Enrichment and differentiation of human germ-like cells mediated by feeder cells and basic fibroblast growth factor...

Human embryonic stem cells (hESCs) have recently demonstrated the potential for differentiation into germ-like cells in vitro. This provides a novel model for understanding human germ cell development and human infertility. Mouse embryonic fibroblast (MEF) feeders and basic fibroblast growth factor (bFGF) are two sources of signaling that are essential for primary culture of germ cells, yet their role has not been examined in the derivation of germ-like cells from hESCs. Here protein and gene expression demonstrated that both MEF feeders and bFGF can significantly enrich germ cell differentiation from hESCs. Under enriched differentiation conditions, flow cytometry analysis proved 69% of cells to be positive for DDX4 and POU5F1 protein expression, consistent with the germ cell lineage. Importantly, removal of bFGF from feeder-free cultures resulted in a 50% decrease in POU5F1- and DDX4-positive cells. Quantitative reverse transcription-polymerase chain reaction analysis established that bFGF signaling resulted in an upregulation of genes involved in germ cell differentiation with or without feeders; however, feeder conditions caused significant upregulation of premigratory/migratory (Ifitm3, DAZL, NANOG, and POU5F1) and postmigratory (PIWIL2, PUM2) genes, along with the meiotic markers SYCP3 and MLH1. After further differentiation, >90% of cells expressed the meiotic proteins SYCP3 and MLH1. This is the first demonstration that signaling from MEF feeders and bFGF can induce a highly enriched population of germ-like cells derived from hESCs, thus providing a critically needed model for further investigation of human germ cell development and signaling. Disclosure of potential conflicts of interest is found at the end of this article.