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Stem Cell Research (2010) 4, 25–37

REGULAR ARTICLE

Pluripotency genes overexpressed in primateembryonic stem cells are localized on homologues ofhuman chromosomes 16, 17, 19, and XAhmi Ben-Yehudah a,b, Christopher S. Navara a,b,1, Carrie J. Redinger a,Jocelyn D. Mich-Basso a, Carlos A. Castro a,b, Stacie Oliver a,Lara J. Chensny c, Thomas J. Richards c,Naftali Kaminski c, Gerald Schatten a,b,d,⁎

a Pittsburgh Development Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USAb Department of Ob/Gyn and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USAc Department of Medicine and the Dorothy P. and Richard P. Simmons Center for Interstitial Lung Disease & LungTranslational Genomics Center, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School ofMedicine, Pittsburgh, PA 15213, USAd Department of Cell Biology and Physiology and Bioengineering, University of Pittsburgh School of Medicine, Pittsburgh,PA 15213, USA

Received 7 August 2009; received in revised form 10 September 2009; accepted 11 September 2009

Abstract While human embryonic stem cells (hESCs) are predisposed toward chromosomal aneploidities on 12, 17, 20, and X,rendering them susceptible to transformation, the specific genes expressed are not yet known. Here, by identifying the genesoverexpressed in pluripotent rhesus ESCs (nhpESCs) and comparing them both to their genetically identical differentiatedprogeny (teratoma fibroblasts) and to genetically related differentiated parental cells (parental skin fibroblasts from whomgametes were used for ESC derivation), we find that some of those overexpressed genes in nhpESCs cluster preferentiallyon rhesus chromosomes 16, 19, 20, and X, homologues of human chromosomes 17, 19, 16, and X, respectively. Differentiatedparental skin fibroblasts display gene expression profiles closer to nhpESC profiles than to teratoma cells, which are geneticallyidentical to the pluripotent nhpESCs. Twenty over- and underexpressed pluripotency modulators, some implicatedin neurogenesis, have been identified. The overexpression of some of these genes discovered using pedigreed nhpESCs derivedfrom prime embryos generated by fertile primates, which is impossible to perform with the anonymously donated clinicallydiscarded embryos from which hESCs are derived, independently confirms the importance of chromosome 17 and X regions inpluripotency and suggests specific candidates for targeting differentiation and transformation decisions.

© 2009 Elsevier B.V. All rights reserved.

⁎ Corresponding author. 204 Craft Avenue Pittsburgh, PA 15213, USA. Fax: +1 412 641 2410.E-mail address: schattengp@upmc.edu (G. Schatten).

1 Current address: Department of Biology, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249-0662, USA.

1873-5061/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.scr.2009.09.003

26 A. Ben-Yehudah et al.

Introduction

Human embryonic stem cell (hESC) research is invaluable indiscovering early developmental mechanisms and funda-mental principles in pluripotency and differentiation. Inaddition, hESCs hold great promise for clinically relevantinsights into the causes of diseases and perhaps evenimproved treatment strategies (Dhara and Stice, 2008;Menendez et al., 2006; Mountford, 2008; Unger et al.,2008; Wang et al., 2008; Xu et al., 2006; Ben-Yehudah et al.,2004). Notwithstanding the great significance of the currentbasic and translational investigations, the origins of, andstrength of, evidence for hESCs differ strikingly from theirmurine counterparts. Mouse ESCs were derived from blas-tocysts obtained from genetically pedigreed fertile mice(Evans and Kaufman, 1981; Tremml et al., 2008; Bryja et al.,2006). The pluripotency of mESCs was unequivocally dem-onstrated by mESC contributions to mouse offspring,including germ-line transmission (Tremml et al., 2008;Bryja et al., 2006) through the transfer of chimeric embryosgenerated by mixing mESCs with mouse morulae or blas-tocysts (Bryja et al., 2006). In contrast, existing human ESCswere derived from embryos surplus to requirements forassisted reproduction (Menendez et al., 2006; Mountford,2008; Yu and Thomson, 2008; Hyun et al., 2008). To ensurepatient confidentiality, the established hESC lines areanonymous, so neither pedigree analysis nor investigationsbetween or among closely related lines have been possible.

Pluripotency with hESCs has been demonstrated bydetection of specific markers as well as in vitro and in vivodifferentiation in teratomas; intraspecific chimera investi-gations, on the other hand, raise significant ethical concerns(Hyun et al., 2008; Deb et al., 2008; Pera et al., 2000; Talbotand Blomberg le, 2008). However, though hESCs are similarin their global characteristics, heterogeneity exists in geneexpression among the various hESC lines (Allegrucci andYoung, 2007). Regardless of these hESC limitations, vitalinsights into the particular genes expressed during pluripo-tency and the expression profile switches during differenti-ation are swiftly emerging with profound implications forinduced pluripotency (iPS) cells (Lowry et al., 2008;Takahashi et al., 2007; Yu et al., 2007; Brambrink et al.,2008; Park et al., 2008). Recently, patient-specific plurip-otent stem cells (PSCs) have been established (Choi et al.,2009), providing the foundation both for in vitro clinicaldisease models and for potentially immune matched lines fortransplantation.

