Resetting the Epigenome beyond Pluripotency in the Germline
2009; Elsevier BV; Volume: 4; Issue: 6 Linguagem: Inglês
10.1016/j.stem.2009.05.007
ISSN1934-5909
AutoresKatsuhiko Hayashi, M. Azim Surani,
Tópico(s)CRISPR and Genetic Engineering
ResumoGerm cells undergo comprehensive epigenetic reprogramming toward acquiring fitness for pluripotency and totipotency. Notably, the full extent of the epigenetic reprogramming experienced by germ cells remains unmatched by attempts to experimentally restore pluripotency in somatic cells. We propose that the defects present in experimentally generated cells are corrected upon differentiation into the germ cell lineage, as has been observed in cases of germline transmission. Unraveling the mechanisms responsible for germ cell-specific epigenetic reprogramming will likely have important implications for both basic and clinical stem cell research. Germ cells undergo comprehensive epigenetic reprogramming toward acquiring fitness for pluripotency and totipotency. Notably, the full extent of the epigenetic reprogramming experienced by germ cells remains unmatched by attempts to experimentally restore pluripotency in somatic cells. We propose that the defects present in experimentally generated cells are corrected upon differentiation into the germ cell lineage, as has been observed in cases of germline transmission. Unraveling the mechanisms responsible for germ cell-specific epigenetic reprogramming will likely have important implications for both basic and clinical stem cell research. Epigenetic reprogramming of somatic cells, for example, by nuclear transplantation into an oocyte, frequently leads to defects in the resulting conceptus and cells (Hochedlinger and Jaenisch, 2002Hochedlinger K. Jaenisch R. Curr. Opin. Cell Biol. 2002; 14: 741-748Crossref PubMed Scopus (97) Google Scholar, Tamashiro et al., 2003Tamashiro K.L. Wakayama T. Yamazaki Y. Akutsu H. Woods S.C. Kondo S. Yanagimachi R. Sakai R.R. Exp. Biol. Med. (Maywood). 2003; 228: 1193-1200PubMed Google Scholar). Many of these defects are eliminated upon transmission through the germline, suggesting that they are epigenetic in nature and reversible (Shimozawa et al., 2002Shimozawa N. Ono Y. Kimoto S. Hioki K. Araki Y. Shinkai Y. Kono T. Ito M. Genesis. 2002; 34: 203-207Crossref PubMed Scopus (81) Google Scholar, Tamashiro et al., 2002Tamashiro K.L. Wakayama T. Akutsu H. Yamazaki Y. Lachey J.L. Wortman M.D. Seeley R.J. D'Alessio D.A. Woods S.C. Yanagimachi R. Sakai R.R. Nat. Med. 2002; 8: 262-267Crossref PubMed Scopus (301) Google Scholar). Cells derived via recent advances in reprogramming, including experimentally induced pluripotent stem cells (iPSCs), require critical evaluation of their properties with respect to the events in the germline, given that these cells may lack all of the attributes of an authentic pluripotent state. Here, we discuss our growing knowledge of the mammalian germ cell lineage and the implications of these findings to the experimental manipulation of epigenetic states. Primordial germ cells (PGCs) originate during development from postimplantation epiblast cells, which, in turn, arise from the pluripotent primitive ectoderm cells of the inner-cell mass (PEct/ICM) of blastocysts (McLaren and Lawson, 2005McLaren A. Lawson K.A. Differentiation. 2005; 73: 435-437Crossref PubMed Scopus (67) Google Scholar). Development of the postimplantation epiblast is accompanied by epigenetic modifications that are generally irreversible, including X inactivation when this chromosome switches from early to late replication (Takagi et al., 1982Takagi N. Sugawara O. Sasaki M. Chromosoma. 1982; 85: 275-286Crossref PubMed Scopus (143) Google Scholar); this alteration is perhaps a hallmark of genome-wide irreversible epigenetic changes and may involve DNA methylation. Other changes, including histone modifications and DNA methylation, also ensue during differentiation of the epiblast and appear to be required in that their absence in the wake of mutation to several key epigenetic regulators results in early embryonic lethality (Surani et al., 2007Surani M.A. Hayashi K. Hajkova P. Cell. 2007; 128: 747-762Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar). Thus, PGCs originate from epiblast cells that have initiated the process of differentiation toward somatic cell lineages (Ohinata et al., 2005Ohinata Y. Payer B. O'Carroll D. Ancelin K. Ono Y. Sano M. Barton S.C. Obukhanych T. Nussenzweig M. Tarakhovsky A. et al.Nature. 