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

ABSTRACT: 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 naïve-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 naïve-state stem cells. The FL6i medium also assisted generation of naïve-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.

SUBMITTER: Yuan Y

PROVIDER: S-EPMC6579764 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Yuan Ye Y Park Jinkyu J Tian Yuchen Y Choi Jungmin J Pasquariello Rolando R Alexenko Andrei P AP Dai Aihua A Behura Susanta K SK Roberts R Michael RM Ezashi Toshihiko T

Cell death discovery 20190617

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 naïve-like state and developed a medium called FL6i. In addition to FGF2 and LIF, this medium contained inhibitors ...[more]

PMID: 31240131

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Project description: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.

Dataset Information

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

Publications

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

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