Modeling primordial germ cell formation in vitro

In the mouse embryo, specification of the germ lineage occurs by a unique combination of signals that converge upon the proximal epiblast at embryonic day (E) 6.25, and by E7.25, approximately 40 cells are lineage restricted to the germ line, termed primordial germ cells (PGCs). Stella expression has been identified as a reliable molecular marker for these primordial germ cells (PGCs) after E7. Here, we describe and characterize the formation of PGCs in vitro during murine embryonic stem cell (ESC) differentiation using a StellaGFP transgene. These Stella-positive cells erase their imprints in a time-dependent manner and possess transcriptional profiles that overlap with in vivo-derived germ cells. Germ cells must reset genomic imprints to reflect their sex, and PGCs erase paternally and maternally-inherited imprints, making imprint erasure a process unique to these cells.;Derivation of PGCs from ESCs in vitro opens up new possibilities for studying genes relevant to the germ lineage. We hypothesized that a combined strategy using RNA interference (RNAi) against target gene expression during EB differentiation and ESC lines harboring genetic mutations abrogating individual gene function would demonstrate the role of candidate genes in germ cell allocation and development and/or imprint erasure. RNAi-mediated knockdown of Blimp1 during EB differentiation of ESCs prevents the formation of PGCs and EGCs in vitro and eliminates ESC contribution to the germ line in chimeric mouse hosts. In combination, these results demonstrate that in vitro germ cell formation from ESCs closely recapitulates in vivo PGC development and describe a system poised for further analysis of germ cell biology and associated epigenetic phenomena. In particular, we have demonstrated that the Stella transgene provides a robust means of monitoring and identifying PGCs and serves as a valuable tool for further investigations into the mechanisms of PGC development and imprint erasure.