2005; 436: 207-213Crossref PubMed Scopus (717) Google Scholar), as reflected in their transcriptional profile. Whereas the majority of epiblast cells continue to develop toward diverse somatic fates, this trend is arrested in a few epiblast cells destined to form the PGCs (Figure 1). Blimp1/Prdm1, a transcriptional repressor and the key germ cell determinant in mammals, initiates the reversion of differentiating epiblast cells by repressing the somatic program and initiating the germ cell program at embryonic day (E) 6.25 (Ohinata et al., 2005Ohinata Y. Payer B. O'Carroll D. Ancelin K. Ono Y. Sano M. Barton S.C. Obukhanych T. Nussenzweig M. Tarakhovsky A. et al.Nature. 2005; 436: 207-213Crossref PubMed Scopus (717) Google Scholar). Additional changes follow, including re-expression of pluripotency genes such as Nanog and Sox2 (Yabuta et al., 2006Yabuta Y. Kurimoto K. Ohinata Y. Seki Y. Saitou M. Biol. Reprod. 2006; 75: 705-716Crossref PubMed Scopus (214) Google Scholar, Yamaguchi et al., 2005Yamaguchi S. Kimura H. Tada M. Nakatsuji N. Tada T. Gene Expr. Patterns. 2005; 5: 639-646Crossref PubMed Scopus (230) Google Scholar) in nascent PGCs, but not in the other differentiating epiblast cells. These adjustments suggest a trend toward reversion to an earlier ICM-like epigenetic state (Figure 1), although nascent PGCs still possess some characteristics of epiblast cells, such as an inactive X chromosome (Chuva de Sousa Lopes et al., 2008Chuva de Sousa Lopes S.M. Hayashi K. Shovlin T.C. Mifsud W. Surani M.A. McLaren A. PLoS Genet. 2008; 4: e30Crossref PubMed Scopus (123) Google Scholar, de Napoles et al., 2007de Napoles M. Nesterova T. Brockdorff N. PLoS ONE. 2007; 2: e860Crossref PubMed Scopus (71) Google Scholar, Sugimoto and Abe, 2007Sugimoto M. Abe K. PLoS Genet. 2007; 3: e116Crossref PubMed Scopus (144) Google Scholar). This epigenetic memory of the cell's initial trajectory toward a somatic fate is erased progressively in PGC precursors. Epigenetic reprogramming events commence immediately after PGC specification at E7.25, which is marked by the detection of Stella/Dppa3 (Saitou et al., 2002Saitou M. Barton S.C. Surani M.A. Nature. 2002; 418: 293-300Crossref PubMed Scopus (679) Google Scholar, Sato et al., 2002Sato M. Kimura T. Kurokawa K. Fujita Y. Abe K. Masuhara M. Yasunaga T. Ryo A. Yamamoto M. Nakano T. Mech. Dev. 2002; 113: 91-94Crossref PubMed Scopus (198) Google Scholar). These epigenetic changes are accompanied by downregulation of genes implicated in DNA methylation and changes in histone modifications (Yabuta et al., 2006Yabuta Y. Kurimoto K. Ohinata Y. Seki Y. Saitou M. Biol. Reprod. 2006; 75: 705-716Crossref PubMed Scopus (214) Google Scholar). Notably, genes, including Glp and Dnmt3b, are downregulated. As a result, a global loss of histone H3 lysine 9 (H3K9me2) methylation is observed in PGCs between E7.5 and E8.5, whereas the Ezh2-dependent H3K27me3 modification is accentuated (Hajkova et al., 2008Hajkova P. Ancelin K. Waldmann T. Lacoste N. Lange U.C. Cesari F. Lee C. Almouzni G. Schneider R. Surani M.A. Nature. 2008; 452: 877-881Crossref PubMed Scopus (482) Google Scholar, Seki et al., 2005Seki Y. Hayashi K. Itoh K. Mizugaki M. Saitou M. Matsui Y. Dev. Biol. 2005; 278: 440-458Crossref PubMed Scopus (376) Google Scholar). Although the specific loci remodeled by these global histone modifications in PGCs remain to be elucidated, the overall effect in PGCs is to shift them toward the ICM/ESC-like epigenetic state. Indeed, with these changes, PGCs acquire the potential to dedifferentiate into pluripotent embryonic germ (EG) cells that are virtually identical to ESCs (see below). There are no published reports on the derivation of EG cells from PGCs prior to E8.5, which may indicate the significance of resetting the epigenome between E7.5 and E8.5 in nascent PGCs (Figure 1). Recent evidence demonstrates that Prdm14, another PR domain-containing transcriptional regulator that is detected shortly after Blimp1/Prdm1 in PGC precursors, plays a pivotal role in regulating epigenetic changes in nascent PGCs. Gene disruption of Prdm14 hampers both the loss of H3K9me2 and the enhancement of K3K27me3. Consequently, PGC development and the derivation of EG cells are impaired in the Prdm14 mutant embryos (Yamaji et al., 2008Yamaji M. Seki Y. Kurimoto K. Yabuta Y. Yuasa M. Shigeta M. Yamanaka K. Ohinata Y. Saitou M. Nat. Genet. 