Embryonic pluripotent stem cells (ePSCs) can generate all somatic cell types, as well as functional gametes. In mouse and rat, derivation of ePSCs from the early epiblast is promoted by the double inhibition (“2i”) of mitogen-activated protein kinase kinase (MAP2K), antagonizing fibroblast growth factor signaling (FGF), and glycogen synthase kinase 3 (GSK3), stimulating the WNT pathway. However, it has remained unclear whether this culture regime is applicable to nonrodent livestock species. Here we report the generation of bovine ePSCs under minimal conditions. Inner cell masses (ICMs) were immunosurgically isolated from in vitro fertilized bovine blastocysts and cultured feeder-free in 2i medium. Dual kinase inhibition primed bovine ICMs for stem cell derivation and sustained expression of epiblast-specific pluripotency markers SOX2 and NANOG, while repressing the hypoblast marker GATA4. Following mechanical passage, 2i supported limited proliferation for several weeks. Continuously cultured ePSC lines expressed discriminatory markers of naïve pluripotency and primordial germ cells, but not of primed epiblast stem cells. In female ePSCs, most OCT4-positive cells lacked epigenetically silenced X-chromosomes, displaying a diagnostic feature of naïve pluripotency. Bovine ePSCs maintained a normal karyotype and differentiated into derivatives of all three germ layers in suspension culture. This culture system provides a screening platform for factors that maintain long-term proliferation of pluripotent embryonic cattle cells without genetic intervention.
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