Recently, we established 10 pedigreed nonhuman primateESC (nhpESC) lines from prime embryos conceived usinggametes from fertile breeders (Navara et al., 2007). Incontrast to hESCs but similar to mESCs, this panel of nhpESCsis extremely homogeneous in its gene expression profiles(N97% identity between and among lines). These pedigreed,partially inbred, well-characterized nhpESCs were then usedto assess a more comprehensive list of stemness geneswithout the interference of unknown or heterogeneousgenetic backgrounds. Due to our knowledge of the parentalpedigrees of these ESCs, we were able to isolate skinfibroblast from the monkeys from which gametes wereobtained for establishing these nhpESCs.

hESCs have been found to display predispositions towardaneploidities on chromosomes 12, 17, 20, and X (Gertow et

al., 2007; Imreh et al., 2006; Draper et al., 2004; Lefort etal., 2008; Spits et al., 2008; Hanson and Caisander, 2005),though the expressed genes are not yet known. Thesepredispositions render hESCs susceptible to transformation(Werbowetski-Ogilvie et al., 2009), which jeopardizes theirutility as biological resources for in vitro investigations andundermines their clinical reliability in patients after trans-plantation. This investigation sought to identify the genes onthe homologous nonhuman primate (NHP) chromosomes.Using a battery of ESCs from pedigreed fertile nonhumanprimates, in this study we compared the gene expressionprofiles of these fully differentiated cells to nhpESCs as wellas to fibroblasts isolated from teratomas derived from thesenhpESC, i.e., genetically identical fully differentiated cells.We found that some of the genes overexpressed clusteredpreferentially on rhesus chromosomes 16, 19, 20, and X,homologues of human chromosomes 17, 19, 16, and X,respectively (Gibbs et al., 2007). The overabundance ofoverexpressed genes on chromosomes 17 and X indepen-dently confirms the importance of these chromosome regionsin pluripotency and suggests specific candidates for targetingtransformation decisions.

Results

To overcome the genetic background heterogeneity whichmight mask some aspects of the actual stemness signature,we utilized a newly established bank of pedigreed nhpESCs(Navara et al., 2007) to generate a unique nonhuman primate“stemness gene” list generated by the comparison of nhpESCgene expression to two different sources of geneticallyidentical or genetically related differentiated fibroblasts.The first were grown out of skin explants taken from thenonhuman primate parents from which gametes were used togenerate the stem cell lines. The second were teratomaexplants. The pedigree of the different types of cells isdepicted in Fig. 1A. For Figs. 1B and 1C, RNA was isolatedfrom all three types of cells: (1) skin fibroblasts; thesesamples are indicated by the letter M (monkey) before themonkey identification number and sample label (a–f; 2)nhpESCs denoted by C (cell line) followed by the cell line andsample; (3) fibroblast explants from teratomas generated bythe injection of nhpESC into immune-compromised mice.Since there were a number of teratomas for every stem cellline, we designated the nomenclature as follows: T (terato-ma)–number of teratoma from the line–line name–sample.Therefore, TA31b denotes that this is the second sample ofthe first teratoma from line 3106.

The accuracy of findings in stem cell research is subject toconsiderations regarding the purity of the cellular popula-tions. We verified the quality of our cellular populations inseveral ways. First, we showed that the nhpESC lines cangenerate teratomas that consist of tissues from all threegerm layers (Supplementary Fig. 1). We also analyzed theteratoma sections for OCT-4 detection; the observations thatthey did not react with OCT-4 antibodies, while controlseminomas did, demonstrate that they did not harborpluripotent cells. Were they to contain pluripotent stemcells as a contaminant, our ability to detect pluripotent geneexpression would have been compromised. We neveridentified OCT-4-positive cells within teratomas derived