2008; 40: 1016-1022Crossref PubMed Scopus (384) Google Scholar). Thus, Blimp1/Prdm1 and Prdm14 appear to work in tandem to repress the somatic program and initiate the germ cell-specific epigenetic program. Coupled with the global epigenetic modifications that are observed in PGCs, some key pluripotency genes are also re-expressed. Included in this list are Nanog and Sox2 (Yabuta et al., 2006Yabuta Y. Kurimoto K. Ohinata Y. Seki Y. Saitou M. Biol. Reprod. 2006; 75: 705-716Crossref PubMed Scopus (214) Google Scholar, Yamaguchi et al., 2005Yamaguchi S. Kimura H. Tada M. Nakatsuji N. Tada T. Gene Expr. Patterns. 2005; 5: 639-646Crossref PubMed Scopus (230) Google Scholar), which are normally repressed in postimplantation epiblast cells and are the very factors involved in the reversion of somatic cells to pluripotent stem cells (Silva et al., 2006Silva J. Chambers I. Pollard S. Smith A. Nature. 2006; 441: 997-1001Crossref PubMed Scopus (285) Google Scholar, Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (16979) Google Scholar). A recent study also reveals that the binding of Nanog, Oct4, and Sox2 to a regulatory element of Xist, a noncoding RNA, may help to induce reactivation of the X chromosome (Navarro et al., 2008Navarro P. Chambers I. Karwacki-Neisius V. Chureau C. Morey C. Rougeulle C. Avner P. Science. 2008; 321: 1693-1695Crossref PubMed Scopus (251) Google Scholar). However, reactivation of the X chromosome occurs in a protracted manner in PGCs compared to PEct; X reactivation commences at around E7.0 and is not completed until E12.5 in PGCs, but it occurs within a day in PEct (Mak et al., 2004Mak W. Nesterova T.B. de Napoles M. Appanah R. Yamanaka S. Otte A.P. Brockdorff N. Science. 2004; 303: 666-669Crossref PubMed Scopus (382) Google Scholar, Okamoto et al., 2004Okamoto I. Otte A.P. Allis C.D. Reinberg D. Heard E. Science. 2004; 303: 644-649Crossref PubMed Scopus (583) Google Scholar). This difference may be because Xist repression in PEct involves histone modifications, but the late replication of the X chromosome in epiblast cells (Takagi et al., 1982Takagi N. Sugawara O. Sasaki M. Chromosoma. 1982; 85: 275-286Crossref PubMed Scopus (143) Google Scholar) may be coupled with DNA methylation, which is initially inherited by nascent PGCs. As described above, reprogramming in early germ cells results in PGCs from E8.5–E11.5 embryos being in a permissive state with respect to their potential to give rise to pluripotent EG cells. EG cells are virtually identical to ESCs, except for the loss of DNA methylation from imprinted gene loci in EG cells (Shovlin et al., 2008Shovlin T.C. Durcova-Hills G. Surani A. McLaren A. Dev. Biol. 2008; 313: 674-681Crossref PubMed Scopus (44) Google Scholar, Tada et al., 2001Tada M. Takahama Y. Abe K. Nakatsuji N. Tada T. Curr. Biol. 2001; 11: 1553-1558Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar). Both ESCs and EG cells have two active X chromosomes in female cells, can contribute to chimeras and the germline, and have transcriptomes that are very similar (Sharova et al., 2007Sharova L.V. Sharov A.A. Piao Y. Shaik N. Sullivan T. Stewart C.L. Hogan B.L. Ko M.S. Dev. Biol. 2007; 307: 446-459Crossref PubMed Scopus (86) Google Scholar). Despite their similarities, EG cells and ESCs have distinctive origins from PGC and PEct, respectively. PGCs are the founders of a unipotent lineage that generates sperm and eggs only, whereas PEct give rise to all of the somatic fetal tissues, as well as to germ cells (Figure 1). To restrict their cell fate, PGCs exhibit lineage-specific gene expression, including Blimp1 and Nanos3 (Ohinata et al., 2005Ohinata Y. Payer B. O'Carroll D. Ancelin K. Ono Y. Sano M. Barton S.C. Obukhanych T. Nussenzweig M. Tarakhovsky A. et al.Nature. 2005; 436: 207-213Crossref PubMed Scopus (717) Google Scholar, Tsuda et al., 2003Tsuda M. Sasaoka Y. Kiso M. Abe K. Haraguchi S. Kobayashi S. Saga Y. Science. 