Three-dimensional suspension induction of porcine EPSCs into PGCs

Primordial germ cells (PGCs), the precursors of germ cells, originate from the post-implantation epiblast under the regulation of signals from other germ layers. Elucidating the origin and differentiation mechanisms of PGCs is essential for understanding embryonic development and germ cell formation. This study delineated the cell fate characteristics and key molecular expression patterns during the differentiation of porcine extended pluripotent stem cells (pEPSCs) into epiblast-like cells (EpiLCs) and subsequently into PGCs, while also revealing the phenomenon and regulatory mechanisms of spontaneous PGC specification during this process. pEPSCs were first induced into EpiLCs using N2B27 medium with Activin A and bFGF, then further differentiated via 3D suspension culture. Analysis of key markers revealed a complex cell fate transition. During EpiLC induction, the core marker OTX2 was highly expressed, alongside upregulation of other lineage markers. Notably, even without exogenous BMP4, the PGC marker PRDM1 was detectable at 36 h, indicating spontaneous PGC specification. In subsequent embryoid bodies (EBs), PRDM1 expression increased alongside pluripotency gene reactivation, confirming advanced PGC differentiation. This work establishes that pEPSCs can differentiate into PGCs via an "EpiLCs→EB" pathway and demonstrates spontaneous PGC specification during the EpiLC stage. These findings provide a foundation for understanding the regulatory mechanisms of PGC differentiation in vitro and for optimizing PGCs induction systems.