Nanog reverses the effects of organismal aging on mesenchymal stem cell proliferation and myogenic differentiation potential

TISSUE-SPECIFIC STEM CELLS

Nanog Reverses the Effects of Organismal Aging on Mesenchymal

Stem Cell Proliferation and Myogenic Differentiation Potential

JUHEE HAN,aPANAGIOTIS MISTRIOTIS,

aPEDRO LEI,

aDAN WANG,

bSONG LIU,

bSTELIOS T. ANDREADIS

a,c,d

aDepartment of Chemical and Biological Engineering; cDepartment of Biomedical Engineering; and dCenter of

Excellence in Bioinformatics and Life Sciences, University at Buffalo, State University of New York, Amherst,

New York, USA; bDepartment of Biostatistics, Roswell Park Cancer Institute, Buffalo, New York, USA

Key Words. Mesenchymal stem cells • Aging • Nanog • Smooth muscle • Contractility • Cardiovascular regeneration

ABSTRACT

Although the therapeutic potential of mesenchymal stemcells (MSCs) is widely accepted, loss of cell function due todonor aging or culture senescence are major limiting factorshampering their clinical application. Our laboratoryrecently showed that MSCs originating from older donorssuffer from limited proliferative capacity and significantlyreduced myogenic differentiation potential. This is a majorconcern, as the patients most likely to suffer from cardiovas-cular disease are elderly. Here we tested the hypothesis thata single pluripotency-associated transcription factor,namely Nanog, may reverse the proliferation and differen-tiation potential of bone marrow-derived MSC (BM-MSC)from adult donors. Microarray analysis showed that adult(a)BM-MSC expressing Nanog clustered close to Nanog-

expressing neonatal cells. Nanog markedly upregulatedgenes involved in cell cycle, DNA replication, and DNA dam-age repair and enhanced the proliferation rate and clono-genic capacity of aBM-MSC. Notably, Nanog reversed themyogenic differentiation potential and restored the con-tractile function of aBM-MSC to a similar level as that ofneonatal (n)BM-MSC. The effect of Nanog on contractilitywas mediated—at least in part—through activation of theTGF-b pathway by diffusible factors secreted in the condi-tioned medium of Nanog-expressing BM-MSC. Overall, ourresults suggest that Nanog may be used to overcome theeffects of organismal aging on aBM-MSC, thereby increas-ing the potential of MSC from aged donors for cellular ther-apy and tissue regeneration. Stem Cells 2012;30:2746–2759

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

Mesenchymal stem cells (MSCs) provide a promising cellsource for tissue regeneration that is already under investiga-tion in several clinical trials [1–3]. However, previous studiesshowed that MSC suffer from several drawbacks hamperingclinical applications including: (a) decreased number andquality of cells with donor age [4–6] and (b) loss of prolifera-tion and differentiation potential upon expansion in vitro[7–9]. Previously, we reported our findings on the effects ofdonor aging on bone marrow-derived smooth muscle cells(BM-SMCs) originating from ovine BM-MSCs [10]. Usingneonatal (n)BM-MSCs and adult (a)BM-MSCs, we found thataging affected not only proliferation and clonogenic potentialbut also the myogenic differentiation and contractile proper-ties of aBM-MSC significantly. In addition, culture senes-cence limits the culture time of aBM-MSC to approximately8–10 passages, thereby preventing their expansion to the num-bers required for cellular therapies. These observations pose asignificant challenge that must be overcome in order to enablecellular therapies for older patients, the population mostly inneed for tissue replacement.

Nanog is a homeodomain transcription factor that inhibitsdifferentiation and maintains pluripotency of embryonic stemcells (ESCs) along with Oct4 and Sox2 [11, 12]. Nanog isexpressed in ESCs, embryonic germ, and pluripotent cellsfrom preimplantation and early postimplantation embryos anddisappears rapidly upon differentiation [13]. Nanog overex-pression enabled ESCs to maintain pluripotency independentof feeder cells [14] or leukemia inhibitory factor (LIF) [15],while downregulation of Nanog induced ESC differentiationtoward extraembryonic lineages [16]. While some studiesreported expression of pluripotency-related factors in MSC,the literature lacks consensus on this issue and the function ofthese factors in the context of adult stem cells has not beendetermined [17–20].

Ectopic expression of Nanog was shown to accelerate thegrowth of somatic cells such as NIH3T3 [21, 22] and BM-MSCs [23, 24]. The effects, however, of Nanog on differen-tiation potential varied depending on the cell type and the dif-ferentiation lineage. Forced expression of Nanog had no effecton terminal differentiation of myogenic progenitors into mus-cle fibers, but significantly impaired their transdifferentiationinto the osteogenic lineage [25]. However, combined expres-sion of Nanog with Oct4 inhibited terminal differentiation of

Author contributions: J.H. and P.M.: collection and/or assembly of data, data analysis and interpretation, and manuscript writing; P.L.:data analysis and interpretation and manuscript writing; D.W. and S.L.: statistical analysis of microarray data; S.T.A.: conception anddesign, data analysis and interpretation, manuscript writing, and final approval of manuscript.

Correspondence: Stelios T. Andreadis, Ph.D., Bioengineering Laboratory, 908 Furnas Hall, Department of Chemical and BiologicalEngineering, University at Buffalo, State University of New York, Amherst, New York 14260-4200, USA. Telephone: 716-645-1202;Fax: 716-645-3822; e-mail: [email protected] Received January 31, 2012; accepted for publication August 5, 2012; first publishedonline in STEM CELLS EXPRESS September 4, 2012. VC AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.1223

STEM CELLS 2012;30:2746–2759 www.StemCells.com

myoblast progenitors [26]. Conversely, ectopic expression ofNanog in human BM-MSC enhanced the differentiationpotential along the chondrogenic and osteogenic lineages butabated adipogenic differentiation [23, 24].

In this study, we examined the hypothesis that ectopicexpression of Nanog might restore the function of BM-MSCfrom older donors. Using BM-MSCs from neonatal and adultdonors, we discovered that Nanog reversed the aging-medi-ated loss of proliferation and myogenic differentiation poten-tial of aBM-MSC to a similar level as those of nBM-MSC. Inaddition, we identified a signaling pathway that mediated—atleast in part—the effects of Nanog on the contractile functionof BM-MSC that were coaxed to differentiate along the myo-genic lineage.