Figure 1 Genes expressed in pluripotent ESCs differ significantly from their genetically identical differentiated fibroblasts. (A)Experimental design. Gene expression profiles of three different cell types were compared: pluripotent nhpESCs (lines C3106 andC3806), their pedigreed parents, and their differentiated progeny in teratomas denoted by the letter T followed by the teratomanumber and cell line from which it was derived. Teratoma images are panoramic views at a 4× magnification after H&E staining. (B)Heat map of clustering described in C. Thirty-nine (6 stem cell samples, 24 teratoma and 9 skin fibroblast) were analyzed for geneexpression using Affymetrix Rhesus gene expression chips. We applied agglomerative hierarchical clustering to the gene expressionvalues, using the minimum variance incremental sum of squares method of Ward (Gordon, 1999), with dissimilarity measured by oneminus the squared Pearson correlation. Yellow, relatively overexpressed; purple, relatively underexpressed. Color scale is in log ratioto the overall mean of the row. (C) Clustering analysis of 29 000 genes with an Entrez gene ID described in B. The three cell typesclustered together with their cellular subtype: pluripotent nhpESCs were significantly different from both types of differentiatedfibroblasts. In addition, skin fibroblasts clustered together and separated from the teratoma-derived lines in two distinct nodes.

27Chromosome localization of pluripotency genes

Table 1 Genes significantly overexpressed or underexpressed in pluripotent nhpESCs compared to differentiated parental orprogeny fibroblasts

a Pluripotent marker; b membrane protein; c transcription factor; d associated with neuronal development or function. Red, yetuncharacterized gene. Yellow, overexpressed. Pink, underexpressed genes in nhpESC compared to fibroblast.

28 A. Ben-Yehudah et al.

from line 3106, and very infrequently (b1% and only in oneminor cell type) could we find Oct-4-positive cells interatomas derived from line 3806 (Supplementary Fig. 2).Furthermore we examined cellular karyotypes, to ensurethat the cells we investigated were euploid. SupplementaryFigs. 3 (line 3106) and 4 (line 3806) show that both thenhpESC lines and the teratoma fibroblasts have normalkaryotypes and that the teratoma karyotypes fit the nhpESClines from which they have been derived. While the teratomaand nhpESC have an identical genome, it is only 50% identicalto the parental skin fibroblast. We investigated both male(3106) and female (3806) nhpESC lines to eliminate genetic

background, as well as to examine the effects of sex on thedifferences in gene expression.

After mRNA isolation and hybridization to Affymetrixrhesus arrays, we examined the gene expression profiles ofthese cells. A heat map (Fig. 1B) was generated using clusteranalysis and, as expected, the nhpESC displayed a highlydifferent gene expression profile from both types offibroblasts. When clustering the single samples using all∼53 000 probes on the array (Fig. 1C), we found that thethree cell types clustered together with their cellularsubtype: pluripotent nhpESCs were significantly differentfrom both types of differentiated fibroblasts. In addition, we

29Chromosome localization of pluripotency genes

found that the skin fibroblasts clustered together andseparately from the teratoma-derived lines in two distinctnodes. These results indicate that although both werefibroblasts, they were not identical cells. In addition, Fig.1C shows the internal quality of our results since the threeindependent samples from each cell sample usually clusteredtogether with its replicates. Out of the triplicates used foreach experiment, we omitted one outlier of the ES samples(C3806c) since its gene expression differed extensively fromall other nhpESC samples, as shown in the heat mapillustrated in Supplementary Fig. 5.

We next analyzed the genes that are significantlydifferent between nhpESC and both types of fibroblasts.We found that out of the 28 895 annotations with an entrezgene ID, 8252 annotations differ between the two groups. Nosignificant changes were detected between male and femalecells (data not shown), indicating that the differences weregreater between cell types (nhpESC versus fibroblast) thanbetween sexes. As expected, we found that Oct-4, Nanog,and Sox-2 that have been associated with pluripotency wereamong the 20 most up regulated genes in nhpESC comparedto both types of fibroblasts (depicted in Table 1 and Fig. 2A).However, many of the genes that were either up regulated ordown regulated are still not characterized in the rhesus, andtheir study will lead to further insights into pluripotencycharacteristics. In addition, to our surprise, we found that15% of the most up regulated genes were associated to

Figure 2 Differentially expressed genes are detected by geneexpression arrays and qPCR. (A) Plotting gene expression levelson the microarray. Black bars denote stem cells, white bars skinfibroblast, and gray bars teratoma fibroblast. For each expres-sion level at least 5 samples were used. (B) For verification ofarray information, we used quantitative-PCR assay. Resultsdenote at least 3 experiments for at least 2 cell lines. Blue, Sox2;Red, Oct4; Yellow, Nanog. Error bars, SD.

neurogenesis (according to OMIM). No apoptosis-associatedproteins were observed, and consequently it seems unlikelythat contact inhibition or apoptosis was significantly differ-ent between the cell types.

We confirmed these differences in pluripotent markersbetween cell lines using quantitative PCR (qPCR; Fig. 2). Forthese experiments, we used the same RNA extracted for thegene arrays for consistency. We used two samples from eachcell type and averaged results of the samples are shown inFig. 2B. While Nanog was not detected in either group offibroblasts, OCT-4 was expressed in low levels, and SOX2 wasexpressed in low levels in the teratoma fibroblasts; this issimilar to the gene array expression differences betweenboth types of fibroblasts (Fig. 2A).

