205 Establishment of bovine extra-embryonic endoderm stem cells

Mammalian blastocysts are composed of trophectoderm and inner cell mass (ICM). The ICM further differentiates into epiblast (i.e. future fetus) and hypoblast (i.e. primitive endoderm, future yolk sac). The stem cells of these three blastocyst lineages have been established in mice: trophoblast stem cells, embryonic stem cells (ESCs), and extra-embryonic endoderm stem cells (XENs), respectively. Despite the importance of early development, our knowledge of hypoblast development, particularly in ruminant species, is limited owing to in vivo experimental feasibility and the lack of in vitro models. Here, we report that a chemical cocktail enables de novo derivation and long-term culture of bovine XENs (bXENs) from blastocysts. The characterization of bXENs revealed that (1) bXENs maintained stable colony morphology and a normal diploid number of chromosomes (60) after long-term in vitro culture (>30 passages as of now); (2) bXENs highly expressed hypoblast transcriptional factors (GATA6, SOX17, and GATA4), as well as novel hypoblast lineage markers identified from single cell transcriptomic data set of Day 12 bovine embryos (CTSV, FETUB, APOA1, APOE), but not epiblast and trophoblast markers; (3) bXENs resembled human and mouse XENs, while being distinct from ESCs of bovine, human, and mouse at the transcriptome level; and (4) bXENs enriched gene markers (e.g. CDH1, IHH, LAMB1, SPARC) and pathways (e.g. migration, TNF, and Hippo pathways) of extra-embryonic visceral endoderm and parietal endoderm markers, indicating their developmental capacity toward to yolk sac. We also identified signaling that is critical for the maintenance of bXENs. Specifically, FGF4 and BMP4 were indispensable for bXEN proliferation, while activation of Wnt signaling by adding CHIR99021 activated the epiblast transcriptional network (e.g. SOX2, GBX2, and KLF2) and directed the bXENs to epiblast-like cells, suggesting the plasticity of bovine hypoblast and epiblast cell fate. Additionally, using a three-dimensional co-culture of bXENs and bEPSCs, we found that bovine ESCs had a faster doubling time in the presence of bXENs while still maintaining pluripotency, suggesting that bXENs could efficiently guide the progression of the epiblast. The bXENs established in this study will provide a model to study bovine hypoblast development and early pregnancy failure.