Generation of stable integration-free pig induced pluripotent stem cells under WNT-inhibited condition

Genome integration-free pig induced pluripotent stem cells (iPSCs) bring tremendous value in preclinical testing of regenerative medicine, as well as conservation and exploitation of endangered or rare local pig idioplasmatic resources. However, due to a lack of appropriate culture medium, efficient induction and stable maintenance of pig iPSCs with practical value remains challenging. Here, we established an efficient induction system for exogenous gene-independent iPSCs under WNT-inhibited condition previously used for generation of stable pig pre-gastrulation epiblast stem cell lines (pgEpiSCs). WNT suppression was found to play an essential role in establishment of exogenous gene-independent iPSCs. Strikingly, stable integration-free pig iPSCs could be reprogrammed from pig somatic cells using episomal vectors in this cultured condition. The iPSCs had pluripotency features and transcriptome characteristics approximating pgEpiSCs. More importantly, this induction system may be used to generate integration-free iPSCs from elderly disabled rare local pig somatic cells, and the iPSCs could be gene-edited and used as donor cells for nuclear transfer. Our results provide novel insights into potential applications for genetic breeding of livestock species and pre-clinical evaluation of regenerative medicine. Pig iPSCs were obtained in 3i/LAF system. Characterization assays were carried out to prove the pluripotency of pig iPSCs.