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Cell Journal، جلد ۱۶، شماره Suppl ۱، صفحات ۱۲۰-۱۲۰
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عنوان انگلیسی Ps-89: Generation of Porcine Induced Pluripotent Stem Cells from mCherry Expressing Fibroblasts Mediated through PiggyBac Transposon
چکیده انگلیسی مقاله Objective: Despite advances in understanding of human Induced Pluripotent Stem (iPS) cells, their safety for potential cell therapies has to be proved in appropriate animal models. A limitation of murine models for preclinical assessments of innovative cell therapies is the short life span, small size, and the high level of inbreeding in this species. The pig is an attractive large animal model for preclinical testing of safety and efficacy of cell based therapies. Porcine organs are largely similar in size and physiology to their human counterparts rendering the domestic pig a suitable model for cardiovascular disease, muscular dystrophies, atherosclerosis, wound repair, diabetes and ophthalmological diseases. Therefore, the present study was carried out to derive porcine iPS cells from transgenic fetuses systemically expressing mCherry through a non-viral piggy- Bac transposon. Flurophore (mCherry)-labelled porcine iPS cells may turn out as important tool for blastocyst complementation assays. Materials and Methods: Porcine fetal fibroblasts isolated from mCherry porcine fetuses at passage 2 described by Garrels et al., 2011, were co-electroporated with a PB transposon carrying a multigene cassette consisting of the cDNAs for Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 separated by self-cleaving 2A peptide sequences and a helper plasmid expressing the pCMVPB transposase. On day 6 post electroporation morphology of fibroblasts started change to round structure and on day 9 cell colonies appeared. These putative iPS cells were cultured, propagated and characterized as described by. Results: In this study, the morphology of generated iPS cells was similar to ES cells and they were clonally propagated up to passage 30 and then cryopreserved for future used. The iPS cells were characterized through immunostaining and found positive for AP, OCT4, NANOG, SOX-2, SSEA-1 and SSEA-4. Expression of stemness genes OCT4, SOX-2, NANOG, c-MYC, KLF, UTF, E-CADHERIN, CHD, STELLA, TDH and GAPDH was detected by RT-PCR. In vitro differentiation potential was assessed by Embryoid Bodies (EBs) formation. The formed EBs exhibited the expression of mCherry in their cells and expressed genes like NESTIN, TUJI, GATA4 and AFP. To test their tumorigenic potential, 1 ×106 porcine iPS cells were injected under the skin of immune deficient nude mice. A visible tumor growth was observed 6 weeks later. Conclusion: This study indicates that piggyBac transposon containing six transcription factors is able to reprogram the porcine fetal fibroblast into iPS cells. These cells could be cultured and maintained in vitro for a prolonged period, exhibit characteristics of stem cells and offer a potential source for future blastocyst complementation experiments
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نشانی اینترنتی http://celljournal.org/journal/article/abstract/414
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