Using Ingenuity software we found that a large number ofpathways were differentially expressed between stem cellsand both types of fibroblast. Ingenuity constructs systemsnetworks by analyzing our data together with its scientificliterature-based software. Among the networks which aredifferentially expressed between pluripotent and differen-tiated cells are mitochondria dysfunction gene networks, aswell as those involved in the G2/M transition (SupplementaryFig. 6). It has been established that stem cells are bettermaintained in a low oxygen environment (Ezashi et al.,2005). In addition, stem cells appear to have less stringentcell cycle checkpoints. Both phenomena might be explainedby the enriched pathways in stem cells compared tofibroblast depicted in Supplementary Fig. 6.

To test whether genes over- or underexpressed in nhpESCslie more—or less—often on certain macaque chromosomesthan expected by chance we used chi-squared to test thegoodness of fit (GOF) of the observed counts of differentiallyexpressed genes on all 22 chromosomes to the expectedcounts based on the distribution among chromosomes of allprobes on the chip with chromosomal attributions. Supple-mentary Table 1 shows a representative analysis of confidencevalues for the respective chromosomes using chi-squared“goodness of fit (GOF)” with a P value of 10-4. Additionalstatistical analyses using other models and P values were alsoperformed. Having obtained statistically significant resultswith the omnibus GOF test, we used Bonferroni to control thefamilywise error rate at 0.05 in binomial proportions testing todetect over- or underabundances of differentially expressedgenes on chromosomes (Fig. 3A). We found that there is anoverabundance of overexpressed genes on chromosomes 16,19, 20, and X that correlate to the human chromosomes 17,19, 16, and X, respectively. Interestingly, of these 4chromosomes 2 have previously been shown to undergoaneploidities in prolonged hESC tissue culture conditions.Rhesus chromosomes 6 and 12 were shown to be under-expressed in our experiments (Fig. 3B). These results are inagreement with previous published results showing chromo-somal instability in rhesus preimplantation embryos (Dupontet al., 2009a,b). When global gene expression patterns werecharacterized, we found that the nhpESC gene expression wascloser to skin fibroblasts rather than to the teratomafibroblasts, although genetically they share their genomewith teratoma fibroblasts and only half with skin fibroblasts.

We identified a stem cell signature that would define apluripotent cell by its relative average gene expression for allgenes. We examined the average log relative gene expres-sion of both the 29 000 genes with a known gene Entrez gene

Figure 3 Chromosome overabundance of gene expression. Genes overexpressed in nhpESC are clustered on rhesus chromosomes 16,19, 20, and X and underabundant are clustered on rhesus chromosomes 6 and 12. (A) Ratio of overexpressed genes observed divided bythe overexpressed genes expected calculated by the ratio of annotations for each gene on the array is shown by the bars. Significanceis indicated by the orange graph (-log (P value)). Only differentially expressed genes with an Entrez gene ID and a chromosome locationwere analyzed. (B) Ratio of underexpressed calculated as in A.

30 A. Ben-Yehudah et al.

ID (Fig. 4A) as well as the 8200 genes that differed betweennhpESC and fibroblasts (data not shown). We found that theoverall relative gene expression of the fibroblast was indeedsignificantly higher than in the nhpESCs. Interestingly, therelative expression of the skin fibroblast was closer to thenhpESC than to the teratoma fibroblasts. Using BRBArrayTools we could visualize a 3-D relationship among the3 groups of cells illustrated in Fig. 4B, in which nhpESC werecloser in gene expression to skin fibroblasts than to teratomafibroblasts. This 3-D analysis permits rotation of the datasetsso that their interrelationships can be viewed from variousperspectives. These effects were independent of sex.

Finally, we have compiled system networks inferred toregulate the pluripotent ↔ differentiation transition innonhuman primate stem cells (Fig. 5). It is important tonote that this list is generated totally independently of thelist of “stemness genes” which have been identified in themore heterogeneous human ESCs. Anticipated candidategenes include SOX2, OCT-4, and NANOG, while unanticipatedgenes include up regulation of IL17. Interestingly, we foundthat many genes, such as members of LHX and DLX families ofgenes, were down regulated in nhpESCs. Both gene familieshave been shown to be involved in fetal development (Hardtet al., 2008; Kraus and Lufkin, 2006; Woodside et al., 2004).

