogenic and osteogenic differentiation of humanbone marrow multipotent mesenchymal stromal cells in vitrordquoJournal of Cellular Biochemistry vol 109 no 2 pp 406ndash4162010
[22] L Zou X Zou L Chen et al ldquoMultilineage differentiation ofporcine bonemarrow stromal cells associated with specific geneexpression patternrdquo Journal of Orthopaedic Research vol 26 no1 pp 56ndash64 2008
[23] A Karystinou F DellrsquoAccio T B A Kurth et al ldquoDistinct mes-enchymal progenitor cell subsets in the adult human synoviumrdquoRheumatology vol 48 no 9 pp 1057ndash1064 2009
[24] E Warlich J Kuehle T Cantz et al ldquoLentiviral vector designand imaging approaches to visualize the early stages of cellularreprogrammingrdquoMolecularTherapy vol 19 no 4 pp 782ndash7892011
[25] A Haase R Olmer K Schwanke et al ldquoGeneration of inducedpluripotent stem cells from human cord bloodrdquo Cell Stem Cellvol 5 no 4 pp 434ndash441 2009
[26] B Ruster S Gottig R J Ludwig et al ldquoMesenchymal stemcells display coordinated rolling and adhesion behavior onendothelial cellsrdquo Blood vol 108 no 12 pp 3938ndash3944 2006
[27] S Kern H Eichler J Stoeve H Kluter and K BiebackldquoComparative analysis of mesenchymal stem cells from bonemarrow umbilical cord blood or adipose tissuerdquo StemCells vol24 no 5 pp 1294ndash1301 2006
[28] M Sgodda S Mobus J Hoepfner et al ldquoImproved hepaticdifferentiation strategies for human induced pluripotent stemcellsrdquo Current Molecular Medicine vol 13 no 5 pp 842ndash8552013
[29] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006
[30] AMDiMarino A I Caplan andT L Bonfield ldquoMesenchymalstem cells in tissue repairrdquo Frontiers in Immunology vol 4article 102 2013
[31] K-R Yu and K-S Kang ldquoAging-related genes in mesenchymalstem cells a mini-reviewrdquo Gerontology vol 59 no 6 pp 557ndash563 2013
[32] C L Radtke R Nino-Fong B P Esparza Gonzalez HStryhn and L A McDuffee ldquoCharacterization and osteogenicpotential of equine muscle tissue- and periosteal tissue-derivedmesenchymal stem cells in comparison with bone marrow-and adipose tissue-derived mesenchymal stem cellsrdquo AmericanJournal of Veterinary Research vol 74 no 5 pp 790ndash800 2013
[33] S J Morrison and A C Spradling ldquoStem cells and nichesmechanisms that promote stem cell maintenance throughoutliferdquo Cell vol 132 no 4 pp 598ndash611 2008
[34] A Wilson and A Trumpp ldquoBone-marrow haematopoietic-stem-cell nichesrdquo Nature Reviews Immunology vol 6 no 2 pp93ndash106 2006
[35] F Arai A Hirao M Ohmura et al ldquoTie2angiopoietin-1signaling regulates hematopoietic stem cell quiescence in thebone marrow nicherdquo Cell vol 118 no 2 pp 149ndash161 2004
[36] K W Orford and D T Scadden ldquoDeconstructing stem cellself-renewal genetic insights into cell-cycle regulationrdquo NatureReviews Genetics vol 9 no 2 pp 115ndash128 2008
[37] J Zhang C Niu L Ye et al ldquoIdentification of the haematopoi-etic stem cell niche and control of the niche sizerdquo Nature vol425 no 6960 pp 836ndash841 2003
[38] T Sugiyama H Kohara M Noda and T Nagasawa ldquoMainte-nance of the hematopoietic stem cell pool by CXCL12-CXCR4chemokine signaling in bone marrow stromal cell nichesrdquoImmunity vol 25 no 6 pp 977ndash988 2006
14 Stem Cells International
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
