Abstract：Although the isolation of porcine (Sus scrofa) embryonic stem cells (pESCs) has been reported, the self-renewal and differentiation potential of pESCs were limit. In this study, the isolation of pESCs from parthenogenetic embryos (PA), somatic cell nuclear transfer embryos (SCNT) and in vivo-produced embryos (IVP) based on the different culture media were researched. The culture condition and key pluripotent genes in PA and SCNT embryos were analyzed by the qRT-PCR, and the effects of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), inhibitors of glycogen synthase kinase 3β (CHIR99021), and mitogen-activated protein kinase 1 (PD0325901) on pESCs isolation were investigated. The results showed that PZM3 and NCSU23 media were suitable for PA and SCNT embryos development in vitro, respectively. The 4 octamer-binding transcription factor 4(OCT4) expression was significantly increased in PA and SCNT embryos compared with NANOG and SOX2 in PA and SCNT embryos. Basic fibroblast growth factor receptor(bFGFR) gene expression was also significantly increased, suggesting that growth factor bFGF was needed for pESCs isolation from PA and SCNT embryos. Furthermore, addition of bFGF in medium could elevate the efficiency of pESCs isolation and cloning from in vivo-produced embryos, which showed morphology of the epiblast-like stem cells (EpiSCs), expressed NANOG and SOX2 proteins by immunochemistry, and had alkaline phosphatase(AP) activity. The results may contribute to a new method to isolate pESCs.
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