MATERIALS AND METHODS

Cultivation of BM-MSCs and LentivirusTransduction

Pools of three neonatal (nBM-MSC) and three adult (aBM-MSC) MSCs were prepared. Isolation of individual cellderived from BM of three neonatal lambs (<3 days old) andthree adult sheep (4–4.5 years old) were described previously[27]. Isolation of human hair follicle-derived-MSCs (HF-MSCs) was performed as described previously [28]. Briefly,human scalp of a 73-year-old male containing hair follicleswas obtained from the Cooperative Human Tissue Network(Philadelphia, PA, http://www.chtn.nci.nih.gov). The samplewas washed extensively and subsequently cut into smallpieces. To dissociate hair follicles from the surrounding ma-trix, the samples were treated with 0.5% collagenase type I(Invitrogen, Carlsbad, CA, http://www.invitrogen.com) for 4hours at 37�C. Next, single hair follicles were unpluggedfrom the dermis and plated each in a well of a 24-well plate.Hair follicles were cultured in Dulbecco’s modified Eagle’smedium (DMEM; Gibco, Grand Island, NY, http://www.invi-trogen.com) supplemented with 10% fetal bovine serum(FBS; Gibco). HF-MSCs migrated out of the follicle and werefurther expanded. Human adult BM-MSCs from two donors(22 years old female and 29 years old male) were obtainedfrom Stem Cell Technologies (Vancouver, Canada, http://www.stemcell.com). MSCs were cultured in DMEM contain-ing 10% FBS supplemented with 2 ng/ml basic fibroblastgrowth factor (bFGF; BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). Nanog was expressed from a lenti-viral vector, pSIN-EF2-Nanog-Puro (Addgene, Cambridge,MA, http://www.addgene.org), under the control of EF1a pro-moter. The internal ribosome entry site was placed right afterNanog to allow expression of puromycin phosphotransferaseunder the same promoter. For lentivirus production, 293T/17cells (ATCC, Manassas, VA, http://www.atcc.org) werecotransfected with the following three plasmids as describedrecently [29]: Nanog lentiviral vector, psPAX2-gag/pol/tat/rev, and pMD2.G-VSVG. Neonatal and adult MSCs weretransduced with viral supernatant and were selected with 0.5lg/ml puromycin until all noninfected cells were killed.

Proliferation and Clonogenic Assays

Neonatal and adult BM-MSCs were seeded at 1.0 � 105 cellsper well in six-well plates and cultured in DMEM containing10% FBS plus bFGF (2 ng/ml). After reaching near conflu-ence, the cells were trypsinized, counted, and the doublingtime was calculated. Clonogenic assay was previouslydescribed in detail [28]. In short, nBM-MSC and aBM-MSC

were seeded (500 cells per dish) in a 100 mm culture dishand cultured for 10–15 days. The cells were then fixed andstained with trypan blue. Each dish was photographed at aconstant distance using a gel documentation imaging system(UVP, Upland, CA, http://www.uvp.com). Images were ana-lyzed using the NIH software ImageJ (version 1.43u; NationalInstitutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij)to determine the area and effective diameter of each clone.

Senescence-Associated-b-Galactosidase Assay

The senescence-associated-b-galactosidase (SA-b-gal) activitywas detected using b-Gal Staining Kit (Invitrogen) accordingto the manufacturer’s recommendations except that citricacid/sodium phosphate buffered-staining solution (pH 6.0)was used as described before [30]. Cells were photographedusing the EVOS phase-contrast microscope (Advanced Mi-croscopy Group, Bothell, WA, http://www.amgmicro.com).SA-b-gal-positive cells were counted in five randomlyselected fields of view to determine the percentage of b-galþ

cells (total of >400 cells were counted).

Immunostaining and Western Blots

Immunostaining for alpha smooth muscle actin (aSMA) andCalponin was performed as described previously [27].

Western blots (WBs) were performed as described previ-ously [31, 32] using antibodies against Nanog (1:1,000 in 5%milk; BD Biosciences), aSMA (1:1,000 in 5% milk; Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com), Calpo-nin (1:1,000 in 5% milk; Santa Cruz Biotechnology, SantaCruz, CA, http://www.scbt.com), p-Smad2 (1:1,000 dilutionin 5% bovine serum albumin (BSA); Cell Signaling, Danvers,MA, http://www.cellsignal.com), and Smad2 (1:1,000 dilutionin 5% BSA; Cell Signaling).

Vascular Contractility of MSCs on TissueEquivalents

Cylindrical vascular tissue equivalents containing MSC infibrin hydrogels (106 cells per milliliter) were cultured arounda 6.0 mm mandrel of poly(dimethyl siloxane) as describedpreviously [33, 34]. The medium containing DMEM and 10%FBS supplemented with insulin (2 lg/ml; Sigma-Aldrich),TGF-b1 (2 ng/ml, BioLegend, San Diego, CA, http://www.biolegend.com), ascorbic acid (300 lM; Sigma-Aldrich),and e-amino-n-caproic acid (2 mg/ml; Sigma-Aldrich) waschanged every 2 days. After 2 weeks in culture, the tissueconstructs were photographed and the wall thickness andlength were measured using ImageJ. Then the tissues werereleased from the mandrel and mounted on two stainlesshooks through the lumen while one side was fixed, and theother side was connected to a force transducer. Each con-structs resided in an isolated tissue bath filled with Krebs–Ringer solution. The tissues were continuously bubbled with94% O2, 6% CO2 to obtain a pH of 7.4, a PCO2

of 38 mmHg,and a PO2

>500 mmHg at 37�C. Tissues were equilibrated ata basal tension of around 1.0 g and constant length for 30–60minutes. After equilibration, endothelin-1 (20 nM; Sigma-Aldrich), the thromboxane A2 mimetic U46619 (1 lM;Sigma-Aldrich), or potassium chloride (KCl: 118 mM; Sigma-Aldrich) was added to the tissue bath and isometric contrac-tion was recorded using a PowerLab data acquisition unit andanalyzed with Chart5 software (ADInstruments, CO Springs,CO, http://www.adinstruments.com). Contractility was nor-malized by the tissue area applying force and expressed inkPa.

