Abstract
ABSTRACT
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.