Discussion

Pluripotent stem cell (PSC) research, which emerged fromthe intensive investigations on embryonic stem cells, has

tremendous importance for understanding fundamentalconcepts of early development, differentiation decisions,and cell proliferation controls. Now with the intense focus onhuman PSCs, this previously fundamental science is rapidlymoving toward more clinically relevant arenas. These fieldsinclude PSC differentiation for cell replacement, PSCprogenitors of cancer, patient-specific and/or disease-specific induced PSCs for mechanistic and drug discoveries.These proposed medical implications of pluripotent stemexpression patterns in human PSCs now demand increasedscrutiny to assure that the discoveries are indeed accurateand unaffected by inherent biological and genetic limita-tions, i.e., the heterogeneity of the existing hESC lines; theirsuboptimal origins from clinically discarded embryos con-ceived in vitro by infertility patients; their genetic anonym-ity; their extensive propagation in vitro; among others. Toaddress these concerns, we have conducted a completelyindependent analysis of “stemness” gene networks usingpedigreed rhesus ESCs derived from fertile primates (Navaraet al., 2007). Because these datasets do not rely on theexisting hESC stemness gene expression patterns, it isnoteworthy that the most prominent pluripotency genes,i.e., SOX2, OCT-4, and NANOG, emerge as dominantregulatory factors. In addition, unexplored candidate genesmay be worthy targets for further research attention.

To ensure that we did not have contaminations ofdifferentiated cells within our undifferentiated cultures, orundifferentiated cells within our differentiated cultures, weroutinely stained our undifferentiated cultures with “stem-ness markers,” i.e., OCT-4, NANOG, SSEA-3, and -4, as well

Figure 4 Gene expression averages of skin fibroblast are moreclosely related to pluripotent nhpESCs than genetically identicalfibroblasts differentiated within teratomas. (A) Box plot ofaverage gene expression. Dark solid lines show median expres-sion level off all genes in each group. The interquartile range(IR), or middle 50%, of average gene expression is delimited by abox. Data are expressed on a log base 2 scale. (B) Snap shotsfrom a 3-D analysis using BRB ArrayTool show that nhpESC aredifferent from both types of fibroblast (left), nhpESC are similarto skin fibroblast but different from teratoma fibroblast(middle), and the 3 groups are slightly different from eachother (right). Sex of the cells did not affect results. Red, malenhpESC; green, female nhpESC; pink, male teratoma fibroblast;pink, female teratoma fibroblast; cyan, male skin fibroblast;blue, female skin fibroblast.

31Chromosome localization of pluripotency genes

as neuronal markers (data not shown). In addition, asdepicted in Supplementary Fig. 2, we could not find OCT-4-positive cells within the teratomas generated from line 3106,and only very rarely from those generated from line 3806.Since a possibility of “contamination” exists, a purepopulation of FACS sorted cells would be the ideal populationfor these experiments. However, since most nhpESC die in asingle cell population, we refrained from FACS sorting. Inaddition, we grew both skin and teratoma fibroblasts for N4passages before extracting their RNA to ensure as pure apopulation of fibroblasts as possible.

When global gene expression patterns were character-ized, we found that the nhpESC gene expression was closer toskin fibroblasts than to teratoma fibroblasts, althoughgenetically they share their genome with the teratomafibroblast and only half with skin fibroblasts. These resultsmight indicate that the cells isolated from the skin and

characterized as fibroblasts might harbor less well differen-tiated cells closer in their gene expression to authenticundifferentiated cells. Perhaps this result might shed light onthe success rate of skin fibroblast to form iPS cells ascompared to other types of cells (Lowry et al., 2008;Takahashi et al., 2007; Yu et al., 2007; Brambrink et al.,2008; Park et al., 2008).

Although most of the genes depicted in Table 1 areunidentified, some of the differentially expressed genes areassociated with pluripotency, such as Oct-4 and Sox-2. Inaddition, others could be found in future research to playroles in regulating the pluripotency ↔ differentiationtransitions. For example, TACSTD1, also called CD44 or Ep-CAM, is an epithelial adhesion molecule that was originallyidentified as a marker of carcinomas (Baeuerle and Gires,2007; Trzpis et al., 2007). We found this gene to be the mostdifferentially expressed gene between stem cells andfibroblasts, indicating that it might have other functions insignaling rather than solely adhesion. In contrast, decorin(DCN) has an important role in maintaining the balance inPeyronie's disease (PD) where fibrotic plaques are formed.DCN neutralizes one of the causes for plaque formation—transforming-growth-factor β1 (Gonzalez-Cadavid andRajfer, 2005). Consequently it is perhaps not surprisingthat DCN was underexpressed in the pluripotent stem cellswe examined.