Han, Mistriotis, Lei et al. 2747

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Compaction of Fibrin Hydrogels

Fibrin gel compaction assay was previously described indetail [28]. Briefly, BM-MSCs were embedded in fibrin gels(106 cells per milliliter) that contained fibrinogen (2.5 mg/ml)and thrombin (2.5 U/ml). One hour after polymerization, gelswere released from the plate walls and cells were allowed tocompact the gels over time. To quantify gel compaction activ-ity, gels were photographed at the indicated times and theirarea was measured using ImageJ software.

Total RNA Preparation and GeneChip Arrays

Total RNA was isolated from three replicates per group. Cellswere seeded in 100 mm culture dishes under growth condition(DMEM þ 10% FBS þ 2 ng/ml bFGF). When they reachedconfluence, total RNA was isolated using the RNeasy kit (Qia-gen, Chatsworth, CA, http://www.qiagen.com). The quality ofpurified RNA was analyzed on an Agilent Bioanalyzer 2100(Agilent Technologies, Santa Clara, CA, http://www.agilent.com). Intact whole RNA was labeled to obtain biotinylatedcRNA for hybridization to Affymetrix GeneChip Bovine Ge-nome Arrays according to the manufacturer’s protocol. Thisarray contains 24,027 probe sets representing approximately23,000 bovine transcripts and the ovine and bovine genomeshave a higher than 90% genetic similarity. Scanned microarrayimages were imported into GeneChip Command Console Soft-ware (AGCC, Affymetrix, Santa Clara, CA, http://www.affy-metrix.com) to generate raw signal values for each probe.

Microarray Analysis

Affymetrix Bovine GeneChip data files were processed usingthe MAS5.0 algorithm (implemented in the affy R library ofBioconductor package) to generate expression summary valuesfor each probe set. MAS5.0-based ‘‘present calls’’ was used tokeep the probe sets with ‘‘present’’ status across all three sam-ples in at least one of the four groups for downstream analysis.We then performed three separate comparisons based on thefollowing samples characteristics: Nanogþ aBM-MSC(aBM.N) versus control aBM-MSC (aBM.C); Nanogþ nBM-MSC (nBM.N) versus control nBM-MSC (nBM.C); andnBM.C versus aBM.C. The Limma program in the Bioconduc-tor package was used to calculate the level of differential geneexpression. Briefly, a linear model was fit to the data with cellmeans corresponding to the different conditions and a randomeffect for array. For each comparison, we obtained the list ofdifferentially expressed genes (DEGs) constrained by p-value<.01 and at least twofold change. Following single gene-basedsignificance testing, we used the expression value of DEGs tocluster the samples for each comparison. Hierarchical cluster-ing based on the average linkage of Pearson correlation wasused. The list of DEGs was further analyzed for enrichedKyoto Encyclopedia of Genes and Genomes (KEGG) pathwayusing the NCBI DAVID server with default setting. The statis-tical significance was calculated using the Fisher’s exact test inwhich the null hypothesis is that no difference exists betweenthe number of genes falling into a given pathway in the targetDEG list and the genome as a whole.

Quantitative Real-Time Polymerase Chain Reaction

Total RNA was reverse transcribed using a cDNA synthesis kit(Qiagen) according to the manufacturer’s instructions. Quantita-tive polymerase chain reaction (PCR) was performed using theiCycler (Bio-Rad Laboratories, Hercules, CA, www.bio-rad.com). The reaction was carried out in a volume of 25 ll contain-ing 1 ll of cDNA, 0.4 lM of each primer (Sigma Genosys,Woodlands, TX, https://www.sigma-genosys.com), and 12.5 llof 2� IQ TM SYBR Green Supermix (Bio-Rad Laboratories).

The primer sequences for the genes used in this study were listedin Supporting Information Table 5. Each reaction comprises 40cycles each with melting at 95�C for 10 seconds, annealing andextension at 55�C for 30 seconds. The fluorescence intensity wasrecorded during the extension step of each cycle. The specificityof the PCR products was verified using the melting curve gener-ated by MyiQ software and by electrophoresis on 1% agarosegels. The PCR data analysis was performed as described before[35]. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)served as a loading control.

Statistical Analysis

Pairwise statistical analysis of the data was performed using atwo-tailed Student’s t test using Microsoft Excel software.The data were considered statistically different when p < .05.Each experiment was repeated at least three times with tripli-cate samples each time unless indicated otherwise.

RESULTS

Generation of Nanog-Expressing nBM-MSC andaBM-MSC

Previously, we demonstrated that BM-MSC-derived SMCfrom aged donors showed dramatic loss of proliferative anddifferentiation potential when compared with their neonatalcounterparts [10]. Here, we attempted to overcome the aging-induced loss of cellular function by introducing the ESC plu-ripotency-related transcription factor, Nanog. We hypothe-sized that the negative effects of donor aging on BM-MSCproliferation and myogenic differentiation may be reversed byectopic expression of a single pluripotency factor, Nanog. Totest this hypothesis, we generated BM-MSCs overexpressingNanog protein from neonatal (<3 days old) or adult (4–4.5years old) ovine cells using lentiviral vectors encoding forhuman Nanog and puromycin phosphotransferase. Controlcells were modified with the same construct lacking theNanog coding sequence. After puromycin selection expressionof Nanog protein in nBM-MSC and aBM-MSC was confirmedby Western blot analysis (Fig. 1A). In addition, immunostain-ing showed that Nanog was absent from control cells but washighly expressed and localized exclusively in the nucleus oftransduced aBM-MSC and nBM-MSC (Fig. 1B, SupportingInformation Fig. S1). However, Nanog expression did notinduce expression of other pluripotency factors neither at themRNA (Fig. 1C) or the protein level (data not shown).

Nanog-Induced Changes in the Global GeneExpression Profile aBM-MSC and nBM-MSC

Next we used Affymetrix GeneChip Bovine Genome microar-rays to investigate the global molecular effects of Nanogexpression on nBM-MSC and aBM-MSC. A total of 12microarrays representing triplicates of nBM.C, nBM.N,aBM.C, or aBM.N were performed. The data were analyzedusing the following criteria: (a) a gene was included in theanalysis when it was expressed in at least one of the fourgroups; (b) a gene was differentially expressed if its expres-sion changed by at least twofold (fold change (FC) �2) andthe change was statistically significant with p-value less than.01. Using the first criterion, we obtained 11,818 transcriptsfor downstream analysis (Supporting Information Table S1).Among them, 1,671 transcripts were differentially expressed(p < .01, FC �2) in at least one of the following three pairsof conditions: (a) nBM.C versus aBM.C; (b) aBM.N versusaBM.C; and (c) nBM.N versus nBM.C (Supporting

2748 Nanog Restores Function of Aged MSC

Information Table S2). DEGs in the first group are likely tobe affected by the donor age, while genes in the second andthird groups are likely affected by Nanog expression in adultand neonatal cells, respectively.

