Generation of human oogonia from induced pluripotent stem cells in culture

This protocol describes how to differentiate human induced pluripotent stem cells into oogonia in vitro. It is suitable for investigating the mechanisms of human primordial germ cell specification and epigenetic reprogramming. The human germ-cell lineage originates as human primordial germ cells (hPGCs). hPGCs undergo genome-wide epigenetic reprogramming and differentiate into oogonia or gonocytes, precursors for oocytes or spermatogonia, respectively. Here, we describe a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into oogonia in vitro. hiPSCs are induced into incipient mesoderm-like cells (iMeLCs) using activin A and a WNT pathway agonist. iMeLCs, or, alternatively, hPSCs cultured with divergent signaling inhibitors, are induced into hPGC-like cells (hPGCLCs) in floating aggregates by cytokines including bone morphogenic protein 4. hPGCLCs are aggregated with mouse embryonic ovarian somatic cells to form xenogeneic reconstituted ovaries, which are cultured under an air-liquid interface condition for 4 months for hPGCLCs to differentiate into oogonia and immediate precursory states for oocytes. To date, this is the only approach that generates oogonia from hPGCLCs. The protocol is suitable for investigating the mechanisms of hPGC specification and epigenetic reprogramming.