[39] L Milazzo F Vulcano A Barca et al ldquoCord blood CD34+ cellsexpanded onWhartonrsquos jelly multipotent mesenchymal stromalcells improve the hematopoietic engraftment in NODSCIDmicerdquo European Journal of Haematology vol 93 no 5 pp 384ndash391 2014
[40] S Nishiwaki T Nakayama S Saito et al ldquoEfficacy and safetyof human adipose tissue-derived mesenchymal stem cells forsupporting hematopoiesisrdquo International Journal of Hematol-ogy vol 96 no 3 pp 295ndash300 2012
[41] D Jing A-V Fonseca N Alakel et al ldquoHematopoietic stemcells in co-culture with mesenchymal stromal cellsmdashmodelingthe niche compartments in vitrordquoHaematologica vol 95 no 4pp 542ndash550 2010
[42] M B Sharma L S Limaye and V P Kale ldquoMimicking thefunctional hematopoietic stem cell niche in vitro recapitulationof marrow physiology by hydrogel-based three-dimensionalcultures of mesenchymal stromal cellsrdquo Haematologica vol 97no 5 pp 651ndash660 2012
[43] W Wagner C Roderburg F Wein et al ldquoMolecular andsecretory profiles of human mesenchymal stromal cells andtheir abilities to maintain primitive hematopoietic progenitorsrdquoStem Cells vol 25 no 10 pp 2638ndash2647 2007
[44] A Keating ldquoMesenchymal stromal cells new directionsrdquo CellStem Cell vol 10 no 6 pp 709ndash716 2012
[45] A Uccelli L Moretta and V Pistoia ldquoMesenchymal stem cellsin health and diseaserdquo Nature Reviews Immunology vol 8 no9 pp 726ndash736 2008
[46] B R Blazar W J Murphy and M Abedi ldquoAdvances ingraft-versus-host disease biology and therapyrdquo Nature ReviewsImmunology vol 12 no 6 pp 443ndash458 2012
[47] Y Liu R Yang and S Shi ldquoSystemic infusion of mesenchymalstem cells improves cell-based bone regeneration via upregula-tion of regulatory T cellsrdquo Tissue Engineering Part A vol 21 no3-4 pp 498ndash509 2015
[48] M Giuliani N Oudrhiri Z M Noman et al ldquoHuman mes-enchymal stem cells derived from induced pluripotent stemcells down-regulate NK-cell cytolytic machineryrdquo Blood vol118 no 12 pp 3254ndash3262 2011
[49] R Meisel A Zibert M Laryea U Gobel W Daubenerand D Dilloo ldquoHuman bone marrow stromal cells inhibitallogeneic T-cell responses by indoleamine 23-dioxygenase-mediated tryptophan degradationrdquo Blood vol 103 no 12 pp4619ndash4621 2004
[50] W T Tse J D Pendleton W M Beyer M C Egalka and EC Guinan ldquoSuppression of allogeneic T-cell proliferation byhuman marrow stromal cells implications in transplantationrdquoTransplantation vol 75 no 3 pp 389ndash397 2003
[51] M Krampera S Glennie J Dyson et al ldquoBone marrow mes-enchymal stem cells inhibit the response of naive and memoryantigen-specific T cells to their cognate peptiderdquo Blood vol 101no 9 pp 3722ndash3729 2003
[52] F Saldanha-Araujo R Haddad K C R Malmegrim de Fariaset al ldquoMesenchymal stem cells promote the sustained expres-sion of CD69 on activated T lymphocytes roles of canonicaland non-canonical NF-120581B signallingrdquo Journal of Cellular andMolecular Medicine vol 16 no 6 pp 1232ndash1244 2012
[53] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013
[54] C Nazarov J L Surdo S R Bauer and C-HWei ldquoAssessmentof immunosuppressive activity of human mesenchymal stem
cells using murine antigen specific CD4 and CD8 T cells invitrordquo Stem Cell Research and Therapy vol 4 no 5 article 1282013
[55] A Dorronsoro I Ferrin J M Salcedo et al ldquoHuman mes-enchymal stromal cells modulate T-cell responses throughTNF-alpha-mediated activation of NF-kappaBrdquo European Jour-nal of Immunology vol 44 no 2 pp 480ndash488 2014
