Embryonic and Induced Pluripotent Stem Cells Analogous to Inner Cell Mass-Derived LIF-Dependent Mouse Embryonic Stem Cells Established from the Domestic Pig, Sus scrofa. (2010)

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 piPSC resembled FGF2-dependent human (h) ESC and are likely to advance swine as a model in biomedical research, since grafts could be matched to the animal that donated the cells for re-programming. The piPSC also share similarities with murine ‘epiblast stem cells’ developed from advanced epiblast, which like hESC are characterized by their flattened morphology, dependence on FGF2 and TGF-beta/Activin/Nodal signaling for maintenance of their pluripotency, inactivation of one of the X chromosomes in female cell lines, intolerance to passage as single cells, and lack of competency for producing germ-line chimeras. Indeed, two emerging concepts are, first, that the FGF- and LIF-dependent ESC are not limited to rodents, and, second, that only LIF-dependent lines are competent to contribute to germ-line chimeras and are, therefore, intrinsically valuable for genetic modification. Recent successes in the establishment of LIF-dependent ESC and iPSC from hitherto "difficult" species such as rat, and non-permissive mouse strains such as NOD, suggested possible means for establishing analogous lines in pig and other domestic ungulates. The underlying principle behind such approaches is either to augment the endogenous levels of c-MYC and/or KLF4 ectopically or to supplement the culture medium with kinase inhibitors to influence endogenous signaling pathways. Of the latter, one compound CHIR99021 (CH) inhibits GSK3B and activates WNT signaling, thereby substituting for c-MYC, while a a second, Kenpaullone (KP), inhibits both GSK3B and CDK1 and replaces KLF4 function. The objective here has been to (1) to develop LIF-dependent porcine ESC (pESC) by over-expression of KLF4 in dissociated pig ICM and subsequently culturing the transfected cells in the presence of KP/CH medium, and (2) generate LIF-dependent piPSC by reprogramming somatic cells derived from umbilical cord mesenchyme with OSKM factors and also culturing the transfected cells in presence of the same inhibitors. The lentiviral vectors employed for introducing the re-programming genes were modified for doxycycline-mediated induction of expression, and 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 in vitro-produced blastocysts and four from umbilical cord mesenchyme recovered from fetuses at d 35 of pregnancy. The derived piPSC had been completely reprogrammed as evidenced by activation of their endogenous pluripotency genes and do not require doxycycline to maintain their undifferentiated phenotype. The LIF-dependent pESC and piPSC express markers consistent with pluripotency, are alkaline phosphatase positive and bear a striking resemblance to ICM-derived mESC in colony morphology, culture characteristics, and short cell cycle time. Currently, the transcriptome of the pESC and piPSC is being analyzed by microarray, and the ability of these cells to give rise to teratomas and chimeras is under investigation. Supported by MO Life Sciences Board Grant 00022147 and NIH grant HD21896. (platform)