Protocol of Human Primordial Germ Cell-like Cell Generation from Pluripotent Stem Cells in 2D and 3D Culture Systems

Because human infertility can arise from genetic, molecular, cellular, anatomical, and endocrine abnormalities, in vitro gametogenesis has become an important area of reproductive research. New developing technology involving the generation of human primordial germ cell-like cells (hPGCLCs) from induced pluripotent stem cells (hIPSCs) might assist with understanding early germ cell development (specification, migration, gametogenesis, and epigenetic reconstitution), as well as provide a solution for infertility and hereditary disorders. Given that human primordial germ cells (PGCs) are still not well characterized at a molecular level, we present a practical workflow to robustly and efficiently induce hPGCLCs from human pluripotent stem cells (hPSCs) XX and XY cell lines. This protocol describes the hPGCLC specification of hPSCs through sequential induction with Activin A for 2 days and BMP4 for 6 days in 2D and 3D culture systems. Induction of hPSCs into hPGCLCs demonstrated expression of early primordial germ cell markers, including PRDM1, NANOS3, DAZL, STELLA, SOX17, SSEA1, and cKIT, on the 8th day of hPGCLC generation. We described the protocol for generating early hPGCLCs, which provides an opportunity for further investigations into maturation into late germ cells and a chance to overcome a meiotic block to obtain haploid gametes in vitro.