Comparing the reprogramming efficiency of mouseembryonic fibroblasts, mouse bone marrow mesenchymalstem cells and bone marrow mononuclear cells to iPSCs

Lei Wang & Mingyan Zhu & Qingsong Guo &

Xiangjun Fan & Yuhua Lu & Shajun Zhu & Yao Wang &

Yan Huang & Zhiwei Wang

Received: 19 December 2011 /Accepted: 24 February 2012 /Published online: 29 March 2012 / Editor: T. Okamoto# The Society for In Vitro Biology 2012

Abstract Induced pluripotent stem cells have been derivedfrom various cell types via the ectopic expression of acocktail of transcription factors. Previous studies havereported that induced pluripotent stem cells can be differen-tiated into multiple somatic cells, providing an invaluableresource in regenerative medicine. In this study, we com-pared the reprogramming efficiency of mouse embryonicfibroblasts, mouse bone marrow mesenchymal stem cells,and mouse bone marrow mononuclear cells by counting thenumber of alkaline phosphatase staining positive clones onday 15 after induced pluripotent stem cells induction. Wefound that a very low number of alkaline phosphatase-staining positive clones were derived from mouse bonemarrow mesenchymal stem cells. We then evaluated thepluripotency of the clones by detecting the expression ofembryonic stem cells markers and assessing their ability toform embryoid bodies and teratomas. Mouse bone marrowmesenchymal stem cells population is more homogeneousthan mouse bone marrow mononuclear cells, which includesa variety of cell types. Our study indicated that the extremelylow efficiency of mouse bone marrow mesenchymal stemcells induction implies that mouse bonemarrowmesenchymalstem cells may not be a suitable cell type for the induction of

induced pluripotent stem cells unless the efficiency of induc-tion can be improved.

Introduction

Since Takahashi and Yamanaka (2006) reprogrammedmouse fibroblasts to a pluripotent state by expressing thetranscription factors Oct4, Sox2, Klf4, and c-Myc (Takahashiand Yamanaka 2006; Takahashi et al. 2007), the ectopicexpression of defined factors in somatic cells have been usedto reprogram a variety of cell types, including stomach cells(Aoi et al. 2008), liver cells (Aoi et al. 2008), pancreatic β-cells (Stadtfeld et al. 2008), lymphocytes (Hanna et al. 2008),keratinocytes and neural progenitor cells (Eminli et al. 2008;Kim et al. 2009), and marrow-derived mesenchymal cells(Takahashi and Yamanaka 2006; Park et al. 2008). Theseinduced pluripotent stem (iPS) cells express genes necessaryfor pluripotency and differentiate into all three germ layers inmouse. Bone marrow is a source of cells that can be harvestedeasily. Over the past several years, adult mouse bone marrowmononuclear cells (Kunisato et al. 2010) and human immobi-lized peripheral blood cells (Kunisato et al. 2011) have beendemonstrated to be competent donor cells that can bereprogrammed into ES-like cells. In this study, we com-pared the reprogramming efficiency of mouse bone marrowmesenchymal stem cells (BMSCs), adult mouse bone mar-row mononuclear cells (BM MNCs), and mouse embryonicfibroblasts (MEFs). Our findings reveal that the reprogram-ming efficiency of BMSCs is significantly lower than BMMNCs and MEFs.

Lei Wang and Mingyan Zhu contributed equally to this work.

L. Wang :M. Zhu :Q. Guo :X. Fan : S. Zhu :Y. Wang :Y. Huang : Z. Wang (*)Department of General Surgery,Affiliated Hospital of Nantong University, Nantong University,226001, Nantong, People’s Republic of Chinae-mail: wzw0417012168@163.com

Y. LuDepartment of surgical comprehensive laboratory, AffiliatedHospital of Nantong University, Nantong University,226001, Nantong, People’s Republic of China

In Vitro Cell.Dev.Biol.—Animal (2012) 48:236–243DOI 10.1007/s11626-012-9493-0

Keywords iPSCs . Reprogramming efficiency .

Pluripotency . BMSCs . BMMNCs

Materials and Methods

Cell culture. Mice. This study was performed with theapproval of the Animal Ethics Committee and Gene Recom-bination Experiment Safety Management Committee of theInstitute of Medical Science, Nantong University.