To identify “stemness genes” a comparison between geneexpression of pluripotent cells to their in vitro differentiatedcounterparts was carried out (Sato et al., 2003; Zeng et al.,2004). While genetic background is identical, the presence ofsome undifferentiated or partially differentiated cellscannot be ruled out. In contrast, a range of undifferentiatedcells can be used for the common denominator[s] thatdefines the pluripotent state (Sato et al., 2003; Bhattacharyaet al., 2004, 2005; Cai et al., 2006; Ivanova et al., 2002;Ramalho-Santos et al., 2002; Sperger et al., 2003), or thecomparison of gene expression between and among differentstem cell lines. These experiments usually showed that hESCsare quite different from each other (Allegrucci and Young,2007; Navara et al., 2007; Abeyta et al., 2004; Rao et al.,2004). A more comprehensive study showed that althoughclosely related, the 59 ESC lines showed heterogeneity ingene expression (Adewumi et al., 2007). Interestingly,variations in gene expression were found not only in genescorrelated with the pluripotent state or differentiation, butalso in the expression of housekeeping genes (Synnergren etal., 2007). Therefore, interactions among many genesforming an active network that will allow the pluripotentstate to be maintained are likely at work (Vogel, 2003).

The first two studies that characterized the “stemnessgene” list (Ivanova et al., 2002; Ramalho-Santos et al., 2002)identified about 250 putative genes involved in mESCpluripotency, and many other genes are being studiedtoday (Allegrucci and Young, 2007; Bhattacharya et al.,2004, 2005; Cai et al., 2006). Stemness gene lists have beengenerated by comparing different types of pluripotent cells(Kim et al., 2006) or by comparing differentiated cells todifferentiated cells (Bhattacharya et al., 2005; Cai et al.,2006; Golan-Mashiach et al., 2005; Liu et al., 2006; Shin etal., 2007; Beqqali et al., 2006; Lee et al., 2007). It isinteresting to note that OCT-4 is on chromosome 6p21.33,SOX-2 3q26.3, and NANOG 12p13.31, so none of this

Figure 5 System analysis using ingenuity. Ingenuity analysis shows that while known players of stemness (Nanog and Sox-2) areoverexpressed (red) in nhpESC compared to fibroblast, many other genes are expressed as well indicated by the red tint. Genesdenoted by the green tinge are overexpressed in fibroblast. For this analysis we compared all genes with a known entrez gene ID.

32 A. Ben-Yehudah et al.

important pluripotency triad are represented on the over-expressed chromosomes found here. Further analysis isunderway to identify these pluripotency regulatory clusters.

It has been well established that a number of key proteinsplay an important role in the maintenance of pluripotency inboth mESCs and hESCs, such as OCT-4, NANOG, and SOX2(Sato et al., 2003; Zeng et al., 2004) as well as the existenceof a regulatory network in mESC (Zhou et al., 2007) for SOX2(Masui et al., 2007; Stanton and Bakre, 2007), OCT-4 (Babaieet al., 2007), and NANOG (Pan and Thomson, 2007). Similarresults were shown in hESCs since hESCs treated with RNAiagainst SOX2 cells readily differentiated (Fong et al., 2009).Although there is much information on the cooperationactivity of OCT-4, NANOG, and SOX2, we still lack informa-tion regarding other key players in the maintenance of thepluripotent state.

To better understand the extensive list of genes involvedin the differences between cell lines, research has beenconducted on specific pathways that are differentially

expressed. Rho et al. (Rho et al., 2006) found that theWnt, Hh, Notch pathways but not the JAK/STAT hadoverabundant transcripts in ESC compared to EBs. Thereforethey concluded that self-renewal is a coordinated signaling-specific mechanism. In addition, Li et al. (Li et al., 2006)identified a transcriptome involved in this differentiationprocess. Still, most genes that are differentially expressedhave yet to be identified (ESTs) or have not been correlatedwith pluripotency (Robson, 2004). Studying them willenhance our understanding of the pluripotent state.

In order to achieve an orchestrated pathway in develop-ment, a well coordinated machinery must be activated toturn off pluripotent genes and turn on the expression ofdifferentiation genes. This process is usually carried out bytranscription factors as shown in mESCs (Hailesellasse Seneet al., 2007) and hESCs (Laslett et al., 2007). Thesetranscription factors can together form a hierarchy in acomplex network that maintains the pluripotent state (Kimet al., 2008) and include reprogramming factors used for iPS

33Chromosome localization of pluripotency genes

(OCT-4, SOX2, KLF-4, and c-MYC) as well as NANOG, DAX-1,REX-1, ZPF281, and NAC-1. Other DNA remodeling proteinshave been associated with pluripotency in mESC (Zhou et al.,2007) and both the similarities and the differences betweenthe mouse networks generated by Zhou et al. (Zhou et al.,2007) and ours are informative. Both found genes not yetappreciated as regulators of pluripotency.

