An efficient protocol to derive primordial germ cell like cellsin vitrofrom murine embryonic stem cells

Primordial germ cells (PGCs) are the precursors of germ cells, specified from a subset of epiblast stem cells at the post-implantation stage of mammalian embryonic development. Over the past decade, primordial germ cell-like cells (PGCLCs) have been successfully derivedin vitrofrom mouse and human embryonic stem cells (ESCs). These PGCLCs can further differentiate into mature sperm upon transplantation into neonatal testes. Such advancements offer promising strategies to address male infertility, which affects approximately 15% of couples worldwide. The discovery of induced pluripotent stem cells (iPSCs), which possess similar potential to ESCs, further raises hope for patient-specific therapeutic interventions. However, a major challenge remains: the low efficiency ofin vitroPGCLC differentiation. To overcome this, we developed an optimized PGCLC differentiation strategy using a Dppa3-mCherry knock-in reporter mouse ESC line, TDM11. By testing various culture conditions, we identified ascorbic acid as a potent inducer of PGCLC differentiation from ESCs, providing a potential avenue for enhancingin vitrogerm cell derivation.