Defining core signaling pathways for supporting in vitro maintenance of pig extraembryonic endoderm (XEN) cells

Extraembryonic endoderm (XEN) cells can be derived from blastocyst primitive endoderm (PrE), becoming a useful tool for studying mammalian development, including early lineage segregation and embryo patterning. Establishment of stem cells representing the respective lineages in blastocysts has been robustly attempted in domestic animals, especially pigs, to reconstitute embryogenesis in vitro for comparative studies. Therefore, we developed a serum-free culture system for pig XEN cells by dissecting the signals governing the core gene network of the PrE lineage. The FGF, LIF and WNT signaling pathways and B27 supplements are essential for maintaining a rapid proliferation rate in pig XEN cells. These cells recapitulated the molecular features and differentiation capacity of the PrE lineage. Especially, the XEN cells incorporated into normal development, retaining cellular identity and contributing to the PrE lineage when injected into in vitro-produced porcine blastocysts. In addition, species-specific characteristics of pigs were observed, including the involvement of lipid metabolism and NANOG/GATA co-expression in XEN cells. Taken together, our findings can contribute to the expansion of the understanding of developmental biology and its biomedical applications by enabling reproducible and homogeneous porcine XEN cell culture.