262. Towards derivation of primordial germ cells from murine embryonic stem cells

Abstract

The process of primordial germ cell (PGC) specification begins at the earliest stages of murine embryogenesis. The mechanisms underlying this process are the strong instructive cues generated by the extra-embryonic ectoderm, which, via ligand-receptor signalling in the visceral endoderm, activate pathways in the proximal epiblast to induce the PGC phenotype. We have subjected murine embryonic stem (ES) cells to similar cues in order to drive PGC lineage specification in vitro. ES cells were differentiated as aggregates (embryoid bodies (EBs)), a process that is thought to recapitulate the early stages of embryogenesis by providing an environment conducive to the spontaneous emergence of multiple cell lineages. To date, we have shown that EBs can also support the spontaneous emergence of cells expressing PGC markers. Expression analysis was performed on EBs from 1 to 30 days in culture. PGC markers, including nanog, dazl, fragilis, stella and SSEA1, are expressed in undifferentiated ES cells, but rapidly become undetectable in EBs as the constituent ES cells undergo differentiation. The spontaneous emergence of cells expressing these markers occurred only following long-term EB culture. This indicates a lag in the signalling normally required for PGC specification. In vivo, the lack of BMP4, its receptor (ALK-2) or downstream signalling molecules (Smad 1 and 5), results in the absence of PGCs in embryos. Therefore, in order to enhance PGC specification in our in vitro system, we have added BMP4 into the culture media. Under these conditions, the emergence of cells expressing PGC markers occurs at both an apparently higher efficiency and in a shorter time period. This suggests that BMP4 response pathways are present within the EB context and, when activated, can direct PGC specification. Thus, by recapitulating an in vivo physical and biochemical environment, we are able to direct PGC lineage specification in vitro.