Recently, new mathematical algorithms have beendeveloped to help identify pluripotent genes (Grskovic etal., 2007). Analyses using them showed that every hESC hasits unique signature but shares many genes with other hESClines that help maintain the pluripotent state (Suarez-Farinas and Magnasco, 2007; Suarez-Farinas et al., 2005;Rao and Stice, 2004). However, that analysis was based onthe comparison of three hESC lines that, although similar,still contain many differences (Abeyta et al., 2004).

While many studies have been carried out on mESC andhESC, very few have searched for pluripotent genes innhpESC. A single study has compared rhesus ESC to EBs(Byrne et al., 2006). They identified 367 genes that wereexpressed in 5 nhpESC lines and include CCNB1, GDNF3,LeftB, OCT-4, and NANOG. These 367 genes may representthe stemness core allowing the cells to maintain thepluripotent state.

Conclusion

The prominent stemness triad of SOX2/OCT-4/NANOG foundin pluripotent human stem cells is the dominant regulatorynetwork in primate PSCs. This confirmation is reassuringsince it suggests that the hESC lines' heterogeneous origins—embryos discarded by anonymous infertility patients—do notcompromise the fundamental utility of the existing lines.Needless to say, newer, more robust, and more diverse lineswould accelerate research discoveries. Differences betweengene expression profiles in human lines compared withprimate ones may prove instrumental in discovering thegenetic basis of certain forms of infertility which might havebeen transmitted to the resultant hESCs. Also, since themajority of these lines came from Indian rhesus monkeys, thesubspeciation of this group could be further analyzed.Finally, the preferred clustering of overexpressed genes onthe NHP homologues of human chromosomes 17, 19, 16, andX highlights the centrality of these regions to pluripotencyand suggests that investigating the role of these genes mayfurther enhance our understanding of the mechanisms thatdetermine stemness and pluripotency.

Materials and methods

The overall strategy to identify the genes overexpressed andunderexpressed in pluripotent stem cells and, if clustered onchromosomes, to localize those chromosomes is depicted inFig. 1. Pluripotent pedigreed rhesus ESCs (female ESC lineC3806 – black circle; male ESC line C3106 – black square)were compared to genetically identical differentiatedprogeny grown from their resultant teratomas (five maleteratomas and three female teratomas: ‘T’, i.e., TA31, TB31,TC31, TD31, TE31, and TA38, TB38, TC38, respectively) as

well as to differentiated cells from their macaque parents(‘M’).

Teratoma formation and fibroblast isolation

Stem cell pluripotency potentials were assessed by teratomaformation in SCID mice. Nonhuman primate ESCs (nhpESCs)were isolated by scraping colonies with good morphology andusing a brief 5-min treatment with 0.25% trypsin/EDTA tobreak up large fragments. Cells were pelleted at 800g for10 min and washed twice in sterile PBS, and approximately5×105 – 5×106 cells were injected into the testis of 8- to 12-week-old NOD-SCID mice (Jackson Labs) by modification ofefferent duct injection (Ogawa et al., 1997). The injectionpipette was advanced along the efferent duct and throughthe rete testes into the interstitial space where cells wereinjected (using Eppendorf Femtojet). The injection itselfwas monitored with trypan blue added to the cell suspension.Tumor growth was determined by palpation and was typicallydetectable 10–12 weeks after injection. Teratomas weredissected from euthanized mice placed in tissue culture (forthe isolation of outgrowths) or fixed (for pathologicalexamination). For tissue culture (skin or teratoma), cellswere minced into small fragments and maintained infibroblast media (DMEM containing 10% FBS, penicillin/streptomycin, L-glutamine, nonessential amino acids) for 7days before media replacement to ensure attachment ofcells. In all fibroblast experiments (both skin fibroblast andteratoma fibroblast) cells were cultured for N4 passagesbefore RNA isolation. All RNA was isolated when cells were∼80% confluent to ensure that cells were proliferating, andtherefore unaffected by contact inhibitor. For pathologicalexamination, teratomas were fixed in 4% paraformaldehyde,paraffin embedded, and examined histologically afterHematoxylin and Eosin (H&E) staining. Isolated tumorswere identified as teratomas if tissues derived from allthree germ layers were identified in the sections.

Cytogenetic analysis to assay normal karyotype

Cytogenetic analysis was performed as described previously(Navara et al., 2007).

Gene expression profiling

RNA extractionTotal RNA was extracted using the Trizol protocol (100 μl/10×104 –105 cells) (Navara et al., 2007) and purified usingthe Qiagen RNeasy Micro Kit (Valencia, CA) according to themanufacturer's recommendations. RNA quantity and qualitywere determined using a Nanodrop spectrophotometer andAgilent Bioanalyzer (Navara et al., 2007).