2003; 301: 1239-1241Crossref PubMed Scopus (429) Google Scholar). Of particular note for the maintenance of early unipotent germ cell lineage is the presence of a Blimp1-Prmt5 repressive complex; Prmt5 is a histone H2A/H4 symmetrical arginine 3 demethylase (H2A/H4R3me2s) (Ancelin et al., 2006Ancelin K. Lange U.C. Hajkova P. Schneider R. Bannister A.J. Kouzarides T. Surani M.A. Nat. Cell Biol. 2006; 8: 623-630Crossref PubMed Scopus (353) Google Scholar). During EG cell derivation, Blimp1 is rapidly downregulated (Durcova-Hills et al., 2008Durcova-Hills G. Tang F. Doody G. Tooze R. Surani M.A. PLoS ONE. 2008; 3: e3531Crossref PubMed Scopus (121) Google Scholar), which likely reverses restriction on the germ cell lineage, while Prmt5 assumes another role in promoting pluripotency. The Blipm1-Prmt5 complex translocates to the cytoplasm at E12.5 (Ancelin et al., 2006Ancelin K. Lange U.C. Hajkova P. Schneider R. Bannister A.J. Kouzarides T. Surani M.A. Nat. Cell Biol. 2006; 8: 623-630Crossref PubMed Scopus (353) Google Scholar), precisely when the ability to generate EG cells from PGCs ceases. Thus, the Blimp1-Prmt5 complex may safeguard unipotency of early germ cells, but it may also have a role in epigenetic reprogramming itself. These hypotheses are testable predictions that will be interesting to tackle in the future. It is known that human ESCs that resemble pluripotent epiblast stem cells (EpiSCs) (Brons et al., 2007Brons I.G. Smithers L.E. Trotter M.W. Rugg-Gunn P. Sun B. Chuva de Sousa Lopes S.M. Howlett S.K. Clarkson A. Ahrlund-Richter L. Pedersen R.A. Vallier L. Nature. 2007; 448: 191-195Crossref PubMed Scopus (1426) Google Scholar, Tesar et al., 2007Tesar P.J. Chenoweth J.G. Brook F.A. Davies T.J. Evans E.P. Mack D.L. Gardner R.L. McKay R.D. Nature. 2007; 448: 196-199Crossref PubMed Scopus (1569) Google Scholar) can generate cells with characteristics resembling PGCs (Clark et al., 2004Clark A.T. Bodnar M.S. Fox M. Rodriquez R.T. Abeyta M.J. Firpo M.T. Pera R.A. Hum. Mol. Genet. 2004; 13: 727-739Crossref PubMed Scopus (432) Google Scholar). It is possible that the significance of epigenetic reprogramming in PGCs may become evident by investigating EpiSCs, which are derived from E5.5–E6.5 postimplantation embryos. EpiSCs differ significantly from ESC/EG cells in their overall transcription profile and in their epigenetic state (Brons et al., 2007Brons I.G. Smithers L.E. Trotter M.W. Rugg-Gunn P. Sun B. Chuva de Sousa Lopes S.M. Howlett S.K. Clarkson A. Ahrlund-Richter L. Pedersen R.A. Vallier L. Nature. 2007; 448: 191-195Crossref PubMed Scopus (1426) Google Scholar, Hayashi et al., 2008Hayashi K. Lopes S.M. Tang F. Surani M.A. Cell Stem Cell. 2008; 3: 391-401Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar, Tesar et al., 2007Tesar P.J. Chenoweth J.G. Brook F.A. Davies T.J. Evans E.P. Mack D.L. Gardner R.L. McKay R.D. Nature. 2007; 448: 196-199Crossref PubMed Scopus (1569) Google Scholar), even though this population also exhibits expression of key pluripotency-specific genes (Figure 2A). However, EpiSCs have an inactive X chromosome and possibly hypermethylation of CpG sequences of some pluripotency genes, such as stella and Rex1/Zfp42. EpiSCs can neither contribute to adult chimeras, which precludes their contribution to PGCs in vivo (Brons et al., 2007Brons I.G. Smithers L.E. Trotter M.W. Rugg-Gunn P. Sun B. Chuva de Sousa Lopes S.M. Howlett S.K. Clarkson A. Ahrlund-Richter L. Pedersen R.A. Vallier L. Nature. 2007; 448: 191-195Crossref PubMed Scopus (1426) Google Scholar), nor be easily converted to ESCs (Guo et al., 2009Guo G. Yang J. Nichols J. Hall J.S. Eyres I. Mansfield W. Smith A. Development. 2009; 136: 1063-1069Crossref PubMed Scopus (563) Google Scholar). If PGCs can be derived from EpiSCs in vitro and if these PGCs undergo appropriate epigenetic reprogramming, they may, in turn, be induced to give rise to EG cells. These results would demonstrate that reprogramming in PGCs is a route through which the EpiSC epigenome can be remodeled and perhaps reverted to the ICM/ESC-like pluripotent state (Figure 2A). Thus, the epigenetic barrier that is created during development of the epiblast and EpiScs may be breached during PGC development. The most significant epigenetic reprogramming event in the germline is genome-wide DNA demethylation and extensive histone modifications that take place in gonadal PGCs (Hajkova et al., 2008Hajkova P. Ancelin K. Waldmann T. Lacoste N. Lange U.C. Cesari F. Lee C. Almouzni G. Schneider R. Surani M.A. Nature. 2008; 452: 877-881Crossref PubMed Scopus (482) Google Scholar). Though DNA demethylation does occur to some extent during preimplantation development (Howlett and Reik, 1991Howlett S.K. Reik W. Development. 1991; 113: 119-127Crossref PubMed Google Scholar, Monk et al., 1991Monk M. Adams R.L. Rinaldi A. Development. 