Mouse bone marrow mesenchymal stem cells. Mouse bonemarrow cells were obtained from C57BL/6J mice (6–8 wkof age). The femurs and tibiae were dissected away from theattached muscle and connective tissues and were placed inphosphate-buffered saline (PBS) on ice. The bone marrowof each tibia and femur were washed with Dulbecco's mod-ified Eagle’s medium containing nutrient mixture F-12(DMDM/F12) with 100 units/ml penicillin/streptomycin. Thecells were plated at a density of 2–4×107 cells per 10 cm2 inDMDM/F12 containing 10 % FBS, 2 mM L-glutamine and10 ng/ml bFGF. The suspended cell population was removedafter 72 h by washing three times with PBS. Adherent cellswere then continuously cultured for 3–5 wk. The cells wereisolated by treatment with 0.25 % trypsin/EDTA for 5–6 min at 37°C and diluted 1:2 at each passage. BMSCs(passage 3) were collected and examined for six surfaceantigens (CD29, CD44, Sca-1, CD14, CD45, CD34)according to our previously described method (Xu et al.2007). Nearly all of the BMSCs (passage 3) were CD29+,CD44+, Sca-1+, CD14-, CD45−, and CD34−. These BMSCswere then used for lentiviral infection.

Mouse bone marrow mononuclear cells. Mouse bone mar-row cells were isolated from C57BL/6J mice (6–8 wk ofage). Mononuclear cells were collected by centrifugationthrough a Histopaque density gradient.

Feeder cells. MEFswere isolated from the embryos of femaleC57BL/6J mice that were 13.5 d pregnant. After removing thehead, visceral tissues and gonads, the remaining bodies werewashed and dissociated with 0.05 % trypsin/EDTA. MEFswere cultured in Dulbecco's modified Eagle’s medium(DMEM) containing 10 % FBS and 2 mM L-glutamine. Thenext day, floating cells were removed by washing with PBS.DMEM containing 10 μg/ml mitomycin C was added to theMEFs (passage 3) and incubated at 37°C for 2.5 h. Then, thecells were washed five times with PBS and harvested by0.05 % trypsin/EDTA. The cells were plated at a density of6×104/cm² for culturing iPS cells.

Viral constructs. The cDNA fragments of mouse Oct4,Sox2, Klf4, and c-myc were inserted downstream of EF-1alpha promoter and upstream of IRES-EGFP of lentivirusvector. 293FT cells (Invitrogen, Carlsbad, CA) were trans-fected at a confluence of 80–90 % with 10 μg each of thelentivirus vectors along with 5 μg of pVSVG and 7.5 μg of

Δ8.91 plasmids using Fugene (Roche, Basel, Switzerland)according to the manufacturer's directions. After 12–24 h,the viral supernatants were harvested, filtered through a0.45-μm filter, and the viral titer was determined for futureexperiments.

Lentiviral infection and the generation of iPS cells. In total,5×104 BMSCs or BM MNCs were infected with lentivirus(Lv-ef1a-mOct4-ires-eGFP, Lv-ef1a-mSox2-ires-eGFP, Lv-ef1a-mKlf4-ires-eGFP, Lv-ef1a-mc-myc-ires-eGFP) at amultiplicity of infection (MOI) 5, 10, and 20. After 12 h, themedium containing lentiviruses was removed and replacedwith BMSC medium. After 24 h, the cells were digested by0.25 % trypsin/EDTA, counted and seeded on inactivatedMEFs at 5×104 cells in a six-well plate. On day 2, the BMSCmediumwas replaced with mESCmedium (knockout DMEMsupplemented with 20 % FBS, 1 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 0.1 mMNEAA, 1,000 U/ml LIF). Similarly,infected MEFs were seeded on inactivated MEFs at 5×104

cells in a 6-well plate, and MEF medium was replaced withmESC medium on day 2. The media was replenished everyother day. By days 11–13, the clones were picked and disso-ciated in 0.25 % trypsin/EDTA. The cells derived from eachclone were transferred separately onto MEFs in two 96-wellplates. Three days later, AP staining was performed. The APpositive clones were further expanded in 24-well plates, six-well plates, and culture flasks.

