Long-term Culture of Human Primordial Germ Cell-like Cells

Primordial germ cells (PGCs) are common precursors of all male and female germline cells. PGCs arise in peri-implantation mammalian embryos and become gender-specific germ cell precursors upon sexual differentiation of the gonadal anlage. The in vitro model commonly known as PGC-Like Cells (PGCLCs), generated from human pluripotent stem cells, is a useful surrogate of the human embryonic PGCs, providing a unique opportunity to explore human gametogenesis in vitro. The protocol presented here supports long-term in vitro expansion of human PGCLCs. Freshly isolated PGCLCs are maintained initially on a STO feeder layer and then expanded in a feeder-free condition on basement membrane extract. The long-term culture PGCLCs (LTC-PGCLCs) can be Fluorescence-Activated Cell Sorting (FACS)-enriched as CD38-positive cells and are readily amplified to tens of millions of cells as pure, feeder-free, and serum-free monotonous culture without apparent limitations such as senescence. It is practically feasible to obtain a pure population of more than 1 million human PGCLCs from a few thousand freshly isolated PGCLCs. The human LTC-PGCLC cell culture is a useful and convenient in vitro model to study human germ cell biology and differentiation into the downstream germline cells.