1991; 112: 189-192PubMed Google Scholar), there is no process equivalent to the reprogramming observed in germ cells reported in cells of other lineages, and this process accounts for the complete erasure and resetting of the epigenome in the germline. This specialized reprogramming may account for the rare incidences of transgenerational inheritance of epimutations (Whitelaw and Whitelaw, 2008Whitelaw N.C. Whitelaw E. Curr. Opin. Genet. Dev. 2008; 18: 273-279Crossref PubMed Scopus (134) Google Scholar). The targeted modifications in germ cells also ensure that imprinted genes and retrotransposons, such as LINE1, are remodeled appropriately (Hajkova et al., 2002Hajkova P. Erhardt S. Lane N. Haaf T. El-Maarri O. Reik W. Walter J. Surani M.A. Mech. Dev. 2002; 117: 15-23Crossref PubMed Scopus (925) Google Scholar). Notably, there is no strictly equivalent phenomenon in PEct/ICM, in ESCs, or in other experimentally derived pluripotent cells. The lack of a similar remodeling of the genome in the experimentally generated pluripotent cells may affect their functional properties (see below). Evidence suggests that remodeling of retrotransposon-associated and imprinted genes in germ cells also contributes to totipotency by restoring the potential for subsequent fetal and placental development. Genes associated with retrotransposable elements include Peg10 and Rtl1 (also known as Peg11), which are essential for placental development (Ono et al., 2006Ono R. Nakamura K. Inoue K. Naruse M. Usami T. Wakisaka-Saito N. Hino T. Suzuki-Migishima R. Ogonuki N. Miki H. et al.Nat. Genet. 2006; 38: 101-106Crossref PubMed Scopus (274) Google Scholar, Sekita et al., 2008Sekita Y. Wagatsuma H. Nakamura K. Ono R. Kagami M. Wakisaka N. Hino T. Suzuki-Migishima R. Kohda T. Ogura A. et al.Nat. Genet. 2008; 40: 243-248Crossref PubMed Scopus (216) Google Scholar). Several human placental genes, such as Endothelin B receptor, Insl4, Leptin, Midline1, and Pleiotrophin, are also associated with retrotransposable elements (Rawn and Cross, 2008Rawn S.M. Cross J.C. Annu. Rev. Cell Dev. Biol. 2008; 24: 159-181Crossref PubMed Scopus (165) Google Scholar). It is likely that genome-wide DNA demethylation exclusively in gonadal PGCs contributes to resetting these genes and others that are critical for development of the conceptus after fertilization. Remodeling of the epigenetic status of retrotransposable elements, however, may cause mutagenesis in the genome by active transposition. Recent studies elegantly demonstrate that small RNA pathways, such as piRNA (also known as gsRNA) and endogenous siRNA, play a crucial role in suppressing the expression of retrotransposons (Aravin et al., 2006Aravin A. Gaidatzis D. Pfeffer S. Lagos-Quintana M. Landgraf P. Iovino N. Morris P. Brownstein M.J. Kuramochi-Miyagawa S. 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Recent studies also reveal characteristic remodeling of the epigenetic status of Rhox genes, which were initially identified as a gene cluster of the reproductive homeobox on the X chromosome. Detailed analysis clearly revealed that expression of these genes commences exclusively in PGCs at E12.5 PGCs (Daggag et al., 2008Daggag H. Svingen T. Western P.S. van den Bergen J.A. McClive P.J. Harley V.R. Koopman P. Sinclair A.H. Biol. Reprod. 2008; 79: 468-474Crossref PubMed Scopus (25) Google Scholar), when massive DNA demethylation occurs. Comparison of the epigenetic status of the Rhox gene cluster in PGCs and in fetal and placental tissues will clarify the significance of remodeling these genes in PGCs. Apart from the germline, epigenetic reprogramming also takes place during the establishment of pluripotent cells in the ICM, as exemplified by reactivation of the inactive paternal X chromosome (Mak et al., 2004Mak W. Nesterova T.B. de Napoles M. Appanah R. Yamanaka S. Otte A.P. 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If substantiated, this hypothesis may explain why mouse ESCs differ from human ESCs; for example, mouse female ESCs express two active X chromosomes, whereas the vast majority of human female ESCs retain an inactive X chromosome (Dhara and Benvenisty, 2004Dhara S.K. Benvenisty N. Nucleic Acids Res. 2004; 32: 3995-4002Crossref PubMed Scopus (67) Google Scholar, Shen et al., 2007Shen J. Riggs P.K. Hensley S.C. Schroeder L.J. Traner A.R. Kochan K.J. Person M.D. DiGiovanni J. Mol. Carcinog. 2007; 46: 331-340Crossref PubMed Scopus (11) Google Scholar). In addition, hESCs more closely resemble mouse EpiSCs than ESCs, and both of the former require bFGF/Activin for their self-renewal, whereas mouse ESCs/EG cells require LIF/STAT3 signaling to retain their pluripotent state (Niwa et al., 1998Niwa H. Burdon T. Chambers I. Smith A. Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1192) Google Scholar). Whether the observed differences in patterns of X inactivation and the signaling requirements of mouse and human ESCs are functionally connected remains to be determined. However, it is possible that the ICM/PEct in human embryos may continue development toward a postimplantation epiblast-like stage during the derivation of hESC from blastocysts. Based on the available evidence, it does appear that the extensive epigenetic reprogramming observed in mouse germ cells may also occur in the human, given that extensive DNA demethylation would be required to reset the imprints and for X reactivation. That these modifications occur seems particularly likely because human EG cells, unlike hESC, are dependent on LIF/STAT signaling (Figure 2A) (Shamblott et al., 1998Shamblott M.J. Axelman J. Wang S. Bugg E.M. Littlefield J.W. Donovan P.J. Blumenthal P.D. Huggins G.R. Gearhart J.D. Proc. Natl. Acad. Sci. USA. 1998; 95: 13726-13731Crossref PubMed Scopus (1222) Google Scholar). Spermatogonia-derived human pluripotent stem cells are also similar to mouse ES/EG cells (Conrad et al., 2008Conrad S. Renninger M. Hennenlotter J. Wiesner T. Just L. Bonin M. Aicher W. Buhring H.J. Mattheus U. Mack A. et al.Nature. 2008; 456: 344-349Crossref PubMed Scopus (401) Google Scholar). The nature of epigenetic reprogramming events in human germ cells could be examined in PGCs derived from hESC, and parallel experiments may also be possible with mouse EpiSCs in vitro. A classical approach to restore totipotency/pluripotency in somatic nuclei is by transplantation into an oocyte (SCNT) (Campbell et al., 1996Campbell K.H. McWhir J. Ritchie W.A. Wilmut I. Nature. 1996; 380: 64-66Crossref PubMed Scopus (1398) Google Scholar, Wakayama et al., 1998Wakayama T. Perry A.C. Zuccotti M. Johnson K.R. Yanagimachi R. Nature. 1998; 394: 369-374Crossref PubMed Scopus (1884) Google Scholar). The transferred somatic nuclei are exposed to reprogramming factors in the oocyte, and as a result, they may acquire totipotency. Furthermore, during subsequent development of such reconstituted embryos to the blastocyst stage, donor nuclei may also undergo reprogramming in the ICM. In spite of this two-step reprogramming of somatic nuclei in early embryos, the resulting conceptuses often show both fetal and placental abnormalities, suggesting that neither the oocyte nor the ICM has the comprehensive potential to reset the epigenetic state of the somatic nucleus (Bao et al., 2005Bao S. Miyoshi N. Okamoto I. Jenuwein T. Heard E. Surani M.A. EMBO Rep. 2005; 6: 748-754Crossref PubMed Scopus (51) Google Scholar, Bortvin et al., 2003Bortvin A. Eggan K. Skaletsky H. Akutsu H. Berry D.L. Yanagimachi R. Page D.C. Jaenisch R. Development. 2003; 130: 1673-1680Crossref PubMed Scopus (376) Google Scholar). Many of these defects are, however, corrected upon transmission through the germline (Shimozawa et al., 2002Shimozawa N. Ono Y. Kimoto S. Hioki K. Araki Y. Shinkai Y. Kono T. Ito M. Genesis. 2002; 34: 203-207Crossref PubMed Scopus (81) Google Scholar, Tamashiro et al., 2002Tamashiro K.L. Wakayama T. Akutsu H. Yamazaki Y. Lachey J.L. Wortman M.D. Seeley R.J. D'Alessio D.A. Woods S.C. Yanagimachi R. Sakai R.R. Nat. Med. 2002; 8: 262-267Crossref PubMed Scopus (301) Google Scholar), demonstrating the comprehensive nature of epigenetic reprogramming upon passage through the germ cell lineage (Figure 2B). Though some of the epigenetic defects could be erased during epigenetic reprogramming in the ICM, rather than in germ cells, any defects present in the trophectoderm and other extraembryonic lineages would remain uncorrected. The experimentally generated human pluripotent stem cells (hESCs) and iPSC may show even greater defects compared to the mouse because, as discussed above, it is unclear whether human cells experience an equivalent reprogramming event as observed in the ICM of the mouse. In any case, it seems that the extensive epigenetic reprogramming and resetting of the epigenome observed in the germline, including genome-wide DNA demethylation as well as wide-ranging histone modifications, do not occur in the oocyte or in the ICM. The most important recent advance toward restoring pluripotency in somatic cells comes from Yamanaka's work using transcription factors, including Oct4, Sox2, Klf4, and c-Myc, to convert somatic cells into iPSCs that appear overtly equivalent to pluripotent ESCs (Takahashi and Yamanaka, 2006Takahashi K. Yamanaka S. Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (16979) Google Scholar). Considerable attention has been paid to the low frequency of the derivation of iPSCs, the protracted nature of the process, and some of the key properties of these cells (Hochedlinger and Plath, 2009Hochedlinger K. Plath K. Development. 2009; 136: 509-523Crossref PubMed Scopus (437) Google Scholar). From our perspective, however, the most important remaining question is how closely do iPSCs truly resemble ESCs derived from normal blastocysts. To generate iPSCs, the pluripotency-specific transcriptional factors introduced into somatic cells probably help to establish a new genetic network that evidently approximates the authentic pluripotency network, although it cannot be excluded that subtle yet important differences may be present. However, the generation of iPSCs from somatic cells does not appear to require systematic reversal of the entire developmental program that originally resulted in all of the diverse somatic cells with distinct phenotypes. Differentiation of somatic cells requires robust silencing of genes and regulatory elements that are not required in specific cell types. It is possible that epigenetic modifications associated with such silent genes that are robustly repressed in specific differentiated cells by DNA methylation may be difficult to reverse, as is the case with methylation of imprinted genes, which could make some key regulatory elements inaccessible to tissue-specific binding factors (Xu et al., 2007Xu J. Pope S.D. Jazirehi A.R. Attema J.L. Papathanasiou P. Watts J.A. Zaret K.S. Weissman I.L. Smale S.T. Proc. Natl. Acad. Sci. USA. 2007; 104: 12377-12382Crossref PubMed Scopus (90) Google Scholar). This hypothesis is plausible, given that pluripotency is the property being selected for in these experiments rather than the ability of the newly established iPSC to undergo terminal differentiation to a specific lineage. Thus, traces of residual epigenetic memory could exist in iPSCs, in that some genes that are silenced in specific cell types may remain silenced, and the residual memory marks may only be erased upon transmission through the germline. Therefore, it is essential to carry out comprehensive epigenetic analysis of iPSCs, for example, by using genome-wide methylation analysis by methylDip (Farthing et al., 2008Farthing C.R. Ficz G. Ng R.K. Chan C.F. Andrews S. Dean W. Hemberger M. Reik W. PLoS Genet. 2008; 4: e1000116Crossref PubMed Scopus (265) Google Scholar), in carefully controlled experiments to rigorously address the hypothesis that iPSCs retain tissue restriction patterns from their parental cells. The most stringent test to establish whether cells, such as iPSCs, are indeed pluripotent is to demonstrate that full-term embryos can be derived exclusively from the putative pluripotent cells. To do so, the candidate donor cells are introduced into “tetraploid” host blastocysts, in which the donor cells do not contribute to placental development. Though it remains possible that iPSC do possess this potential, no live young have yet been reported from iPSCs in any such experiments, even though they have in some instances reached an advanced stage of E14 of gestation (Meissner et al., 2007Meissner A. Wernig M. Jaenisch R. Nat. Biotechnol. 2007; 25: 1177-1181Crossref PubMed Scopus (636) Google Scholar, Woltjen et al., 2009Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hamalainen R. Cowling R. Wang W. Liu P. Gertsenstein M. et al.Nature. 2009; 458: 766-770Crossref PubMed Scopus (1372) Google Scholar). Indeed, one could predict that iPSCs might display relatively greater defects at later stages during terminal differentiation of somatic lineages, with the notable exception of germ cells and gametes, given that all epigenetic modifications are erased and reset in this lineage (Figure 2B). Very careful systematic epigenetic analysis, together with a comprehensive examination of differentiation and precise distribution of iPSCs into all of the tissues in chimeric adults, could reveal their true developmental potential. Following this line of reasoning, one might also expect that the nature of the residual epigenetic memory would depend on the original somatic cells used in these experiments, notwithstanding the stochastic nature of the process. It is crucial also to confirm rigorously whether