Comparison between aBM-MSC and nBM-MSC showedthat 5.3% of genes might be affected by organismal aging.Specifically, 623 transcripts were differentially expressed(p < .01, FC �2) between neonatal and adult BM-MSC; 243of them were upregulated and 380 genes were downregulatedin nBM-MSC. Nanog expression affected gene expression ofaBM-MSC to a larger extent than that of nBM-MSC: 967genes (8.2%) and 678 genes (5.7%) were affected by Nanogin aBM-MSC and nBM-MSC, respectively. Compared to theircorresponding control, 387 genes were upregulated and 580genes were downregulated in aBM.N, while 283 genes wereupregulated and 395 genes were downregulated in nBM.N.

Hierarchical clustering showed that the Nanog-expressingcells, nBM.N and aBM.N, were clustered with each otherinstead of their control counterparts (Fig. 2A). Interestingly, asshown in the Venn diagrams (Fig. 2B) among 132 commonDEGs between the first group (Donor age; nBM.C vs. aBM.C)and the second group (Nanog in adult; aBM.N vs. aBM.C),the vast majority (128 out of 132) changed in the same direc-tion for both groups (35 were upregulated and 93 downregu-lated). Conversely, among 107 DEGs shared between the firstand the third group (Nanog in neonate; nBM.N vs. nBM.C),only a small number of genes changed in the same direction(two up and 11 downregulated), and the remaining genes wereregulated in opposite directions (Supporting Information TableS2). These results suggest that Nanog-induced gene expressionchanges might have driven aBM-MSC closer to nBM-MSC,thereby supporting the hypothesis that Nanog might havereversed the effects of aging in aBM-MSC.

To identify direct targets of Nanog in BM-MSC, we com-pared the DEGs from our analysis with the list of 1,687 geneswhose promoters were found to contain Nanog binding sitesin hESC [12]. Our analysis identified 75 genes in aBM-MSCand 56 genes in nBM-MSC that might be directly affected byNanog (Supporting Information Table S3). The small numberof genes that might be direct targets of Nanog may be causedby the limited accessibility of promoters in somatic versusESCs or potential differences in the regulatory targets ofNanog in ovine versus human cells.

Pathway Analysis. We also performed pathway analysis(Fig. 2C) to identify pathways that were significantly enrichedwith DEGs in each of the three sets that were compared(nBM.C vs. aBM.C, aBM.N vs. aBM.C, and nBM.N vs.nBM.C). As shown in Supporting Information Table S4, afew pathways were found to be common between thesegroups. For example, the chemokine-signaling pathway wassignificantly changed in nBM-MSC as well as in aBM-MSCupon Nanog expression. Similarly, the PPAR signaling path-way was significantly changed in both nBM-MSC and aBM-MSC upon Nanog expression, in agreement with a previousstudy, which reported that Nanog inhibited the adipogenicpotential of hBM-MSC [24].

Conversely, Nanog affected some pathways differentiallyin aBM-MSC compared to nBM-MSC. In particular, Nanoginduced changes in cell cycle, DNA synthesis/replication, andnucleotide mismatch/repair pathways in aBM-MSC and to alesser extent in nBM-MSC. Only a small subset (4 out of 26)of cell cycle-related genes were upregulated by Nanog in neo-natal cells (nBM.N vs. nBM.C), possibly because nBM-MSCswere proliferating fast even in the absence of Nanog. Instead,

Figure 1. Ectopic expression of Nanog in neonatal and adult BM-MSC. (A): The presence of Nanog in aBM-MSC and nBM-MSC was meas-ured by Western blot; b-actin served as a loading control. (B): Immunostaining showed that Nanog was present in the nucleus of aBM-MSC(green). Cell nuclei were counterstained with Hoechst 33342 (blue). (C): Reverse transcription-polymerase chain reaction (PCR) analysis of en-dogenous (endo) and exogenous (exo) Nanog as well as endogenous Oct4 or Sox2. Genomic DNA (gDNA) from BM-MSC was used as a posi-tive control template for PCR of endogenous genes; GAPDH served as a loading control. Abbreviations: aBM-MSC, adult bone marrow-derivedmesenchymal stem cell; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; nBM-MSC, neonatal BM-MSC.


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Figure 2. Gene expression analysis by microarray. Gene expression profiles of control and Nanog-expressing nBM-MSC and aBM-MSC using AffymetrixGeneChip Bovine GenomeArrays (n¼ 3 each group). A total of 1,670 DEGswere obtained that were at least twofold upregulated (p< .01) or twofold downre-gulated (p< .01) in at least one of the three groups (aBM.N vs. aBM.C; nBM.N vs. nBM.C; and nBM.C vs. aBM.C). (A):Heatmap for hierarchical cluster anal-ysis of the total DEGs. Red represents upregulation and green represents downregulation. (B): Venn diagrams for total DEGs or upregulated or downregulatedDEGs in the three comparisons. (C):KEGG pathway analysis of genes that are differentially regulated upon Nanog expression in aBM-MSC. The numbers inthe parentheses denote the number of genes that were upregulated or downregulated (up, down) in the corresponding pathway. (D): Verification of microarrayresults by real-time qRT-PCR for the cell cycle-related genes that were differentially expressed upon Nanog overexpression in aBM-MSC. GAPDH served as aloading control. Triplicate independent mRNA samples were used in qRT-PCR experiments. Abbreviations: aBM.C, control aBM-MSC; aBM.N, Nanogþ

aBM-MSC; aBM-MSC, adult bone marrow-derived mesenchymal stem cell; DEG, differentially expressed gene; FC, fold change; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; nBM.C, control nBM-MSC; nBM.N, Nanogþ nBM-MSC; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.


in nBM-MSC, Nanog upregulated several genes in the TGF-band cancer signaling pathways including TGFB1, TGFB2,TGFB3, FGF2, FGF10, VEGFC, HGF, and BMP2.

