Authentic embryonic stem cells (ESC) may never have been successfully derived from the inner cell mass (ICM) of pig and other ungulates, despite over 25 years of effort. Recently, porcine induced pluripotent stem cells (piPSC) were generated by reprogramming somatic cells with a combination of four factors OCT4, SOX2, KLF4 and c-MYC (OSKM) delivered by lentiviral transduction. The established piPSC are analogous to FGF2-dependent human (h) ESC and murine “epiblast stem cells,” and are likely to advance swine as a model in biomedical research. Here, we report for the first time, the establishment of LIF-dependent, so called naïve type pluripotent stem cells (1) from the inner cell mass (ICM) of porcine blastocysts by up-regulating the expression of KLF4 and POU5F1; and (2) from umbilical cord mesenchyme (Wharton's jelly) by transduction with OSKM factors and subsequent culture in the presence of LIF-based medium with inhibitors that substitute for low endogenous expression of c-MYC and KLF4 and promote pluripotency. The 2 compounds that have been used in this study are, CHIR99021 (CH), which substitutes c-MYC by inhibiting GSK3B and activating WNT signalling and Kenpaullone (KP), which inhibits both GSK3B and CDK1 and supplants KLF4 function. The lentiviral vectors employed for introducing the re-programming genes were modified for doxycycline-mediated induction of expression (tet-on) and are ‘floxed’ for Cre-mediated recombination and removal of transgenes following complete reprogramming. Two LIF-dependent cell lines have been derived from the ICM cells of late d 5.5 in vitro produced blastocysts and four from umbilical cord mesenchyme recovered from fetuses at d 35 of pregnancy. The derived stem cell lines are alkaline phosphatase-positive, resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile and expression of pluripotent markers, such as POU5F1, SOX2 and surface marker SSEA1. They are dependent on LIF signalling for maintenance of pluripotency, can be cultured over extended passage (>50) with no senescence. Of importance, the ICM-derived lines have been successful in their ability to form teratomas. The cells could be cultured in feeder free conditions on a synthetic matrix in the presence of chemically defined medium and can be coaxed to differentiate under xeno-free conditions. Currently, the piPSC lines are being investigated for their ability to give rise to teratomas and to produce a live offspring by nuclear transfer.
Supported by Addgene Innovation Award, MO Life Sciences Board Grant 00022147 and NIH grant HD21896.
Development of Endothelial Cell Lines From Embryonic Stem Cells