iPSC-derived differentiated cells are equivalent to normal terminally differentiated cells. The proposed use of terminally differentiated iPSCs in cell therapy and disease models requires them to be phenotypically and physiologically identical to the in vivo terminally differentiated cells. We predict that any residual epigenetic memory in iPSCs would be erased as the epigenome is reset in the germline (Figure 2B). Notably, transdifferentiation of closely related cell types would be predicted to be less affected by the residual epigenetic memory because these cells would share many of their key epigenetic properties (Zhou et al., 2008Zhou Q. Brown J. Kanarek A. Rajagopal J. Melton D.A. Nature. 2008; 455: 627-632Crossref PubMed Scopus (1542) Google Scholar). It is known from several studies that mouse ESCs exhibit functional and epigenetic heterogeneity (Enver et al., 2009Enver T. Pera M. Peterson C. Andrews P.W. Cell Stem Cell. 2009; 4: 387-397Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, Graf and Stadtfeld, 2008Graf T. Stadtfeld M. Cell Stem Cell. 2008; 3: 480-483Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, Hayashi et al., 2008Hayashi K. Lopes S.M. Tang F. Surani M.A. Cell Stem Cell. 2008; 3: 391-401Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar). It is possible that such heterogeneity may be prevalent in experimentally generated pluripotent cells, which may be accentuated in human iPSCs, as they lack the ICM-like reprogramming event that we have described here. Under ideal conditions, following reprogramming, each cell should exhibit an equivalent epigenetic state and identical potential for pluripotency, which may not be the case. For example, during establishment of iPSCs, only a minority of transduced cells are converted to pluripotency and selected under the applied culture conditions, and it is unknown whether all of these cells acquire identical properties. Indeed, even the process of rederiving “secondary” iPSCs using conditional reprogramming vectors remains inefficient (Jaenisch and Young, 2008Jaenisch R. Young R. Cell. 2008; 132: 567-582Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar, Maherali and Hochedlinger, 2008Maherali N. Hochedlinger K. Cell Stem Cell. 2008; 3: 595-605Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). It is possible that the protracted nature of this procedure could result in daughter cells with diverse epigenetic states. This possibility can also be evaluated by rigorous clonal analysis of iPSCs for their phenotypic and epigenetic properties. Resetting of the epigenome in the germline may not only be extensive, but it also benefits from rigorous in vivo selection, as seen during spermatogenesis (Ueno et al., 2009Ueno H. Turnbull B.B. Weissman I.L. Proc. Natl. Acad. Sci. USA. 2009; 106: 175-180Crossref PubMed Scopus (37) Google Scholar). Based on the existing evidence, we suggest that the most comprehensive process of epigenetic reprogramming that ensures authentic pluripotency occurs upon passage through the germline. The wide-ranging erasure of epigenetic modifications, including DNA demethylation, ensures removal of most, if not all, of the extraneous epigenetic information. This conversion apparently does not occur in the experimentally restored pluripotent state. In the mouse, the form of reprogramming that takes place in the ICM provides a possibility for approaching the authentic pluripotent state, but even so, the result is often variable, owing to the stochastic nature of the process (Bortvin et al., 2003Bortvin A. Eggan K. Skaletsky H. Akutsu H. Berry D.L. Yanagimachi R. Page D.C. Jaenisch R. Development. 2003; 130: 1673-1680Crossref PubMed Scopus (376) Google Scholar). hESCs may be more compromised in terms of their functional pluripotency, as these populations evidently lack the ICM-specific reprogramming step. This distinction sets hESCs apart from mESCs and may account for the relatively heterogeneous nature of hESCs, as has been observed with mEpiSCs. Greater understanding of the process of germline-specific reprogramming may thus provide additional tools to advance stem cell research. Uncovering the details of this process is particularly important, given that the use of experimentally generated differentiated cells from pluripotent cells, including iPSCs, has been proposed for widespread use in cell therapy and to develop disease models for drug discovery.
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