Validation of Microarray Results with Quantitative ReverseTranscription-PCR. Next we performed quantitative reversetranscription-PCR (qRT-PCR) to validate the microarray datafor a selected set of genes. To this end, we selected DEGsfrom cell cycle pathways including CCNA2, CDC2, CCNE2,CCNB1, CCNB2, CDKN1C, PCNA, and TP53. The expres-sion level of each gene was normalized to GAPDH and theratio of aBM.N to aBM.C was compared to the fold changeobtained from microarray data. In all cases, qRT-PCR analy-sis showed similar trend as well as quantitatively similarresults with microarray analysis (Fig. 2D).

Nanog Increased Proliferation in Neonatal andAdult BM-MSC

Upregulation of cell cycle-related genes suggested thatNanog might enhance the proliferation potential of aBM-MSC. Indeed, experiments measuring the clonogenic poten-tial and cell proliferation rate confirmed this hypothesis. Tomeasure clonogenicity, cells were seeded at clonal density(500 cells per 100 mm dish) and cultured for 9 days (nBM-MSC) or 18 days (aBM-MSC). Only colonies with diameterlarger than 2 mm were counted, as these were more likely tohave originated from highly proliferative stem cells.Although control neonatal cells showed high colony formingpotential, the number of colonies larger than 2 mm in diame-ter was significantly higher in Nanog-expressing cells (Fig.3A, 3B). In addition, proliferation assays showed that thedoubling time of Nanog-expressing cells was approximately12-hour shorter than that of control cells (Fig. 3C). Interest-ingly, Nanog had a more profound effect on aBM-MSC. Spe-cifically, the Nanog-expressing aBM-MSCs formed fourtimes more colonies than their control counterparts and thecell density was significantly higher in Nanogþ colonies (Fig.3A, 3B). Nanog expression also decreased the doubling timeof aBM-MSCs from 70 hours to approximately 50 hours(Fig. 3C).

Notably, serial passaging showed that expression ofNanog delayed culture senescence significantly. Specifically,aBM-MSC stopped dividing after 14 population doublings(estimated after passage 2) while Nanog-expressing aBM-MSC maintained their proliferation potential up to 39 popula-tion doublings. Indeed, while more than 70% of passage 8control aBM-MSC stained positive for SA-b-gal, only 12% ofNanog-expressing cells was SA-b-gal positive even at passage20 (Fig. 3D, 3E). In addition, a gene that is strongly associ-ated with cellular senescence, the cyclin-dependent kinase in-hibitor 2A or p16INK4a, was highly upregulated (> sevenfold)in aBM-MSC but decreased significantly in Nanog-expressingcells (> 30-fold) (Fig. 3F).

Consistently, the p16INK4a activator mesenchyme homeo-box 2 (MEOX2) was significantly decreased in Nanog-express-ing aBM-MSC and nBM-MSC (Supporting InformationFig. S2A), while the p16INK4a inhibitor EZH2 was significantlyupregulated by Nanog in aBM-MSC (Supporting InformationFig. S2B). Finally, increased proliferation was not accompa-nied by chromosomal abnormalities as demonstrated by karyo-type analysis showing that both Nanog-expressing nBM-MSCand aBM-MSC possessed 54 diploid chromosomes as expectedfor normal ovine cells with no signs of transformation(Supporting Information Fig. S3).

Nanog Improved Myogenic Differentiationof aBM-MSC and nBM-MSC

When expressed in MSC, Nanog was found to promote differ-entiation toward osteogenic and chondrogenic lineages butprevented adipogenesis [23–26]. However, the effect ofNanog on myogenic differentiation and especially in the con-text of organismal aging has not been investigated. To addressthis question, we first examined the expression of SMC-spe-cific markers such as aSMA and calponin after induction ofmyogenic differentiation for 5 days. Immunostaining showedthat both nBM-MSC and aBM-MSC increased expression ofboth proteins, which also assumed fibrillar organization indic-ative of contractile SMC (Fig. 4A, 4B).

Next, we compared the expression level of aSMA and cal-ponin between control and Nanogþ BM-MSCs under myo-genic differentiation conditions using WB. As shown in Fig-ure 4C, Nanog did not alter the protein level of aSMA orcalponin in neonatal or adult BM-MSCs. We also performedreal-time RT-PCR for other smooth muscle contractility-related genes including SM22, smoothelin, and caldesmon forwhich ovine-specific antibodies were not available (Fig. 4D).Although the expression levels of SM22 and caldesmon weresimilar between Nanog-expressing and control nBM-MSCs,SM22 increased slightly but significantly and caldesmonincreased by 7.5-fold in Nanog-expressing aBM-MSCs ascompared to their control counterparts. Conversely, smoothe-lin expression was significantly increased by Nanog expres-sion in both neonatal and adult BM-MSCs. These resultsprompted us to examine the ability of Nanog-expressing cellsto generate force by measuring the compaction of three-dimensional fibrin hydrogels.

To this end, nBM-MSCs and aBM-MSCs were embeddedin fibrin gels (106 cells per milliliter), and 1 hour after poly-merization, the gels were released from the plate wall andallowed to compact in the presence of TGF-b1 (2 ng/ml). Atthe indicated times, the area of each gel was measured usingImageJ and normalized to its initial area. As shown in Figure5, control nBM-MSC showed significant contractility but therate as well as the final extent of gel compaction was higherin Nanog-expressing nBM-MSC (Fig. 5A, 5B). Conversely,aBM-MSC showed limited gel compaction activity but similarto nBM-MSC, Nanog enhanced the force generation ability ofaBM-MSC significantly. Specifically, Nanog-expressing aBM-MSC compacted gels to half of their original gel size within50 hours when compared with 70 hours for control cells (Fig.5A, 5B). These data suggested that Nanog enhanced the con-tractility of nBM-MSC and aBM-MSC that were coaxed todifferentiate to SMC.

Nanog Restored Age-Related Loss of VascularContractility of aBM-MSC

Next, we evaluated the effect of Nanog on vasoreactivity ofvascular constructs prepared from nBM-MSC or aBM-MSC.To this end, we prepared cylindrical tissue equivalents byembedding cells in fibrin hydrogels that were polymerizedaround cylindrical mandrels. After 2 weeks in culture in myo-genic differentiation medium, the cells compacted the hydro-gels down to � 5% of their original volume yielding cylindri-cal constructs with wall thickness of less than 500 lm. Atthat time, vascular rings were placed in isolated tissue bathsto measure isometric tension generated in response to receptoror non-receptor-mediated vasoagonists.

