Generation of Feeder-Free Pig Induced Pluripotent Stem Cells without Pou5f1

Abstract

The pig represents an ideal large-animal model, intermediate between rodents and humans, for the preclinical assessment of emerging cell therapies. As no validated pig embryonic stem (pES) cell lines have been derived so far, pig induced pluripotent stem cells (piPSCs) should offer an alternative source of undifferentiated cells to advance regenerative medicine research from bench to clinical trial. We report here for the first time the derivation of piPSCs from adult fibroblast with only three transcription factors: Sox2 (sex determining region Y-box 2), Klf4 (Krüppel-like factor 4), and c-Myc (avian myelocytomatosis viral oncogene homolog). We have been able to demonstrate that exogenous Pou5f1 (POU domain class 5 transcription factor 1; abbreviated as Octamer-4: Oct4) is dispensable to achieve and maintain pluripotency in the generation of piPSCs. To the best of our knowledge, this is also the first report of somatic reprogramming in any species without the overexpression, either directly or indirectly, of Oct4. Moreover, we were able to generate piPSCs without the use of feeder cells, approaching thus xeno-free conditions. Our work paves the way for the derivation of clinical grade piPSCs for regenerative medicine.

Porcine pluripotent stem cells: progress, challenges and prospects

“…Lines of iPSCs have been generated from some domesticated ungulates, such as sheep [90,91] , pigs [87,92,93] and cattle [94,95] . Porcine iPSCs have been produced in many laboratories using various induction methods and show pluripotency to some degree [59,93,96,97] .…”

“…They show a large, flat and round (polygonal in rare cases) shape with compact colonies and distinct borders, with relatively small diameters and a high nucleus-to-cytoplasm ratio, a single nucleus with multiple nucleoli, and are sensitive to tryptase. These cells grow more slowly and have limited capability to be integrated into host blastocysts [92,120,122,134,141,143] . Another type of porcine PSCs was shown to have a mouse ESCs morphology, i.e., small, not flat and with a compact, glistening, doom shaped appearance.…”

Progress, problems and prospects of porcine pluripotent stem cells

“…Livestock iPSCs such as porcine iPSCs (piPSCs) are the first induced pluripotent stem cell line established from livestock [21,[50][51][52][53][54][55][56][57] and these cells have been described by different research groups who used diverse strategies for reprogramming somatic cells to livestock pluripotent cells, such as piPSCs (Table 1), bovine iPSCs (Table 2), and iPSCs in sheep and goats (Table 3). To generate such cells, either human or mouse pluripotent factors Oct4, Sox2, c-Myc and Klf4 were combined.…”

“…Combination pluripotent factors and microRNAs including miR-302a, miR-302b and miR-200c have been reported to improve the efficiency of porcine somatic cell reprogramming [65]. Lentiviral and retrovirally mediated reprogramming methods have been used more frequently to generate piPSCs [50,51,52,55]. Livestock iPSCs have only been derived from differentiated cells via overexpression of transcriptional mouse, human, or livestock factors.…”

Derivation of Induced Pluripotent Stem (iPS)-like Cells from Microminipig Somatic Cells by Sendai Viral Transduction of 4 HumanG, OCT4, SOX2, KLF4, and c-MYC

“…However, despite these breakthroughs, porcine cells in general are more difficult to culture compared with cells obtained from mice and humans. In addition, porcine iPSCs are unable to silence the inserted transgenes, and cannot self-renew when the transgenes are repressed, suggesting that they are incompletely reprogrammed [5,[7][8][9][10][11].…”