One-Step Induction of Oogonia-Like and Pro-Spermatogonia-Like Cells From Porcine Pluripotent Stem Cells.

Pigs share a germ cell regulatory network highly similar to that of humans, yet lack a robust system for inducing and expanding germ cells from pluripotent stem cells, impeding large-animal gametogenesis research. Here, we established a modular platform capable of efficiently inducing and long-term expanding porcine germ cell-like cells directly from pluripotent stem cells. We first developed a one-step method to induce pro-spermatogonia-like cells (activating SOX17) and oogonia-like cells (activating SOX17/TFAP2C) from porcine pre-gastrulation epiblast stem cells (pgEpiSCs) harboring a NANOS3-tdTomato (NT) reporter, via activation of exogenous transcription factors. Single-cell transcriptome profiling revealed a differentiation trajectory from pgEpiSCs through primordial germ cell-like cells (PGCLCs) to pro-spermatogonia/oogonia-like cells, with concomitant differentiation of gonadal somatic cells. Functional analyses demonstrate that EGF activates core PGC network and is essential for porcine PGCLCs survival in a xenogeneic reconstituted ovary, BMP4 drives NANOS3 and DDX4 expression, and IWR1 enhances germ cell differentiation by suppressing mesodermal programs. Finally, to address the challenge of cell quantity, we established a long-term expansion module based on FGF2, IWR1, and EGF that sustains NT-positive cells for up to 120 days while preserving their early PGC and pro-spermatogonia-like molecular characteristics. Our study provides new insights into porcine gametogenesis and reproductive development across species.