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New Insights into the Control of Stem Cell Pluripotency

2008; Elsevier BV; Volume: 2; Issue: 1 Linguagem: Inglês

10.1016/j.stem.2007.12.007

1934-5909

Paul J. Gokhale, Peter W. Andrews,

CRISPR and Genetic Engineering

Nanog is a transcription factor that is expressed by mouse and human embryonic stem (ES) cells and by primordial germ cells. New research published recently in Nature (Chambers et al., 2007Chambers I. Silva J. Colby D. Nichols J. Nijmeijer B. Robertson M. Vrana J. Jones K. Grotewold L. Smith A. Nature. 2007; 450: 1230-1234Crossref PubMed Scopus (1088) Google Scholar) points to an unexpected role for Nanog in the maintenance of pluripotency in mouse ES cells. Nanog is a transcription factor that is expressed by mouse and human embryonic stem (ES) cells and by primordial germ cells. New research published recently in Nature (Chambers et al., 2007Chambers I. Silva J. Colby D. Nichols J. Nijmeijer B. Robertson M. Vrana J. Jones K. Grotewold L. Smith A. Nature. 2007; 450: 1230-1234Crossref PubMed Scopus (1088) Google Scholar) points to an unexpected role for Nanog in the maintenance of pluripotency in mouse ES cells. ES cells combine the capacity for indefinite growth in an undifferentiated state with the ability to differentiate into all tissues of an adult mouse. When they were first derived by explanting early mouse embryos into culture, success depended crucially upon the use of inactivated fibroblasts as feeder cells. However, the nature of the molecular machinery that maintained the two key features of ES cells, their capacity for self renewal and pluripotency, and the role of the feeder cells were obscure. The first clue came from studies that showed that a single growth factor, leukaemia inhibitory factor (LIF), was able to replace the need for the feeder cells and allow culture of mouse ES cells indefinitely without the use of feeders (Smith et al., 1988Smith A.G. Heath J.K. Donaldson D.D. Wong G.G. Moreau J. Stahl M. Rogers D. Nature. 1988; 336: 688-690Crossref PubMed Scopus (1410) Google Scholar). Building on this discovery, further work revealed that LIF appeared to act by balancing two signaling cascades within ES cells, one through STAT3 that promoted self-renewal and one through the ERK pathway that appeared to promote differentiation. Next came the discovery that the precise level of a transcription factor, Oct4, which is normally downregulated upon ES cell differentiation, is crucial for the maintenance of the undifferentiated state. If the levels of Oct4 were forcibly reduced by genetic manipulation, differentiation to trophectoderm occurred, whereas if the levels were raised, differentiation to endoderm is promoted (Niwa et al., 2000Niwa H. Miyazaki J. Smith A.G. Nat. Genet. 2000; 24: 372-376Crossref PubMed Scopus (2784) Google Scholar). However, both LIF and the correct levels of Oct4 were required to maintain the undifferentiated state. Other genes also showed marked developmental regulation in ES cells, and the question arose as to whether any of these might act upstream of Oct4 or LIF to maintain a pluripotent state. One of these, Nanog, initially appeared to be the key gene required to maintain the pluripotent state (Mitsui et al., 2003Mitsui K. Tokuzawa Y. Itoh H. Segawa K. Murakami M. Takahashi K. Maruyama M. Maeda M. Yamanaka S. Cell. 2003; 113: 631-642Abstract Full Text Full Text PDF PubMed Scopus (2453) Google Scholar, Chambers et al., 2003Chambers I. Colby D. Robertson M. Nichols J. Lee S. Tweedie S. Smith A. Cell. 2003; 113: 643-655Abstract Full Text Full Text PDF PubMed Scopus (2537) Google Scholar). Overexpression of Nanog prevented differentiation of ES cells and allowed their maintenance in the absence of LIF. However, continued expression of Oct4 was also necessary for maintenance of an undifferentiated stem cell state. A model of pluripotency emerged in which Oct4 and Nanog appeared to act at the same level to both promote self-renewal and suppress differentiation. ChIP-on-chip studies further revealed a close relationship between Oct4 and Nanog and another pluripotency-associated factor, Sox2, all of which appear to interact in feedback regulatory circuits (Boyer et al., 2005Boyer L.A. Lee T.I. Cole M.F. Johnstone S.E. Levine S.S. Zucker J.P. Guenther M.G. Kumar R.M. Murray H.L. Jenner R.G. et al.Cell. 2005; 122: 947-956Abstract Full Text Full Text PDF PubMed Scopus (3285) Google Scholar). Together with other reports, these observations pointed to Nanog, Oct4, and Sox2 acting together as master regulators of pluripotency. Nevertheless, when Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (16937) Google Scholar recently described that four genes, Oct4, Sox2, Klf4, and c-Myc, were sufficient to reprogram adult somatic cells to an ES cell-like state, so-called induced pluripotent stem (iPS) cells, Nanog was not among them. New results reported in Nature (Chambers et al., 2007Chambers I. Silva J. Colby D. Nichols J. Nijmeijer B. Robertson M. Vrana J. Jones K. Grotewold L. Smith A. Nature. 