Although nBM-MSC-based constructs showed high vaso-reactivity in response to receptor (Endothelin-1, U46619) ornon-receptor (KCl)-mediated pathways, Nanog expressionincreased contractility significantly (Fig. 6A, 6B). Conversely,


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Figure 3. Ectopic expression of Nanog significantly improved clonogenic and proliferative potential in BM-MSC. Cells were plated onto 10-cmdishes at clonal density (500 cells per dish) and cultured for 9 days (nBM-MSC, n ¼ 3) or 18 days (aBM-MSC, n ¼ 3). (A): Photographs of representa-tive cell culture dishes and colonies. Bar ¼ 200 lm. (B): The number of colonies larger than 2 mm in diameter was quantified for control and Nanog-expressing cells. The symbol (*) denotes p< .05 between the indicated samples and their corresponding control. (C): To measure proliferation rate, cellswere seeded at 10,000 cells per square centimeter. After 6 days (nBM-MSC, n ¼ 3) or 9 days (aBM-MSC, n ¼ 3), the number of cells was counted anddoubling time was calculated assuming exponential cell growth. (D): Representative images of SA-b-gal staining for control aBM-MSC (passage 8) orNanogþ aBM-MSC (passage 20). Scale bar ¼ 400 lm. (E): Quantitation of the percentage of SA-b-gal-positive cells. *, p < .05 between the indicatedsample and corresponding control. (F): Real-time quantitative RT-PCR for p16INK4a. The results were normalized to GAPDH and plotted as average 6SE (n ¼ 3 independent experiments). *, p < .05 between the indicated samples. Abbreviations: aBM-MSC, adult bone marrow-derived mesenchymalstem cell; nBM-MSC, neonatal BM-MSC; SA-b-gal, senescence-associated-b-galactosidase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4. Effects of Nanog on myogenic differentiation of BM-MSC. Control and Nanog-expressing (A) nBM-MSC or (B) aBM-MSC werecultured under growth (DMEM/10% FBS, 2 ng/ml bFGF) or myogenic differentiation medium (DMEM/10% FBS, 2 ng/ml TGF-b1, and 30 lg/ml heparin). On day 3, the cells were fixed and immunostained for aSMA or calponin (red). Cell nuclei were counterstained with Hoechst 33342(blue). Scale bar ¼ 100 lm. (C, D): Control and Nanog-expressing nBM-MSCs or aBM-MSCs were cultured in the presence of TGF-b1 (2 ng/ml) and heparin (30 lg/ml) for 3 days. (C): Western blot for aSMA and calponin. (D): Real-time quantitative RT-PCR for SM22, caldesmon,and smoothelin; *, p < .05 (n ¼ 3). Abbreviations: aBM-MSC, adult bone marrow-derived mesenchymal stem cell; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; nBM-MSC, neonatal BM-MSC.


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constructs generated with aBM-MSC showed very low levelof vascular reactivity. Notably, Nanog enhanced vascular con-tractility of aBM-MSC by more than 10-fold restoring it tosimilar level as that of Nanogþ nBM-MSC and higher thancontrol nBM-MSC. There was no statistically significant dif-ference in the response of Nanogþ aBM-MSC (n ¼ 10) versusNanogþ nBM-MSC (n ¼ 11) to any of the three agonists (p¼ .8, .65, and .87 for Endothelin-1, U46619, and KCl, respec-tively). Collectively, our data show that forced expression ofNanog restored the aging-related loss of vascular function ofaBM-MSC to a similar level as that of nBM-MSC.

Nanog Increased Vascular Contractilityof Human aMSCs

These results prompted us to hypothesize that Nanog mayexert a similar effect on the contractile function of humanaMSC. To this end, we used MSCs from three donors (twomales and one female) and two different anatomic locations,that is, BM and hair follicle (HF-MSCs). Cylindrical tissueequivalents were prepared with Nanog-expressing or controlMSCs and the vascular reactivity was measured in response tothe same vasoactive agonists. Our results clearly demonstratethat Nanog enhanced the contractility of all MSCs irrespectiveof gender, anatomic location, or species (Fig. 6C--6E).

Nanog-Induced Increase in Fibrin CompactionWas Mediated in Part Through the TGF-b/SmadSignaling Pathway

Microarray analysis showed that Nanog upregulated expres-sion of TGF-b1 in nBM-MSC and to a lesser extent in aBM-MSC. Real-time qRT-PCR verified this result and showedthat TGF-b1 expression increased by approximately fivefoldin nBM-MSC and approximately threefold in aBM-MSC uponNanog expression (Supporting Information Fig. S4). SinceTGF-b1 is known to increase SMC contractility, we examinedwhether Nanog enhanced contractility through the TGF-b1pathway. Indeed, Nanog induced Smad2 phosphorylation in

both nBM-MSC and aBM-MSC in the absence of exogenousTGF-b1 (Fig. 7A, 7B), without affecting the expression levelof total Smad2 (Supporting Information Fig. S5). In addition,Smad2 phosphorylation was diminished by treatment with theTGF-b type I receptor kinase inhibitor, SB431542, implicat-ing the TGF-b pathway in this process.

Furthermore, we examined whether increased Smad2phosphorylation and contractility might be induced by solublefactors secreted by Nanog-expressing BM-MSC. To this end,we measured Smad2 phosphorylation in control aBM-MSC ornBM-MSC in the presence of medium that was conditioned(conditioned medium, CM) by Nanogþ aBM-MSC (aBM.N-CM) or Nanogþ nBM-MSC (aBM.N-CM). As control, weused CM from control nBM-MSC (nBM.C-CM) or aBM-MSC (aBM.C-CM), respectively. As shown in Figure 7C,both aBM.N-CM and nBM.N-CM induced Smad2 phospho-rylation, which was completely abolished by SB431542treatment. Interestingly, nBM-MSCs exhibited considerablecompaction in the presence of nBM.N-CM and to a lesserextent aBM.N-CM but failed to contract fibrin gels in thepresence of nBM.C-CM. Similar to Smad2 phosphorylation,the enhanced contractility was diminished by treatment withSB431542. In addition, a TGF-b1 neutralizing antibody inhib-ited nBM.N-CM-induced pSmad2 and hydrogel compactionpartially (Supporting Information Fig. S6). Taken together,these results suggest that Nanog enhanced the myogenic dif-ferentiation potential of aBM-MSC and nBM-MSC by induc-ing expression of TGF-b1 and perhaps also other as yet un-identified factor(s) that activate the TGF-b signaling pathway.

