Objective To establish a stable culture system for porcine induced pluripotent stem cells (piPSCs).Methods Pig fibroblasts were seeded on Matrigel-coated plate,and then infected with lentivirus expressing human Oct4 (hOct4),human kruppel-like factor 4 (hKlf4),human sex determining region Y-box 2 (hSox2) and hc-Myc.Seven to ten days later,piPSCs clones were picked up under invert microscc ope and cultured in KnockoutTM DMEM medium supplemented with 20% KnockoutTM serum replacement.piPSCs clones were detached and dissociated by Accutase digestion.Total mRNA was extracted and pluripotent-associated genes expression of hOct4,hKlf4,hSox2 and hc-Myc and pig Sox2 (pSox2),pNanog and pLin28 was detected by using reverse transcriptase-polymerase chain reaction (RT-PCR).Quantification was performed using the comparative Ct method normalized with the β-actin.The expression of each target gene at each stage was normalized to its level in PF two days after transduction.The immunoflurescence analysis of pluripotency marker (hOct4) and alkaline phosphatase (ALP) activity was also conducted.Results piPSCs were maintained on Matrigel and formed condensed clones with clear borders.Cells in clones had high nucleus/cytoplasm ratio and predominant nuclei.All piPSCs strongly expressed endogenous pOct4,pNanog and pSox2,and the relative expression level was 29.62,2.81 and 7.46,respectively.piPSCs also expressed exogenous hOct4,hc-Myc,hSox2,hKlf4,and the relative expression level was 1.14,0.89,0.74 and 0.86,respectively.piPSCs also expressed ALP activity.All these data suggest the pluripotent state of piPSCs.piPSCs formed embryoid bodies in vitro and teratoma in vivo,indicating the piPSCs proliferated stably and kept high self-renewal and differentiation potency.Conclusion The culture system is suitable for making long-term subculture of piPSCs. Key words: Porcine; Pluripotent stem cells; Culture