2007; 450: 1230-1234Crossref PubMed Scopus (1088) Google Scholar) provide some new and surprising insights into the role of Nanog, and perhaps into the nature of the stem cell state itself. Because earlier studies had indicated that some Oct4(+) cells did not appear to express Nanog, the authors inserted the fluorescent reporter protein eGFP into the Nanog locus of mouse ES cells by homologous recombination. Subsets of eGFP(+) [Nanog(+)] and eGFP(−) [Nanog(−)] cells were found in these transgenic ES cultures, but both expressed other ES markers, SSEA1 and Oct4. Importantly, after isolation by FACS, the eGFP(−) [Nanog(−)] cells gave rise to eGFP(+) [Nanog(+)] cells; the undifferentiated stem cells appeared to oscillate between a Nanog(+) and a Nanog(−) state. In further experiments, the authors derived Nanog null ES cells and showed that these could proliferate as pluripotent stem cells. Thus, they found that expression of Nanog is not essential for maintenance of the undifferentiated stem cell state. In their model, the Nanog(−) cells appear to be more susceptible to differentiation, whereas the expression of Nanog appears to suppress differentiation. In addition, Nanog is normally expressed in primordial germ cells. In contrast with its expendable function in ES cells, however, these cells do not appear to survive in the absence of Nanog. The authors suggest that the common feature might be a role of Nanog in maintaining survival of cells in an epigenetic plastic state. Putting all this together, Nanog appears to be acting in a modulator capacity rather than as a “core” pluripotency determinant. The apparent separation of undifferentiated ES cells into two interchangeable states reflects studies of other stem cell systems in which it is becoming apparent that the stem cell compartment may be often heterogeneous, with the cells capable of adopting various interchangeable substates. For example, within the intestinal crypt, there are suggestions that the stem cell compartment is not composed of a discrete entity but that cells that under some circumstances might be termed progenitor cells can under other circumstances convert back to an apparently true stem cell-like state (Booth and Potten, 2000Booth C. Potten C.S. J. Clin. Invest. 2000; 105: 1493-1499Crossref PubMed Scopus (287) Google Scholar). Likewise substates within the hematopoietic stem cell compartment are becoming evident (Rosu-Myles et al., 2000Rosu-Myles M. Gallacher L. Murdoch B. Hess D.A. Keeney M. Kelvin D. Dale L. Ferguson S.S. Wu D. Fellows F. et al.Proc. Natl. Acad. Sci. USA. 2000; 97: 14626-14631Crossref PubMed Scopus (103) Google Scholar). How does all this relate to human embryonic stem cells? Recently we described substates within the stem cell compartment in human ES cells cultures (Enver et al., 2005Enver T. Soneji S. Joshi C. Brown J. Iborra F. Orntoft T. Thykjaer T. Maltby E. Smith K. Dawud R.A. et al.Hum. Mol. Genet. 2005; 14: 3129-3140Crossref PubMed Scopus (238) Google Scholar). In this case, the phenomenon of culture adaptation provided an experimental situation in which we were able to suggest that human ES cells can fluctuate between two states that express, or do not express, the surface antigen SSEA3 yet retain the undifferentiated state. When human ES cells acquire karyotypic abnormalities and adapt to culture, the cells seem to be trapped within the stem cell compartment, which allows observation of the two SSEA3(+) and SSEA3(−) substates. Intriguingly, adaptation of human ES cells often involves the acquisition of extra copies of chromosome 12, on which includes the locus for Nanog. The emergence of multiple states within the stem cell compartment of pluripotent cells points to more probabilistic rather than deterministic models of the maintenance and exit from the stem cell state. The results also emphasize that elucidation of networks of potential interacting genes by methods such as ChIP on chip must be backed up with functional assays.