Characterization of porcine partially reprogrammed iPSCs from adipose-derived stem cells

Abstract

Partially reprogrammed induced pluripotent stem cells (PiPSCs) have great potential for investigating reprogramming mechanisms and represent an alternative potential material for making genetically modified animals and regenerative medicine. To date, PiPSCs have scarcely been reported in detail when compared with mice and humans. In this study, we obtained PiPSCs from porcine adipose-derived stem cells (pADSCs) by ectopic expression of human transcription factors (OCT4, SOX2, c-MYC, and KLF4) in feeder-free condition. The morphology and proliferation activity of porcine PiPSCs (pPiPSCs) were similar to those of porcine fully reprogrammed iPSCs (pFiPSCs); furthermore, pPiPSCs expressed higher levels of the typical surface molecules (CD29) found in pADSCs. However, pPiPSCs were negative for key proteins (NANOG) connected with stemness and possessed lower differentiation ability in vivo and in vitro. When differentiationinhibiting factors were withdrawn, pPiPSCs-derived cells (pPiPSC-DCs) showed similar features to pADSCs in many aspects, including proliferation, differentiation, and immunosuppression. When both types of cells were used to produce cloned embryos, we found that the blastocyst formation rate of 19DC (one of the pPiPSC-DC cell lines)-derived cloned embryos was obviously higher than that of others. The total cell number of 19DC-derived blastocysts was significantly higher than the 30DC (one pFiPSC-DC cell line)-derived blastocysts. In all, through limited differentiation ability, the proliferation activity of pPiPSCs is similar to that of pFiPSCs, and pPiPSCs can retain several of the features of pADSCs, which are beneficial to cell therapy. Furthermore, the differentiation of pPiPSCs is more favorable for producing high-quality reconstructed embryos. Free Chinese abstract: A Chinese translation of this abstract is freely available at

Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig

“…Compared with pre-iPSC lines, several common characteristics, such as incomplete expression of pluripotent genes, inactive X chromosomes in female cells, and inability to generate germline chimeras, have been observed [ 10 , 20 , 21 , 32 ]. In porcine studies, the majority of pig iPSC lines were partially reprogrammed, showing dependence on transgenes, lack of NANOG expression, failure of epigenetic reprogramming, and inability to form chimeras [ 22 – 27 ]. According to previous studies, the majority of piPSC lines exhibited dependency of continuous transgene expression, and it was impossible to obtain transgene-independent colonies using a transgene-free system such as drug-inducible, episomal or plasmid vectors [ 23 , 26 , 39 , 53 ].…”

“…In human as a non-permissive species, NANOG plays an important role in reprogramming and maintaining pluripotency [ 56 , 57 ]. In some pig studies, NANOG upregulation and epigenetic reprogramming were not achieved [ 26 , 27 ]. Consistent with previous studies, we confirmed that generated iPSCs carrying whole Yamanaka’s factors have no NANOG expression, and that epigenetic alteration relied on the expression of transgenes for maintenance (Figs 3C and 5 ).…”

“…However, if these barriers are not overcome, silencing of transgenes, epigenetic remodeling, and lack of NANOG expression occur, resulting in partial reprogramming of iPSCs (pre-iPSCs) [ 20 , 21 ]. Although various research groups have attempted to generate pig iPSCs (piPSCs), authentic iPSCs have not be obtained, instead showing features of incomplete reprogramming including dependence on transgene expression, epigenetic remodeling, reactivation of pluripotent genes, and chimera formation [ 22 – 27 ]. This may be due to differences in the molecular mechanisms during embryo development between mouse and pig [ 28 ].…”

Hypoxia enhances buffalo adipose‐derived mesenchymal stem cells proliferation, stemness, and reprogramming into induced pluripotent stem cells

“…In particular, biPSCs exhibiting packed dome morphology Because of their multipotent nature, capacity for self-renewal, easy collection, and amplification in vitro, ASCs have become an ideal candidate for regenerative medicine, and donor cells in SCNT and iPSCs research (Abouhamzeh et al, 2015;Ren et al, 2014;Wei et al, 2015;Y. Zhang et al, 2014).…”

Aging adult porcine fibroblasts can support nuclear transfer and transcription factor‐mediated reprogramming

“…Donor cells were prepared as previously described (Wei et al 2015). Briefly, cells of passages 3-7 were trypsinized and re-suspended in TCM199 supplemented with 2% FBS.…”