The Small Molecule Inhibitors PD0325091 and CHIR99021 Reduce Expression of Pluripotency-Related Genes in Putative Porcine Induced Pluripotent Stem Cells

Small molecule inhibitors of the mitogen-activated protein kinase kinase (MEK) and glycogen synthesis kinase 3 (Gsk3) have been essential in the establishment and maintenance of embryonic stem cells (ESCs) from rats and from nonpermissive mouse strains. However, conflicting results have been reported regarding their efficacy in the establishment and maintenance of pluripotent stem cells from other species. Here, we investigated the effects of PD0325091 (PD; a MEK inhibitor) and CHIR99021 (CH; a Gsk3ß inhibitor) on the reprogramming of porcine fetal fibroblasts to induced pluripotent stem cells (piPSCs). Primary cultures treated with the two inhibitors (2i) showed a reduced number of alkaline phosphatase-positive colonies and a lower percentage of OCT4-expressing cells compared with the cultures grown with basic medium, which was supplemented with murine leukemia inhibitory factor (LIF). Moreover, the piPS-like cell lines established under 2i conditions expressed significantly lower levels of pluripotency markers, including OCT4, SOX2, REX1, UTF1, STELLA, TDH, and CHD1, compared with the controls. To test the short-term effects of the small molecule inhibitors, piPS-like cells that had been established in basic culture medium were cultured for five passages in medium supplemented with 2i or PD or CH individually. In accordance with the first experiment, expression levels of most pluripotency genes declined in cultures treated with inhibitors, although the response to each inhibitory molecule varied for the different genes. Results of this study concur with previous reports and cast doubts on the effectiveness of CH and PD in the reprogramming of porcine somatic cells to pluripotency.