Abstract
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. First, 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 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.
Highlights
Pluripotent human embryonic stem cells are crucial for studying the molecular processes governing human germ cell specification
In Vitro Derivation of primordial germ cells (PGCs) Using OCT4-EGFP human embryonic stem cells (hESCs)—To explore the hypothesis that OCT4 is retained in the germ cell population when hESCs differentiate, we generated an OCT4 promoter-EGFP hESC line by the lentiviral system to investigate the process of PGC derivation (Fig. 1, A and B, and supplemental Fig. S1, A–C)
We have presented here an OCT4-EGFP reporter system to track and enrich germ cell-like populations from spontaneously differentiated hESCs
Summary
Pluripotent human embryonic stem cells (hESCs) are crucial for studying the molecular processes governing human germ cell specification. Results: Human germ cells highly expressed epithelial cell adhesion molecule (EpCAM) and the synergistic effects of BMP4/ WNT3A promote hESCs toward germline differentiation. 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. 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. we showed that the pluripotency associated surface marker, epithelial cell adhesion molecule (EpCAM), is expressed in 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 celllike cell population.
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