Alkaline phosphatase staining and immunofluorescence. Al-kaline phosphatase staining was performed according to theinstructions provided by the Alkaline Phosphatase Detectionkit (Millipore, Billerica, MA). For immunofluorescence, iPS

Figure 1. Reprogramming of the MEFs, mBMSCs and mBM MNCsto iPSCs. (A)The morphology of the bone marrow-derived mesenchy-mal stem cells (BMSCs) were isolated and expended in DMDM/F12complete medium. Bright-field images of BMSCs were examinedunder a regular inverted microscope. (A-a) Seven d of passage 2(enlargement ×40). (A-b) Seven d of passage 3 (enlargement ×40).(B) Immunophenotype of BMSCs assayed by flow cytometry. Theexpression rates of six surface antigens were examined. CD29 wasexpressed in 95.6 % of the cells.CD44 was detected in 92.3 % of cells.Sca-1 in 93.8 % of cells and CD14 in only 0.6 % of cells were positive.Notably, two specific surface molecules of hematopoietic stem cells(HSCs), CD45 in 0.2 % of the cells and CD34 in 0.3 % of the cells,were expressed, indicating that these cells differed from the mesenchy-mal stem cells. (C) Scheme for reprogramming of MEFs, BMSCs, andBM MNCs to iPSCs. (D) Examples of 3.5-cm dishes stained for AP onday 15 demonstrating the number of AP positive clones establishedfrom MEFs, mBMSCs, and mBM MNCs at a MOI of 5 and a MOI of20. (E) A comparison of the number of AP positive clones in the sameexperiment (n03), the mean values+SD are shown. (F) Phase contrastimages (top) and fluorescence images (bottom) of OSKC-infectedmBMSCs were shown at days 3, 7, and 11. Scale bars indicate100 μm. (G) Phase contrast images (top) and fluorescence images(bottom) of iPS-M2, iPS-B1, and iPS-BM5 clones established fromMEF, mBMSCs, and mBM MNCs. Scale bars indicate 100 μm.

b

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cells were fixed with 4 % paraformaldehyde in PBS for30 min at room temperature, washed three times with 0.3 %Triton (Sigma, St. Louis, MO), and incubated in blocking

buffer (PBS, 1 % BSA, 4 % normal serum, 0.4 % Triton X-100) for 30 min. The cells were incubated overnight at 4°C inthe following primary antibody solutions: goat anti-Rex-1

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(1:100 Santa Cruz SC-50668), rabbit anti-Nanog (1:100 SantaCruz SC-33760), and mouse anti-SSEA-1 (1:200 Abcamab16285). The stained cells were washed the followingday with 0.3 % Triton and incubated with the appropriatesecondary antibody for 1 h. The cells were then washed threetimes with 0.3 % Triton, and the nuclei were stained with1 μg/ml Hoechst.

Karyotype analysis. iPS cells were treated with mediumcontaining 100 ng/ml colcemid for 3 h and then dissociatedusing 0.05 % trypsin/EDTA. Potassium chloride (0.4 %)was used for hypotonization, and a 1:3 mixture of glacialacidic acid and methanol was then used to fix the cells.Fixed cells were deposited onto a slide and aged overnight.The following day, the fixed cells were stained with Giemsafor 20 min. Imaging and karyotyping analyses were thenperformed.

EB formation. To study embryoid body formation, weharvested iPS cells using 0.25 % trypsin and culturedthem in DMEM supplemented with 10 % FBS using ahanging drop. Three days later, the embryoid bodieswere transferred to a 0.1 % gelatin-coated plated andcultured for an additional 3–5 d in DMEM supplemented with10 % FBS.

Teratoma formation. In total, 2–3×106 iPS cells in 100 μlDMEM with 10 μl Matrigel (BD Biosciences, San Jose,CA) were injected into the dorsal flanks of 6-wk-old non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. Three mice were injected for each cell line.After 4 wk the tumors formed were dissected, and hematox-ylin–eosin staining was performed.

Reverse transcription-polymerase chain reaction and real-time polymerase chain reaction. RNA was isolated usingthe MicroElute Total RNA Kit (OMEGA, Cowpens, SC)according to the manufacturer’s instructions. RNA quanti-ty and quality were determined using a NanoPhotome-terTM (IMPLEN, Westlake Village, CA). First-strandcDNA was synthesized from the RNA of iPS cells orEBs using the RevertAidTM First Strand cDNA SynthesisKit (Fermentas, Glen Burnie, MD). As a negative control,the RNA was allowed to react with the cDNA synthesisreaction mixture in the absence of reverse transcriptase.After cDNA synthesis, the cDNA synthesis reaction mix-ture was used as the template for PCR. Reverse transcrip-tion PCR was performed using DreamTaqTMGreen PCRMaster Mix (2×; Fermentas). Amplification was performedfor 35 cycles of 98°C for 20 s, 60°C for 25 s, and 72°Cfor 25 s. Real-time PCR was performed using FastStartUniversal SYBR Green Master (ROX) following the man-ufacturer’s instructions.

