The Trilineage Coexistence Observed During the Differentiation of Porcine EPSCs

The mammalian early embryo possesses totipotency and can be captured as extended pluripotent stem cells (EPSCs). The first two cell differentiations result in epiblast, primitive endoderm, and trophectoderm, with the trilineage coexisting in a unified uterine microenvironment. Nevertheless, the in vitro counterparts-primed PSCs, trophoblast stem cells (TSCs), and extraembryonic endoderm (XEN) cells-require a distinct culture system. In this study, we successfully derived stable porcine EPSCs from fibroblasts at 35% efficiency, and interestingly observed that these EPSCs differentiated in parallel and gave rise to the transient coexistence (36-84 h) of trilineage cells when cultured in a single system (LCDM: hLIF, CHIR99021, DiM, and MiH). Then, XEN cells gradually predominated and eventually became the sole population in prolonged LCDM culture. However, TSCs and primed PSCs had to be differentiated from EPSCs under their respective culture system. EPSCs can differentiate into PGC-like cells independently of genetic modification and contribute to mouse neurula-stage embryos. Collectively, the trilineage coexistence phenomenon may provide novel insight into an early embryogenesis mechanism and strategy for porcine blastoid construction.