Abstract
The pig is important for agriculture and as an animal model in human and veterinary medicine, yet despite over 20 years of effort, there has been a failure to generate pluripotent stem cells analogous to those derived from mouse embryos. Here we report the production of leukemia inhibitory factor-dependent, so-called naive type, pluripotent stem cells from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. The alkaline phosphatase-positive colonies resulting from reprogramming 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 leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. These cells derived from the inner cell mass of pig blastocysts are clearly distinct from the FGF2-dependent “primed” induced pluripotent stem cells described recently from porcine mesenchymal cells. The data are consistent with the hypothesis that the up-regulation of KLF4, as well as POU5F1, is required to create and stabilize the naive pluripotent state and may explain why the derivation of embryonic stem cells from pigs and other ungulates has proved so difficult.
Highlights
Pluripotency, such as the lack of senescence when cultured in vitro, the ability to differentiate into multiple cell types representing the three germ layers both in vitro and in vivo, and contribution to the germ line in chimeric offspring [6]
We had noted that concentrations of mRNA for endogenous c-MYC and KLF4 in porcine pluripotent stem cells (piPSC) reprogrammed from fetal fibroblasts were extremely low, possibly accounting for the primed phenotype of these cells [17]
The rationale for the experiments was that such cells would likely have many of the desirable features of mESC, including the rapid growth rate, resistance to spontaneous differentiation, ease of genetic manipulation, and ability to incorporate into chimeras that have made mESC such a valuable experimental tool for studies on the mouse model
Summary
A slightly different strategy was employed to generate naive pluripotent stem cells from the NOD strain of mouse, which previously had failed to yield ESC [24]. We rationalized that the previous failures to establish ESC from porcine embryos might have been due to low endogenous levels of c-MYC and KLF4 in ICM cells [27]. We had noted that concentrations of mRNA for endogenous c-MYC and KLF4 in porcine piPSC reprogrammed from fetal fibroblasts were extremely low, possibly accounting for the primed phenotype of these cells [17]. We explored strategies to ectopically overexpress KLF4 in porcine ICM and culture in medium containing KP and CH medium during the reprogramming steps [27]
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