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 similar to 10(6)-fold over a period of 4 months under a defined conditionin vitro. During expansion,hPGCLCs maintain an earlyhPGC-like transcriptome and preserve their genome-wideDNAmethylation profiles, most likely due to retention of maintenanceDNAmethyltransferase activity. These characteristics contrast starkly with those of mousePGCLCs, which, under an analogous condition, show a limited propagation (up to similar to 50-fold) and persist only around 1 week, yet undergo cell-autonomous genome-wideDNAdemethylation. Importantly, upon aggregation culture with mouse embryonic ovarian somatic cells in xenogeneic-reconstituted ovaries, expandedhPGCLCs initiate genome-wideDNAdemethylation and differentiate into oogonia/gonocyte-like cells, demonstrating their germline potential. By creating a paradigm forhPGCLCexpansion, our study uncovers critical divergences in expansion potential and the mechanism for epigenetic reprogramming between the human and mouse germ cell lineage.