BMP4/WNT3A induction and OCT4/EpCAM selection facilitate germ cell differentiation from human embryonic stem cells

Human embryonic stem cells (hESCs) have been proven to be an important platform for studying the molecular, signaling and epigenetic processes of germ cell development. The establishment of an effective germ cell selection/enrichment system from differentiating hESCs is crucial for achieving these goals. In the studies we described in this thesis, we developed a germ cell-enriching system that enables us to identify signaling factors involved in germ cell-fate induction from differentiating hESCs in vitro. Firstly, we demonstrated that selection through an OCT4-EGFP reporter system can successfully increase the percentage of meiotic-competent, germ cell-like cells from spontaneously differentiating hESCs. Furthermore, we showed that the pluripotency associated surface marker, Epithelial Cell Adhesion Molecule (EpCAM), is also expressed in the human fetal gonads and can be used as an effective selection marker for germ cell enrichment from differentiating hESCs. Combining OCT4 and EpCAM selection can further enrich the meiotic-competent germ cell-like cell population. Also, with the percentage of OCT4+/EpCAM+ cells as readout, we demonstrated the synergistic effect of BMP4/pSMAD1/5/8 and WNT3A/β-CATENIN in promoting hESCs toward the germline fate. Combining BMP4/WNT3A induction and OCT4/EpCAM selection can significantly increase the germ cell-like population with meiotic competency. Co-transplantation of these cells with dissociated mouse neonatal ovary cells into SCID mice resulted in more germ cell cluster formation in vivo. The stepwise platform established in this study provides a useful tool to elucidate the molecular mechanisms of human germ cell development, which has implications not only for human fertility research but regenerative medicine in general.