Culture condition modifications to improve pluripotency in porcine pluripotent stem cells

Understanding essential signaling network requirements and adjustment the culture conditions for porcine pluripotent stem cells (pPSC) that allow its full potential are necessary. Here we compared culture conditions of various cytokine and kinase inhibitor combinations by deriving porcine induced pluripotent stem cells (piPSC) and porcine embryonic stem cell (pESC)-like cells. We modified the naïve-type human PSC condition by replacing TGFB1 with TGFB inhibitor and developed a so-called FL6i condition, consisting of FGF2, LIF, p38i, JNKi, TGFBi, GSK3i, MEKi, and BMPi. In such conditions, the primed type lentiviral reprogrammed piPSC (Lv-piPSC) was converted into naïve-like state demonstrating increased expression of endogenous pluripotent genes. By using the FL6i condition, new piPSC lines were generated with non-integrative episomal plasmids (Epi-piPSC). While concurrently generated piPSC in condition with FGF2 and LIF only, and without inhibitors lost the pluripotency within a couple passages of the culture, piPSC in FL6i were successfully established stable lines (> 45 passages) with pluripotent phenotypes. The Epi-piPSC-FL6i lines expressed higher endogenous pPOU5F1, and pSOX2 expressions than Lv-piPSC, however, variable transgene expressions were detected with consistent presence of POU5F1. The teratomas generated from Epi-piPSC-FL6i that developed fully differentiated teratomas while those from Lv-piPSC lines and Epi-piPSC derived in a different condition were limited in differentiation range, suggesting the beneficial role of the FL6i condition supporting elevated pluripotent phenotypes. FL6i conditions were also able to maintain the embryo derived cells with dome-shaped phenotype and extended the culture period that cells showed undifferentiated phenotype than the ones cultured with FGF2 alone. But neither conditions established stable pESC lines. RNAseq analysis with the piPSC and pESC-like cells revealed elevated expressions of early differentiation markers in the lines derived without inhibitors. The pathway analysis suggested that the activation of TGFB and BMP signaling pathways may be responsible for losing pluripotent state in porcine cells. RNAseq results from the piPSC and pESC-like cells were compared to evaluate impacts of the culture conditions of various cytokine and kinase inhibitor combinations onto the gene expressions in the cells lines derived in the respective conditions.