Pluripotent stem cell (PSC) research, which emerged from the intensive investigations on embryonic stem cells, has tremendous importance for understanding fundamental concepts of early development, differentiation decisions and cell proliferation controls. Now with the intense focus on human PSCs, this previously fundamental science is rapidly moving towards more clinically relevant arenas. These fields include PSC differentiation for cell replacement, PSC progenitors of cancer, patient-specific and/or disease-specific induced PSCs for mechanistic and drug discoveries. These proposed medical implications of pluripotent stem expression patterns in human PSCs now demand increased scrutiny to assure that the discoveries are indeed accurate and unaffected by inherent biological and genetic limitations, i.e. the heterogeneity of the existing hESC lines; their sub-optimal origins from clinically-discarded embryos conceived in vitro
by infertility patients; their genetic anonymity; their extensive propagation in vitro
; among others. To address these concerns, we have conducted a completely independent analysis of `stemness' gene networks using pedigreed rhesus ESCs derived from fertile primates [23
]. Because these datasets do not rely on the existing hESC stemness gene expression patterns, it is noteworthy that the most prominent pluripotency genes, i.e. SOX2, OCT-4 and NANOG, emerge as dominant regulatory factors. In addition, unexplored candidate genes may be worthy targets for further research attention.
To insure that we did not have contaminations of differentiated cells within our undifferentiated cultures, or undifferentiated cells within our differentiated cultures, we routinely stained our undifferentiated cultures with “stemness markers”, i.e. OCT-4, NANOG, SSEA-3 and -4, as well as neuronal markers (data not shown). In addition, as depicted in supplementary figure 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 pure population of FACS sorted cells would be the ideal population for these experiments. However, since most nhpESC die in a single cell population, we refrained from FACS sorting. In addition, we grew both skin and teratoma fibroblasts for >4 passages before extracting their RNA to insure as pure a population of fibroblasts as possible.
When global gene expression patterns were characterized, we found that the nhpESC gene expression was closer to skin fibroblasts then to teratoma fibroblasts, although genetically they share their genome with the teratoma fibroblast and only half with skin fibroblasts. These results might indicate that the cells isolated from the skin and characterized as fibroblasts might harbor less well differentiated cells closer in their gene expression to authentic undifferentiated cells. Perhaps this result might shed light on the success rate of skin fibroblast to form iPS cells as compared to other types of cells [17
Although most of the genes depicted in are unidentified, some of the differentially expressed genes are associated to pluripotency, such as Oct-4 and Sox-2. In addition, others be found in future research to play roles in regulating the pluripotency <-> differentiation transitions. For example, TACSTD1, also called CD44 or Ep-CAM is an epithelial adhesion molecule that was originally identified as a marker of carcinomas[41
]. We found this gene to be the most differentially expressed gene between stem cells and fibroblasts, indicating that it might have other functions in signaling rather then solely adhesion. In contrast, decorin (DCN) has an important role in maintaining the balance in Peyronie's disease (PD) where fibrotic plaques are formed. DCN neutralizes one of the causes for plaque formation - transforming-growth-factor β1[43
]. Consequently it is perhaps not surprising that DCN was under expressed in the pluripotent stem cells we examined.
To identify “stemness genes” a comparison between gene expression of pluripotent cells to their in vitro
differentiated counterparts was carried out [44
]. While genetic background is identical, the presence of some undifferentiated or partially differentiated cells can not be ruled out. In contrast, a range of undifferentiated cells can be used for the common denominator[s] that defines the pluripotent state [44
], or the comparison of gene expression between and among different stem cell lines. These experiments usually showed that hESCs are quite different from each other [16
]. A more comprehensive study showed that although closely related, the 59 ESC lines showed heterogeneity in gene expression [54
]. Interestingly, variations in gene expression were found not only in genes correlated with the pluripotent state or differentiation, but also in the expression of housekeeping genes [55
]. Therefore, interactions among many genes forming an active network that will allow the pluripotent state to be maintained is likely at work[56
The first two studies that characterized the “stemness gene” list [49
] identified about 250 putative genes involved in mESC pluripotency, and many other genes are being studied today [16
]. Stemness gene lists have been generated by comparing different types of pluripotent cells [57
] or by comparing differentiated cells to differentiated cells [47
]. It is interesting to note that OCT-4 is on chromosome 6p21.33, SOX-2 3q26.3 and NANOG 12p13.31, so none of this important pluripotency triad are represented on the over expressed chromosomes found here. Further analysis is underway to identify these pluripotency regulatory clusters.
It has been well established that a number of key proteins play an important role in the maintenance of pluripotency both in mESCs and hESCs, such as OCT-4, NANOG and SOX2 [44
] as well as the existence of a regulatory network in mESC [63
] for SOX2 [64
], OCT-4 [66
] and NANOG [67
]. Similar results were shown in hESCs since hESCs treated with RNAi against SOX2 cells readily differentiated [68
]. Although there is much information on the cooperation activity of OCT-4, NANOG and SOX2, we still lack information regarding other key players in the maintenance of the pluripotent state.
To better understand the extensive list of genes involved in the differences between cell lines, research has been conducted on specific pathways that are differentially expressed. Rho et al [69
] found that the Wnt, Hh, Notch pathways but not the JAK/STAT had over-abundant transcripts in ESC compared to EBs. Therefore they concluded that self renewal is a coordinated signaling-specific mechanism. In addition, Li et al [70
] identified a transcriptome involved in this differentiation process. Still, most genes that are differentially expressed have yet to be identified (ESTs) or have not been correlated with pluripotency [71
]. Studying them will enhance our understanding of the pluripotent state.
In order to achieve an orchestrated pathway in development, a well coordinated machinery must be activated to turn off pluripotent genes and turn on the expression of differentiation genes. This process is usually carried out by transcription factors as shown in mESCs [72
] and hESCs [73
]. These transcription factors can together form a hierarchy in a complex network that maintains the pluripotent state [74
] and include reprogramming factors used for iPS (OCT-4, SOX2, KLF-4 and c-MYC) as well as NANOG, DAX-1, REX-1, ZPF281 and NAC-1. Other DNA remodeling proteins have been associated with pluripotency in mESC [63
] and both the similarities and differences between the mouse networks generated by Zhou et al[63
] and ours are informative. Both found genes not yet appreciated as regulators of pluripotency.
Recently, new mathematical algorithms have been developed to help identify pluripotent genes [75
]. Analyses using them showed that every hESC has its unique signature but shares many genes with other hESC lines that help maintain the pluripotent state[76
]. However, that analysis was based on the comparison of three hESC lines that, although similar, still contain many differences[52
While many studies have been carried out on mESC and hESC, very few have searched for pluripotent genes in nhpESC. A single study has compared rhesus ESC to EBs [79
]. They identified 367 genes that were expressed in 5 nhpESC lines and include CCNB1, GDNF3, LeftB, OCT-4 and NANOG. These 367 genes may represent the stemness core allowing the cells to maintain the pluripotent state.