Porcine induced pluripotent cells (iPSC) are considered an important large animal model for developing personalized stem cell therapies. Since the derivation of the first mouse and human iPSC, there have been relatively few reports regarding the reprogramming of pig somatic cells into pluripotency, exclusively with the use of transcription factors from human and mouse origin. To investigate whether using species-specific transcription factors would allow for an efficient reprogramming of porcine somatic cells, we have developed a Sleeping Beauty (SB) transposon system based on the porcine OCT4, SOX2, C-MYC, and KLF4 sequences and have tested it in the reprogramming of mouse and porcine fetal fibroblasts and pig EGC-like cells. Transfection of mouse embryonic fibroblasts with a multicistronic SB-tetO-pOCT4-F2A-pSOX2-T2A-pC-MYC-E2A-pKLF4-ires-Tomato vector resulted in the formation of iPSC colonies, which inactivated the exogenous transcription factors and upregulated their endogenous pluripotency genes. These cells maintained mESC-like morphology, formed embryoid bodies, and differentiated into different cell types in culture, including rhythmically contracting cardiac myocytes. In contrast, porcine fibroblasts and EGC-like cells transfected with the same transposon vector did not proliferate in culture and did not form any iPSC colonies. We then transfected these cells with multiple bi-cistronic vectors SB-Ef1a-pOCT4-ires-Tomato, SB-Ef1a-pSOX2-ires-Neo, SB-Ef1a-pC-MYC-ires-Puro, and SB-Ef1a-pKLF4-ires-Puro. As a result, both cell types formed multiple colonies with mouse ESC-like morphology. Clones established from individually picked colonies from transfected fetal fibroblasts maintained this morphology for 5-6 passages, after which they became flat and epithelial-like. They expressed endogenous SOX2, C-MYC, KLF4, and E-Cadherin, but not OCT4. At the same time, clones derived from EGC-like cells proliferated at accelerated rate and maintained their morphology for over 10 passages at the time of this writing. While the exogenous genes were expressed continuously during this period, the cells expressed also endogenous OCT4, SOX2, TERT, STELLA, TDH, and CHD1; however, C-MYC, KLF4, NANOG, and E-Cadherin expression was relatively low. These cells are currently being characterized for pluripotency. Despite the use of porcine transcription factors, the overall reprogramming of porcine cells appears to be still less efficient compared with mouse fibroblasts. Our results suggest that the species and tissue origin of the somatic cells may play a more important role in the reprogramming to pluripotency than the origin of the transcription factors used. In this respect, optimization of culture conditions may be necessary in order to allow for efficient and complete reprogramming of porcine somatic cells.
This work is supported by a research grant by the Deutsche Forschungsgemeinschaft.