Meiotic competent human germ cell-like cells derived from human embryonic stem cells induced by BMP4/WNT3A signaling and OCT4/EpCAM selection

The establishment of an effective germ cell selection/enrichment platform from in vitro differentiating human embryonic stem cells (hESCs) is crucial for studying the molecular and signaling processes governing human germ cell specification and development. In this study, 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 putative germ cell population with meiotic competency. Co-transplantation of these cells with dissociated mouse neonatal ovary cells into SCID mice resulted in a homogenous 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. Introduction Although the broad differentiation potential of ESCs has long been demonstrated in vitro, evidence of gamete generation from ESCs was hampered by the lack of appropriate markers to track and identify germline differentiation in vitro. In 2003, follicle-like structures were derived from gcOCT4-GFP mouse ESCs by Hubner et al. (1). After this pioneering study, several subsequent reports have demonstrated that primordial germ cells (PGCs), as well as sperm and oocyte-like cells, can be derived from mouse pluripotent stem cells (PSCs) (2-7). Hayashi et al., have lately recreated the complex multistep pathway of germ cell development from mouse PSCs and successfully generated healthy offspring from the by gest on N ovem er 7, 2017 hp://w w w .jb.org/ D ow nladed from