A six-inhibitor culture medium for improving naïve-type pluripotency of porcine pluripotent stem cells

Understanding essential signaling network requirements and making appropriate adjustments in culture conditions are crucial if porcine pluripotent stem cells (PSC) are to achieve their full potential. Here, we first used two protein factors (LIF and FGF2) and kinase inhibitor combinations in attempts to convert primed type lentiviral-reprogrammed porcine induced PSC (Lv-piPSC) into nai\u308ve-like state and developed a medium called FL6i. In addition to FGF2 and LIF, this medium contained inhibitors of MAPK14, MAPK8, TGFB1, MAP2K1, GSK3A and BMP. Crucially, the usual TGFB1 and BMP4 protein components of many stem cell media were replaced in FL6i with inhibitors of TGFB1 and BMP. With this medium, Lv-piPSC were readily transformed from their original primed state into cells that formed colonies with typical features of nai\u308ve-state stem cells. The FL6i medium also assisted generation of nai\u308ve-type piPSC lines from porcine embryonic fibroblasts with non-integrating episomal plasmids (Epi-piPSC). These lines, despite retaining variable amounts of vector DNA, expressed higher endogenous pPOU5F1 and pSOX2 than Lv-piPSC. They have been cultured without obvious morphological change for >45 passages and retained pluripotent phenotypes in terms of upregulation of genes associated with pluripotency, low expression of genes linked to emergence of somatic cell lineages, and ability to generate well differentiated teratomas in immune-compromised mice. FL6i conditions, therefore, appear to support elevated pluripotent phenotypes. However, FL6i was less able to support the generation of embryonic stem cells from porcine blastocysts. Although colonies with dome-shaped morphologies were evident and the cells had some gene expression features linked to pluripotency, the phenotypes were ultimately not stable. Pathway analysis derived from RNAseq data performed on the various cell lines generated in this study suggest the benefits of employing the FL6i medium on porcine cells reside in its ability to minimize TGFB1 and BMP signaling, which would otherwise de-stabilize the stem cell state