Results

BMSCs (passage 3) were collected (Fig. 1A), and six surfaceantigens were examined (CD29, CD44, Sca-1, CD14, CD45,CD34) by fluorescence-activated cell sorting (Fig. 1B).According to the experimental scheme (Fig. 1C), GFP-expressing lentiviruses with the four reprogramming factorswere introduced into BMSCs for 12 h. We reseeded cells ontoaMEF feeder layer after removing the virus from the medium.BMSCs expressed GFP at day 3, and small clones appearedon day 7 (Fig. 1F). We picked and expanded clones ondays 13–15. The iPSCs derived from MEFs, BMSCs, andBM MNCs were able to grow into clones with clear borders(Fig. 1G). We compared the induced efficiency of the threetypes of cells by counting the number of AP positive clones onday 15 (n03; Fig. 1D). The number of AP positive clonesderived from BMSCs is significantly less than that from BMMNCs and MEFs. The number of AP positive clones derivedfrom BMSCs and BMMNCs were 0 and 17 at MOI 5, 2, and45 at MOI 10 and 6 and 107 at MOI 20. We simultaneouslyintroduced viruses into MEFs (5×104) as a control (Fig. 1E).The number of AP positive clones derived fromMEFs is 21 atMOI 5, 62 at MOI 10 and 148 at MOI 20.

To confirm iPSC pluripotency, we selected iPS-M2 derivedfrom MEFs, iPS-B1 derived from BMSCs and iPS-BM5derived from BM MNCs that were passaged 20 times. Weconfirmed the expression of mES cell-specific cell surfacemarkers, including Nanog, SSEA-1 and Rex-1 (Fig. 2A).Real-time and reverse transcription PCR demonstrated thatthe established clones expressed endogenous Oct4, Sox2,Klf4, c-Myc, and Nanog, but exogenous Oct4, Sox2, Klf4,and c-Myc was also expressed (Fig. 2B and C). We found thatthe expression of endogenous Sox2 was weak when we per-formed RT-PCR on the passage 20 cells.

To confirm the pluripotency of each clone, we con-ducted differentiation experiments, which showed thatsuspension cultures of all clones formed EBs both invitro and in vivo (Fig. 3A). After a week of culturingEBs, we examined gene expression. As showed inFig. 3B, all iPS cell lines expressed genes characteristicof all three germ layers, including AFP (endoderm),Sparc (mesoderm), Brachyury (mesoderm), Sox1 (ecto-derm), Pax6 (ectoderm), and Nestin (ectoderm). Howev-er, the expression of Pdx1 (endoderm) was demonstratedto be lower than expected. In addition to the geneexpression analyses, we also injected 2–3×106 iPS cellsinto the dorsal flanks of 6-wk-old NOD/SCID mice.Four weeks after injection, we observed tumor forma-tion and analyzed the tumors by hematoxylin-eosinstaining. We found that tumor samples included tissuederived from all three layers, including neural tissue(ectoderm), keratin epidermal tissue (ectoderm), cartilage(mesoderm), and gut-like epithelial tissue (endoderm;

THE REPROGRAMMING EFFICIENCY OF MEFS, MBMSCS, AND MBM MNCS 239

Figure 2. Assessment of the pluripotency of iPSCs that were estab-lished from MEFs, mBMSCs and mBM MNCs. (A) Immunohisto-chemistry showing the expression of pluripotency markers inexpanded iPS-M2, iPS-B1, and iPS-BM5 clones. Scale bars indicate100 μm. (B) Quantitative reverse transcription-polymerase chain reac-tion (RT-PCR) analyses of total Oct4, Sox2, Klf4, and c-Myc and

endogenous Nanog, Oct4, Sox2, Klf4, and c-Myc expression in iPS-M2, iPS-B1 and iPS-BM5 relative to the parental somatic cell popula-tions. (C) Reverse transcription-PCR analyses of total Oct4, Sox2,Klf4, and c-Myc and endogenous Nanog, Oct4, Sox2, Klf4, and c-Myc in iPS-M2, iPS-B1, and iPS-BM5 compared with the gene ex-pression observed in the parental somatic cell populations.

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Fig. 3C). We also performed karyotype analysis; a normalmouse karyotype contains 40 chromosomes, and in our study,

the iPS-B1 and iPS-BM5 cells possessed the normal karyotype(Fig. 3D).