Preparation of labeled cRNAOne microgram of total RNA was used to start the manualtarget preparation using the Codelink Expression BioarraySystem (Amersham Biosciences/GE Healthcare). Briefly,double-stranded cDNA synthesis was performed with aT7oligo (dT) primer, followed by purification. This cDNAwas used as a template for in vitro transcription with biotin-labeled nucleotides. Fifteen micrograms of the labeled cRNA

34 A. Ben-Yehudah et al.

was hybridized to Affymetrix Rhesus Macaque genome 49format Arrays (GeneChip Rhesus Macaque Genome Array,Cat. No.900656, Affymetrix, Santa Clara, CA, USA), followedby washing and staining with streptavidin phytoerythrin(SAPE) as recommended by the manufacturer. Arrays werescanned on an Affymetrix GeneChip 3000 Scanner. The arrayscontain 52 866 probe sets that represent ∼30 000 humanorthologues and ESTs. Affymetrix GCOS software was usedfor the scanning of the probe arrays and the probe intensityanalysis and normalization was performed using RMA express(Navara et al., 2007).

Microarray data analysisThe gene expression analysis protocol has been previouslydescribed (Kaminski and Friedman, 2002). Statistical analysiswas performed using the Scoregene gene expression package(http://www.cs.huji.ac.il/labs/compbio/scoregenes), anddata visualization was performed using Genomica (http://genomica.weizmann.ac.il) (Segal et al., 2004), SpotfireDecision Site 8.0 (Spotfire Inc. Göteborg, Sweden), Treeview(http://jtreeview.sourceforge.net), and BRB ArrayTools(http://linus.nci.nih.gov/BRB-ArrayTools.html). The RMAoutput for every gene was divided by the geometric meanof all the values for the same gene and was log base 2transformed. In the analysis we only included transcriptswith locus link numbers. To determine the differentiallyexpressed genes we used t test or significance analysis ofmicroarrays (SAM). In the clustering we included only genesthat had a q value=0 in both scoring methods. This criterionwas also employed for assessing over- and underabundantexpression. FDR analysis was carried out as described(Navara et al., 2007).

RNA extraction, quantitative-PCR, and TaqManlow-density arrays

Cells were collected and pelleted. Total RNA was extractedusing Trizol reagent (Invitrogen) and treated with RNase-freekit (Ambion, Austin, TX). PCR mixtures were prepared usingan IMPROM- II reverse transcription system (Promega,Madison, WI) following the manufacturer's instructions.qPCR was performed using an ABI Prism 7700 (AppliedBiosystems Incorporated, Foster City, CA). Taqman geneexpression assays (Applied Biosystems) were used forNANOG, OCT-4, and β actin. Water and no RT sampleswere used as control. All samples were run in triplicate.TaqMan Array human stem cell Pluripotency Panel (AppliedBiosystems) was used following the manufacturer's instruc-tions. Real-time PCR expression data from 96 genes wasanalyzed. Six genes were endogenous controls, and 30 geneswere not included in our analysis due to very pooramplification in any sample. mRNA fold changes werecalculated using the -ΔΔCt method and normalized using βactin expression as endogenous control.

Immunohistochemistry

Paraffin-imbedded tissue was deparaffinized and hydratedby incubating sections in three washes of xylene for 5 mineach, followed by two washes of 100% ethanol for 10 mineach, two washes of 95% ethanol for 10 min each, and two

washes in dH2O for 5 min each. Antigens were retrieved byboiling slides in 10 mM sodium citrate buffer, pH 6.0,maintained at a subboiling temperature for 10 min andcooled for 30 min. For staining, sections were washed indH2O three times for 5 min each, incubated in 3% hydrogenperoxide for 10 min, and washed in dH2O twice for 5 min eachfollowed by washing buffer (1X TBS/0.1% Tween 20 (1XTBST)) for 5 min. Sections were blocked with 400 μl blockingsolution (TBST/5% normal goat serum: to 5 ml 1X TBST add250 μl normal goat serum) for 1 h at room temperature.Primary antibody was added (OCT4; Cell Signaling Technol-ogies) overnight at 4 °C. Antibodies were removed andsections were washed in wash buffer three times for 5 mineach. The amount of 400 μl biotinylated secondary antibodywas added to each section and incubated 30 min at roomtemperature. Sections were washed three times with washbuffer for 5 min each. DAB was added to each section todevelop slides.

Acknowledgments

We thank Angela Palermo and Andre Tartar for theirtechnical assistance in reformatting the manuscript. Thiswork was supported by Grant P01 HD 47675 from the NationalInstitutes of Health.

Appendix A. Supplementary data

Supplementary data associatedwith this article can be found,in the online version, at doi:10.1016/j.scr.2009.09.003.Access to the raw data can be found on Geo by followingthe following link http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18639.

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