Derivation of induced pluripotent stem cells from pig somatic cells.
For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after approximately 22 days, providing an overall reprogramming efficiency of approximately 0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of approximately 17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.
Ezashi
Toshihiko
T
Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA.
Telugu
Bhanu Prakash V L
BP
Alexenko
Andrei P
AP
Sachdev
Shrikesh
S
Sinha
Sunilima
S
Roberts
R Michael
RM
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D003114:Colony-Forming Units Assay; D004622:Embryo, Mammalian / Q000166:cytology; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D007621:Karyotyping; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000201:enzymology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D034421:Sus scrofa; D019098:Telomerase / Q000378:metabolism; D013724:Teratoma / Q000473:pathology
D015415:Biomarkers; D019098:Telomerase