Generation of porcine-induced pluripotent stem cells by using OCT4 and KLF4 porcine factors.

Induced pluripotent stem cells (iPSCs) can be artificially reprogrammed from somatic cells by overexpression of exogenous transcription factors. The pig has increasingly become an important large animal model for preclinical tests and studies of human diseases; thus, the generation of porcine iPSCs will facilitate research into the efficacy and safety of stem cell therapy. A current major problem facing the generation of porcine iPSCs is the failure to silence exogenous transgenes. We hypothesized that this problem can be resolved by reducing the number of transcriptional factors used for porcine iPSCs induction. Here, we report the successful generation of porcine iPSCs using the porcine factors Oct4 and Klf4 in combination with specific small molecules. In comparison with high oxygen conditions (20%), the efficiency of porcine iPSC generation was higher under low oxygen conditions (5%). Porcine iPSCs exhibited a normal karyotype and morphology, like mouse embryonic stem cells (ESCs), and could proliferate in the absence of basic fibroblast growth factor (bFGF) and in the presence of human leukemia inhibitory factor (hLIF) and mouse embryonic fibroblast feeder cells. These iPSCs also expressed ESC-like markers (Oct4, Nanog, Klf4, c-Myc, Bmp4, bFgf). Importantly, the porcine iPSCs showed pluripotency, as evidenced by differentiation into three germ layers in vitro following embryoid body formation, as well as by efficiently forming teratomas containing three germ layers in immunodeficient mice. Thus, pluripotent porcine iPSCs can be generated from somatic stem cells by using only two porcine transcription factors in combination with small molecules. These attempts represent the first step toward generating truly pluripotent porcine iPSCs with fewer exogenous genes and less integration.