Dual kinase inhibition promotes pluripotency in finite bovine embryonic cell lines.

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.

A virus-free poly-promoter vector induces pluripotency in quiescent bovine cells under chemically defined conditions of dual kinase inhibition.

Authentic induced pluripotent stem cells (iPSCs), capable of giving rise to all cell types of an adult animal, are currently only available in mouse. Here, we report the first generation of bovine iPSC-like cells following transfection with a novel virus-free poly-promoter vector. This vector contains the bovine cDNAs for OCT4, SOX2, KLF4 and c-MYC, each controlled by its own independent promoter. Bovine fibroblasts were cultured without feeders in a chemically defined medium containing leukaemia inhibitory factor (LIF) and inhibitors of MEK1/2 and glycogen synthase kinase-3 signaling ('2i'). Non-invasive real-time kinetic profiling revealed a different response of bovine vs human and mouse cells to culture in 2i/LIF. In bovine, 2i was necessary and sufficient to induce the appearance of tightly packed alkaline phosphatase-positive iPSC-like colonies. These colonies formed in the absence of DNA synthesis and did not expand after passaging. Following transfection, non-proliferative primary colonies expressed discriminatory markers of pluripotency, including endogenous iPSC factors, CDH1, DPPA3, NANOG, SOCS3, ZFP42, telomerase activity, Tra-1-60/81 and SSEA-3/4, but not SSEA-1. This indicates that they had initiated a self-sustaining pluripotency programme. Bovine iPSC-like cells maintained a normal karyotype and differentiated into derivatives of all three germ layers in vitro and in teratomas. Our study demonstrates that conversion into induced pluripotency can occur in quiescent cells, following a previously undescribed route of direct cell reprogramming. This identifies a major species-specific barrier for generating iPSCs and provides a chemically defined screening platform for factors that induce proliferation and maintain pluripotency of embryo-derived pluripotent stem cells in livestock.