Long‐term expansion with germline potential of human primordial germ cell‐like cells in vitro.

Human germ cells perpetuate human genetic and epigenetic information. However, the underlying mechanism remains elusive, due to a lack of appropriate experimental systems. Here, we show that human primordial germ cell‐like cells (hPGCLCs) derived from human‐induced pluripotent stem cells (hiPSCs) can be propagated to at least ~106‐fold over a period of 4 months under a defined condition in vitro. During expansion, hPGCLCs maintain an early hPGC‐like transcriptome and preserve their genome‐wide DNA methylation profiles, most likely due to retention of maintenance DNA methyltransferase activity. These characteristics contrast starkly with those of mouse PGCLCs, which, under an analogous condition, show a limited propagation (up to ~50‐fold) and persist only around 1 week, yet undergo cell‐autonomous genome‐wide DNA demethylation. Importantly, upon aggregation culture with mouse embryonic ovarian somatic cells in xenogeneic‐reconstituted ovaries, expanded hPGCLCs initiate genome‐wide DNA demethylation and differentiate into oogonia/gonocyte‐like cells, demonstrating their germline potential. By creating a paradigm for hPGCLC expansion, our study uncovers critical divergences in expansion potential and the mechanism for epigenetic reprogramming between the human and mouse germ cell lineage. Synopsis: In vitro reconstitution of germ‐cell development is a key for analyzing and enhancing reproductive biology. This resource reports a culture method for robust propagation of human primordial germ cell‐like cells (hPGCLCs) under defined conditions, establishing a tractable paradigm for human germ‐cell expansion. Induced pluripotent stem cell‐derived hPGCLCs expand ~106‐fold under forskolin/SCF/bFGF in vitro over a period of four months.During expansion, hPGCLCs maintain an early hPGC(LC) transcriptome and DNA methylome.hPGC(LC) specification and epigenetic reprogramming are genetically dissociable.Expanded hPGCLCs differentiate into oogonia with epigenetic reprogramming in xenogeneic reconstituted ovaries. [ABSTRACT FROM AUTHOR]