Kinetic Analysis of Porcine Fibroblast Reprogramming Toward Pluripotency by Defined Factors

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Induced pluripotent stem cells (iPSCs) are generated from somatic cells through ectopic expression of defined transcription factors. So far, many iPSC lines have been established in various species, including porcine. However, the molecular events during somatic cell reprogramming in pig are largely unknown. The aim of this study was to carry out porcine embryonic fibroblast (PEF) reprogramming by using mouse transcription factors and to monitor morphological and biological progress systematically at an early stage of cell reprogramming. The retrovirus-infected PEF cells retained the four transgenes used and showed morphological changes and alkaline phosphatase staining at day 5 after infection. The endogenous OCT4, NANOG, and TERT genes were activated, and their expression levels were increased significantly. BAX gene expression, a proapoptotic member of the BCL-2 family, was also increased at day 5, suggesting that c-Myc might trigger cell apoptosis. Omission of c-Myc from the cocktail of factors then greatly influenced the reprogramming efficiency and lowered the formation of iPSC colonies. The expression of paternally imprinted DLK1 and DIO3 was slightly downregulated after infection, but then recovered within 2 weeks. However, the expression of maternally imprinted GTL2 was silenced aberrantly at a very early stage of infection and did not recover. Together, these observations illustrated that upregulation of pluripotent-related gene expression, stabilizing the imprinted Dlk1-Dio3 domain, and inhibition of apoptotic events might be conductive to the promotion of the reprogramming process to produce complete porcine iPSCs.