There are still several unanswered questions and problems about the recently claimed possibility of producing functional germ cells in vitro from pluripotent embryonic stem cells (ESCs). In the present paper, we compared by single-cell analysis the capability of putative primordial germ cells (PGCs), produced in vitro from ESCs, and that of endogenous PGCs isolated from embryos, to enter and progress through meiotic prophase I. Using a protocol previously reported to be suitable to produce female germ cells from mouse ESC monolayers, we first identified putative PGCs by analysing the expression pattern of several markers such as SSEA1, APase, OCT4, NANOG, MVH and SCP3 of pre-and post migratory PGCs. Next, after isolation of such cells from culture, we tested their meiotic capability. The evaluation at 2-5 days of culture of the number of cells showing meiotic nuclear SCP3 staining in cytospreads showed that it remained nearly constant in the putative PGCs, whereas it increased markedly in endogenous PGCs. Moreover, we observed that in putative PGCs, the nuclear distribution or expression of SCP3 and other meiotic markers such as DMC1, H2AX and SCP1 were always highly abnormal in comparison to that observed in endogenous cultured PGCs. We conclude that although the formation of cells showing characteristics of PGCs can occur efficiently from ESCs in vitro, these cells possess impaired capability to enter and progress through meiotic prophase I.
KEY WORDS: embryonic stem cell, gametogenesis, meiosis, primordial germ cellFrom the first report by Hübner et al. (2003) showing that XY mouse embryonic stem cells (mESCs) are able to produce follicleenclosed growing oocytes, several studies have described the in vitro differentiation of female and male germ cells from mouse and human ESCs (hESC) in either the monolayer or the embryoid bodies (EBs) culture method (for reviews see Hua and Sidhu, 2008;Nagano, 2007;Aflatoonian and Moore, 2006). The experiments carried out to produce germ cells from ESCs are based on years of intense investigations on the in vivo and in vitro development of mouse primordial germ cells (PGCs), the oocyte and sperm precursors (for reviews see De Felici et al., 2004;De Felici, 2001). In particular the recent progress in elucidating the process of germ line specification in mammals (Ohinata et al., 2009 and references herein) and the possibility to mimic in vitro events of later PGC development including entering into meiosis (Farini et al., 2005), have given precious information for the derivation and characterization of germ cells produced from ESCs.In the mouse embryo, PGC development occurs in about 7Int. J. Dev. Biol. 55: 215-222 (2011) days, from around 5.5 days post coitus (dpc), when the first inductive events of the germ cell lineage occur in the epiblast, to 12.5 dpc when sex differentiation of PGCs into meiotic oocytes in the female and G0-arrested prospermatogoni/gonocytes in maleoccur (for a review, see Western, 2009). Elegant recent studies have shown that m...