Derivation of Porcine Extra-Embryonic Endoderm Cell Lines Reveals Distinct Signaling Pathway and Multipotency States

The inner cell mass of the pre-implantation blastocyst consists of the epiblast and hypoblast from which embryonic stem cells (ESCs) and extra-embryonic endoderm (XEN) stem cells, respectively, can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of its in vivo tissue origin. We have developed a novel approach for deriving porcine XEN (pXEN) cells via culturing the blastocysts with a chemical cocktail culture system. The pXEN cells were positive for XEN markers, including Gata4, Gata6, Sox17, and Sall4, but not for pluripotent markers Oct4, Sox2, and Nanog. The pXEN cells also retained the ability to undergo visceral endoderm (VE) and parietal endoderm (PE) differentiation in vitro. The maintenance of pXEN required FGF/MEK+TGFβ signaling pathways. The pXEN cells showed a stable phenotype through more than 50 passages in culture and could be established repeatedly from blastocysts or converted from the naïve-like ESCs established in our lab. These cells provide a new tool for exploring the pathways of porcine embryo development and differentiation and providing further reference to the establishment of porcine ESCs with potency of germline chimerism and gamete development.