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Discussion

BMSCs were capable of differentiating into adipocytes,chondrocytes, and osteoblasts in vitro as well as osteoblasts/osteocytes in vivo (Baddoo et al. 2003). BM-derived iPS cellsmay be more suitable for clinical applications because theymay undergo fewer genetic mutations compared to terminallydifferentiated somatic cells. We expect that BMSCs willbecome the most suitable cell type for mouse experimentalstudies. In this study, we compared the reprogramming effi-ciency of BMSCs, BMMNCs andMEFs.We initially attemp-ted to introduce the four transcription factors Oct4, Sox2,Klf4, and cMyc using a doxycycline-inducible lentiviral trans-duction system into BMSCs to obtain iPSCs but failed. Wethen introduced LV-Oct4-GFP, LV-Sox2-GFP, LV-Klf4-GFP,LV-cMyc-GFP into BMSCs, BM MNCs, and MEFs. Al-though MEFs show a large number of cell clusters expressingGFP on day 5 as well as AP positive clones on day 15, weobtained fewer completely reprogrammed clones. BMMNCsdemonstrated a greater ability to be reprogrammed relative toMEFs. There are various cell types of BM MNCs such ashematopoietic stem cells, hematopoietic progenitor cells, my-eloid cells (granulocytes and macrophage), B lymphocytes, Tlymphocytes, NK cells, and megakaryocytes. Among thesecells, >85 % of BMMNCs are CD45+ (Kunisato et al. 2010).In this study, six BMSCs surface antigens (CD29, CD44, Sca-1, CD14, CD45, CD34) were examined and only 0.2 % ofBMSCs were positive for CD34 and CD45. Kunisato reportedthat CD45− cells could be more receptive to reprogrammingthan CD45+ cells because they were able to obtain more iPSclones from CD45+/− cells than CD45+ cells (Kunisato et al.2010). Another study from the Kunisato group reported thatmost of the GFP-positive cells infected with GFP virus wereCD45+, CD34+, and CD38+ (Kunisato et al. 2011). Based onour results, we believe that there is a small number of CD45−,CD34− cells in BM MNCs and that the CD45− and CD34−cells are indeed difficult to reprogram into their pluripotentstate. Previous reports demonstrated that mouse iPS cells weregenerated from marrow-derived mesenchymal cells or hema-topoietic stem/progenitor cells (HSPCs) (Okabe et al. 2009),and human iPS cells were generated from mobilized humanCD34+ peripheral blood cells (Loh et al. 2009). BMSCs in

adherent cultures are CD45− and CD34−. Kunisato believedthat iPS cells derived from BM may be hematopoietic pro-genitor cells similar to CD34+ cells (Kunisato et al. 2010).Although we did not compare the reprogramming efficiencyof CD45+ CD34+ cells, CD45+ CD34− cells and CD45−CD34+ cells, it appears that CD34+ cells are easier to repro-gram into iPSCs. However, we can conclude that the reprog-ramming efficiency of BMSCs in adherent culture is lowerthan BMMNCs andMEFs. Due to the low efficiency of iPSCproduction, we expect that complementary molecules can beused to aid reprogramming of BMSCs or BMMNCs in futurestudies. Several studies have shown enhanced reprogrammingefficiency using diverse methods such as the six-factor system(OSKCNL) instead of the four-factor system (OSKC), expres-sion of p53 (Zhao et al. 2008; Hong et al. 2009; Kawamura etal. 2009), knockout serum replacement instead of fetal bovineserum (FBS) in the culture medium (Zhao et al. 2010),vitamin C (Vc) (Esteban et al. 2010), SV40 large T antigen(T) (Mali et al. 2008), different proportions of the fourfactors (OSKC; Papapetrou et al. 2009), synthetic modifiedmRNA (Warren et al. 2010; Anokye-Danso et al. 2011) andthe use of an optimized defined medium, iCD1 (Chen et al.2011). Overall, however, we conclude that BMSCs may notbe suitable for use in the induction of iPS cells if thederived cells are to be used in disease modeling and poten-tial therapeutic applications.

Acknowledgments This research was supported by grants fromNational Natural Science Foundation of China (no. 81070654/H0713),the Science and Technology Innovation Project of Jiangsu Province forPostgraduates (no. CXZZ11_0643) and the Science and TechnologyInnovation Project of Nantong University for Postgraduates (no.YKC11035). We acknowledge the technical assistance from Laboratoryof Gynecology and Obstetrics (Affiliated Hospital of Nantong University),School of Life Sciences (Nantong University), SiDanSai StemCell Technology CO., LTD and Key Laboratory of Neuroregeneration(Nantong University).

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