TY - JOUR
T1 - A six-inhibitor culture medium for improving naïve-type pluripotency of porcine pluripotent stem cells
AU - Yuan, Ye
AU - Park, Jinkyu
AU - Tian, Yuchen
AU - Choi, Jungmin
AU - Pasquariello, Rolando
AU - Alexenko, Andrei P.
AU - Dai, Aihua
AU - Behura, Susanta K.
AU - Roberts, R. Michael
AU - Ezashi, Toshihiko
N1 - Funding Information:
We thank Dr. Jacob Hanna at Weizmann Institute of Science, Israel for his guidance and input in culturing human stem cells under NHSM conditions and Lee Spate and Dr. Randall Prather for provision of porcine blastocysts. We thank Dr. Alexander Jurkevich at the Molecular Cytology Core at University of Missouri for his assistance for immunofluorescence staining imaging. The monoclonal antibodies, MC-480 and MC-813-70 developed by Solter, D./ Knowles, B.B were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. This work was supported by NIH Grants R01HD069979 (to R.M.R., T.E.), R21OD026516 (to T.E.) and the University of Missouri Food for the 21st Century Program (R.M.R.).
Publisher Copyright:
© 2019, The Author(s).
PY - 2019/12/1
Y1 - 2019/12/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85071006586&partnerID=8YFLogxK
U2 - 10.1038/s41420-019-0184-4
DO - 10.1038/s41420-019-0184-4
M3 - Article
AN - SCOPUS:85071006586
SN - 2058-7716
VL - 5
JO - Cell Death Discovery
JF - Cell Death Discovery
IS - 1
M1 - 104
ER -
