Establishment and Characterization of Bovine Trophoblast Stem Cells Growing as Adherent Cells or Vesicles

In ungulates, the development of the trophoblast lineage follows a peculiar process, as implantation is preceded by a relatively long period of trophoblast elongation, the molecular control of which is still not well known. Here, we have established novel bovine trophoblast stem cells (b2iTSCs) growing in a completely defined medium containing inhibitors of GSK3b and MEK, and WNT3 during establishment. These cells can grow either as adherent, or as vesicles in suspension. These fluid-filled vesicles show high proliferative capacity and apical polarity. Transcriptome analysis reveals that b2iTSCs are distinct from bovine embryonic fibroblasts and exhibit gene expression profiles related to lipid metabolism, steroid hormone synthesis, and nutrient absorption. The cells also express different Pregnancy Associated Glycoproteins, such as PAG11. Comparative transcriptome analysis shows that 2iTSCs have a signature intermediate between early trophectoderm and elongating trophoblast, expressing canonical trophoblast markers but not IFNt. Additionally, b2iTSCs can differentiate into binucleate cells in vitro and participate in the formation of bovine blastoïds when cocultured with bovine embryonic stem cells, demonstrating their potential for studying trophoblast development and function.