DISCUSSION

Although the therapeutic potential of MSCs is widelyaccepted, loss of cell function due to donor aging or culturesenescence are major limiting factors hampering their clinicalapplication. In a recent study, we characterized the effects of

Figure 5. Nanog increased the ability of BM-MSC to generate force. BM-MSCs were embedded in fibrin gels, which were allowed to polymer-ize for 1 hour before they were released from the walls and allowed to compact. At the indicated times, the gels were photographed and theirarea was measured using ImageJ software. The ratio of each gel area (A) at the indicated times over its initial area (A0) was plotted as a functionof time. (A): Gel compaction profiles of control or Nanog-expressing nBM-MSC and aBM-MSC in the presence of TGF-b1 (2 ng/ml). (B): Rep-resentative gel pictures at the last time point (t ¼ 67 hours). Abbreviations: aBM-MSC, adult bone marrow-derived mesenchymal stem cell;nBM-MSC, neonatal BM-MSC.


donor aging on MSCs derived from neonatal or adult BM[10] and found that the BM-MSC proliferation and myogenicdifferentiation potential declined significantly with donoraging. The effects of organismal aging are also compounded

by culture senescence limiting the culture time of MSC toapproximately 8–10 passages, thereby preventing their expan-sion to the large-cell numbers required for cellular therapies.This is a major concern, as the patients mostly in need for

Figure 6. Nanog increased receptor-dependent and -independent contractility of nBM-MSC and aBM-MSC. BM-MSCs were embedded infibrin hydrogels and cultured around 6 mm mandrels for 2 weeks in the presence of myogenic medium (2 ng/ml TGF-b1, 2 lg/ml insulin, and300 lM vitamin C). Vascular reactivity was measured using an isolated tissue bath system. (A): Vascular reactivity (kPa) in response to Endothe-lin-1 (20 nM), U46619 (10�6 M), or KCl (118 mM). (B): Representative graph of isometric contraction of control or Nanog-expressing nBM-MSC and aBM-MSC in response to Endothelin-1. (C--E): Vascular reactivity (kPa) of vascular tissue constructs prepared from human MSC. (C):Male aBM-MSC; (D) female aBM-MSC; and (E) male aHF-MSCs. All values are the mean 6 SD of triplicate samples in a representative experi-ment. *, p < .05 between the indicated samples and corresponding control. Abbreviations: aBM-MSC, adult bone marrow-derived mesenchymalstem cell; HF-MSC, human hair follicle-derived-MSCs; nBM-MSC, neonatal BM-MSC.


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Figure 7. Nanog induced gel compaction by activating the TGF-b/Smad signaling pathway. (A): Cells were incubated in low serum medium(DMEM/2% FBS) and then next day they were treated with TGF-b1 (2 ng/ml) or the TGF-b type I receptor inhibitor SB431542 (10 lM) for 1hour. The presence of p-Smad2 in cells was determined by Western blotting; GAPDH served as loading control. (B): The band intensity of p-Smad2 was determined using ImageJ software and normalized to GAPDH. All values are the mean 6 SE of three independent experiments (n ¼3). The symbol (*) denotes p < .05 between the indicated samples. (C): Control and Nanog-expressing neonatal or adult BM-MSCs were cul-tured in low serum medium (DMEM/2% FBS), and 24 hours later, the CM was harvested and filtered. Cells were incubated in low serum me-dium (DMEM/2% FBS) for 24 hours and exposed to CM from control or Nanogþ nBM-MSC or aBM-MSC in the presence or absence ofSB431542 (10 lM). After 1 hour, cells were lysed and the presence of p-Smad2 was measured by Western blot; GAPDH served as loading con-trol. (D): Gel compaction assay was performed with conditioned media in the presence or absence of SB431542 (10 lM). All values are themean 6 SD of triplicate samples in a representative experiment (n ¼ 3). The symbol (*) denotes p < .05 between the indicated samples and cor-responding control at the same time point, n ¼ 3. Abbreviations: aBM-MSC, adult bone marrow-derived mesenchymal stem cell; CM, condi-tioned medium; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; nBM-MSC, neonatal BM-MSC.


cellular therapies in general and for vascular grafts in particu-lar are elderly. Here, we attempted to address this challengeby ectopic expression of pluripotency-associated factors,Nanog or Oct4, into BM-MSC from neonatal and adultanimals.

Some studies showed that MSCs express pluripotency-related factors such as Oct4 and Sox2 [17] or Oct4 and Nanog[36], but their expression decreased over time in culture.Nanog and Oct4 expression correlated inversely with organis-mal aging as human first-trimester fetal MSCs, whichexpressed Nanog and Oct4, had longer telomeres and grewfaster as compared to adult BM-MSCs lacking both factors[18]. Other groups did not observe endogenous expression ofpluripotency factors in MSC but showed that ectopic expres-sion of Nanog, Oct4, or Sox2 increased the proliferation rateand improved osteogenesis and chondrogenesis. Interestingly,while Oct4 promoted Nanog inhibited adipogenesis [23, 24].Similarly, our results show that ovine aBM-MSC or nBM-MSC did not express Nanog endogenously but ectopic expres-sion had profound effects on proliferation and myogenic dif-ferentiation, especially for aBM-MSC.

Specifically, Nanog expression shortened the doublingtime and delayed the senescence of adult MSCs significantly.Previous studies demonstrated that cell cycle and DNA repli-cation/mitosis genes were significantly reduced in senescentor late passage adult cells [37–42]. Interestingly, microarrayanalysis revealed that cell cycle, DNA replication, and DNArepair-related genes were highly upregulated by Nanog inaBM-MSC but not in nBM-MSC, which were already highlyproliferative. Conversely, p16INK4a and the p16INK4a activatorMEOX2 (a suppressor of cell proliferation that is involved insenescence [43]) were significantly decreased in Nanog-expressing aBM-MSC and nBM-MSC. Conversely, thep16INK4a inhibitor EZH2, which was shown to restore prolifer-ation of aged b-cells [44] and hematopoietic stem cells [45]was significantly upregulated by Nanog in aBM-MSC.

