br Results br Discussion Mammalian primordial germ cells

Results
Discussion Mammalian primordial germ EX 527 are considered unipotent, giving rise only to the gametes. Indeed, the sperm and egg represent two of the most overtly differentiated cell phenotypes. Yet, these two specialized cells regain access to the entire embryonic and extraembryonic differentiation programs following fertilization and zygotic reprogramming. Immature cells of the germline can also acquire pluripotency through nonphysiological routes, ex vivo formation of EG cells and multipotent germline stem cells (Kanatsu-Shinohara et al., 2004), or in vivo teratocarcinogenesis. However, those events have previously been obtained at low frequency in complex environments. Here, we demonstrate that 10%–30% of single mouse PGCs can convert to pluripotent EG cells in well-defined conditions. This is comparable to the efficiency of ES cell derivations reported from single epiblast cells (Brook and Gardner, 1997; Rugg-Gunn et al., 2012) and challenges the notion that PGCs are an intrinsically committed unipotent lineage. PGC identity depends on the activity of determinants such as Blimp1 (Ohinata et al., 2005), Prdm14 (Yamaji et al., 2008), and Tcfap2c (Weber et al., 2010). However, PGC specification is also associated with reexpression or upregulation of core pluripotency transcription factors including Nanog, Sox2, and Klf2 (Kurimoto et al., 2008). These factors are thought to be essential in PGCs (Chambers et al., 2007; Kehler et al., 2004; Yamaguchi et al., 2009), although their role remains unclear. Their presence may mean that pluripotency is not extinguished in PGCs as in other postgastrulation lineages (Osorno et al., 2012), but could instead lie dormant. Derivation of EG cell lines from the first specified PGCs at E7.5 is consistent with the idea that latent pluripotency may be a necessary feature of the germline. The efficiency of EG cell generation does decrease during PGC development however, falling by more than an order of magnitude at E11.5. Interestingly, this coincides temporally with widespread epigenome modifications (Hajkova et al., 2008). Nonetheless, a rudiment of pluripotency is retained in later germline development as evidenced by the ability to derive a type of pluripotent stem cell from spermatogonial stem cells (Kanatsu-Shinohara et al., 2004; Ko et al., 2009). In feeder-free cultures with 4Fs and GSK3 inhibition, we observed that PGCs exhibit features of locomotor cells, such as cell extension and lamellopodia (Movie S1). This motile phenotype persists throughout the early proliferative phase of culture for approximately 72 hr. After this time, cell death is progressive, and only cells undergoing conversion to EG cells continue to proliferate extensively. However, cell loss is heterogeneous and occasional cells with PGC morphology survive until much later time points. This raises the intriguing possibility that it may be feasible to sustain PGC proliferation and survival without EG cell formation. In this context, it might be productive to omit LIF while employing GSK3 inhibition. LIF does not appear important for initial PGC culture but is specifically required to drive EG cell conversion. Inhibition of MAPK signaling is also not required for the initial 48 hr of PGC culture, in fact, is deleterious during that period. Our observations suggest that EG cell formation can be divided into two discrete phases: an initial 48 hr period of PGC adaptation to culture that is promoted by bFGF, RA, SCF, and GSK3 inhibition and a subsequent period of fate conversion over 6 days. The second phase is driven by LIF stimulation and MAPK inhibition, which is augmented by inhibition of GSK3. A key goal for future studies will be to elucidate the temporal pattern of STAT3 target gene induction and delineate the synergy with MAPK inhibition that reconstructs the full pluripotency and self-renewal circuit (Nichols and Smith, 2012). The intersection between these two pathways also appears crucial to achieve authentic induced pluripotency by somatic cell reprogramming (Silva et al., 2008; Sridharan et al., 2009; van Oosten et al., 2012; Yang et al., 2010). Elucidating the process of EG cell formation may therefore illuminate generally the acquisition of pluripotency.