KEGG pathway analysis revealed that Nanog downregu-lated adipogenic genes such as CEBPa and PPARc, in agree-ment with previous work showing that ectopic expression ofNanog suppressed adipogenic differentiation [24]. Remark-ably, several studies reported that aging correlated withdecreased MSC capacity for osteogenesis, chondrogenesis,and myogenesis and increased propensity for adipogenesis[10, 46, 47]. Interestingly, the p53 level increased moderatelybut significantly in aBM-MSC upon Nanog expression. It isgenerally accepted that increased p53 results in cell cyclearrest through p21 but p53 was also shown to maintain ge-nome stability and prevent oncogenic transformation [48].Interestingly, the level of p53/p21 was significantly increasedduring reprogramming leading to reduced efficiency ofinduced pluripotent stem cell (iPSC) generation [49, 50].Others reported that p53/p21 expression was considerablydecreased in MSCs from old donors [38] or late passage cells(>30 passages) [37], possibly indicating an increased propen-sity for generating cancer stem cells. Therefore, the increasein p53 by Nanog might help to sustain genomic stability andprevent oncogenic transformation of BM-MSC.

Cellular senescence has been attributed to—among otherfactors—deficiencies in DNA damage repair or genome main-tenance [51–53]. DNA damage has been associated with oxi-dative stress due to endogenous mitochondrial respiration orexogenous stressors such as UV irradiation. Replication-asso-ciated DNA damage also leads to replicative exhaustion anddecreased proliferative capacity of aged cells [53]. DuringDNA synthesis, deficiencies in DNA replication machinery,introduction of errors, or slow base excision repair couldincrease susceptibility of DNA to recombination, eventually

resulting in the onset of replication-arrest [51]. Besides cellcycle genes, our microarray analysis identified DNA replica-tion, nucleotide (purine and pyrimidine) metabolism, baseexcision repair, and mismatch repair-related genes that wereupregulated by Nanog in aBM-MSC. These microarray find-ings suggest that Nanog expression might delay adult stemcell senescence and sustain the proliferation potential ofaBM-MSC by facilitating DNA repair and stabilizing genomeintegrity.

We also observed that Nanog enhanced differentiation ofovine BM-MSC into SMC as demonstrated by the dramati-cally increased contractility, especially of aBM-MSC. In addi-tion, we observed that Nanog significantly increased the con-tractile function of human adult MSCs, independent ofanatomic location or gender. Increased myogenic functioncorrelated with higher expression of SMC markers such asSM22, smoothelin, and caldesmon. Interestingly, Nanog-expressing cells exhibited sustained phosphorylation ofSmad2 in the absence of exogenous TGF-b1, suggesting thatthe TGF-b signaling might be sustained through autocrine sig-nals. Indeed, CM from Nanog-expressing cells inducedSmad2 phosphorylation, which was inhibited by the TGFbtype I receptor inhibitor, SB431542, supporting this hypothe-sis. However, ELISA measurements showed only a smallincrease in the level of TGF-b1 protein in CM of Nanog-expressing cells (data not shown). In addition, TGF-b neutral-izing antibody inhibited Smad2 phosphorylation and contrac-tility induced by CM from Nanog-expressing cells only par-tially (Supporting Information Fig. S6), suggesting that inaddition to TGF-b1 other TGF-b family ligands might also beresponsible for increased myogenic function. Alternatively,part of the TGF-b1 activity might not be in soluble form butmight be bound to extracellular matrix, which was recentlyshown to enhance its bioactivity [54]. Immobilized TGF-b1might not be detected by ELISA and might not be neutralizedby the anti-TGF-b1 antibody, although the TGF-b1 pathwaywould still be blocked by the TGFb receptor inhibitor,SB431542. In other experiments, TGF-b1 was required forhydrogel compaction by control but not by Nanog-expressingBM-MSC. Although addition of exogenous TGF-b1 increasedNanog-induced compaction even further, SB431542 inhibitedcompaction only partially (Supporting Information Fig. S7),suggesting that Nanog-induced contraction might be mediatedby TGF-b1-independent pathways as well. More studies arerequired to uncover these Nanog-activated pathway(s).

Interestingly, another pluripotency-associated transcriptionfactor, Oct4, had different effects on BM-MSC. AlthoughOct4 also enhanced proliferation and clonogenic capacity ofboth neonatal and adult BM-MSCs significantly (SupportingInformation Fig. S8), it had differential effects on the myo-genic differentiation of adult and neonatal cells. Specifically,upon myogenic differentiation, Oct4-expressing aBM-MSCexhibited increased vascular contractility albeit to a muchlower extent than Nanog-expressing cells. Conversely, Oct4significantly decreased contractility of nBM-MSC, suggestingthe Oct4 might inhibit differentiation of nBM-MSC into func-tional SMC. Previous studies showed that Oct4 promoted adi-pogenesis of human adult BM-MSC [24] but failed toenhance osteogenesis [23]. Taken together these results mayindicate that Oct4 and Nanog affect the transcription programof BM-MSC in different ways, despite recent findings show-ing that in ESC they co-occupy a substantial portion of theirtarget genes and collaborate in regulating pluripotency anddifferentiation [12]. Finally, our data may also suggest thatthe effects of Nanog and Oct4 on BM-MSC depend stronglyon donor age, possibly due to differences in target gene acces-sibility in neonatal versus adult stem cells.


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CONCLUSIONS

In summary, this study demonstrated that Nanog expressionenhanced the proliferation and myogenic differentiation poten-tial of nBM-MSC and to a significantly greater extent aBM-MSC, suggesting that Nanog may reverse or prevent aging-induced loss of aBM-MSC function, with no need for reprog-ramming into a pluripotent state. This in turn may allow forexpansion of aBM-MSC to the large-cell numbers requiredfor regenerative medicine, thereby facilitating development ofcellular therapies for the elderly—the patients mostly in needfor tissue replacement.

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutesof Health (R01 HL086582) and the New York State Stem CellScience (NYSTEMContract #C024316) to S.T.A.

DISCLOSURE OF POTENTIAL

CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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Nanog reverses the effects of organismal aging on mesenchymal stem cell proliferation and myogenic differentiation potential