Abstract
The development of porcine expanded potential stem cells (pEPSCs) provides an invaluable tool for investigation of porcine stem cell pluripotency and opens a venue for research in biotechnology, agriculture, and regenerative medicine. Since the derivation of pEPSC from porcine pre-implantation embryos has been demanding in resource supply and technical challenges, it is more feasible and convenient for most laboratories to derive this new type of porcine stem cells by reprogramming somatic cells. In this chapter, we describe the detailed procedures for reprogramming porcine fetal fibroblast cells to EPSCiPSC with the eight reprogramming factors cloned on the piggyBac vectors followed by a selection for pluripotent cells independent of transgene expression using the EPSC media. This technique allows the generation of pEPSCs for stem cell research, genome editing, biotechnology, and agriculture.
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References
Yang L, Guell M, Niu D, George H, Lesha E, Grishin D, Aach J, Shrock E, Xu W, Poci J, Cortazio R, Wilkinson RA, Fishman JA, Church G (2015) Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science 350(6264):1101–1104. https://doi.org/10.1126/science.aad1191
Niu D, Wei HJ, Lin L, George H, Wang T, Lee H, Zhao HY, Wang Y, Kan YN, Shrock E, Lesha E, Wang G, Luo YL, Qing YB, Jiao DL, Zhao H, Zhou XY, Wang SQ, Wei H, Guell M, Church GM, Yang LH (2017) Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science 357(6357):1303–1307. https://doi.org/10.1126/science.aan4187
Yan S, Tu Z, Liu Z, Fan N, Yang H, Yang S, Yang W, Zhao Y, Ouyang Z, Lai C, Yang H, Li L, Liu Q, Shi H, Xu G, Zhao H, Wei H, Pei Z, Li S, Lai L, Li XJ (2018) A Huntingtin knockin pig model recapitulates features of selective neurodegeneration in Huntington’s disease. Cell 173(4):989–1002, e1013. https://doi.org/10.1016/j.cell.2018.03.005
Gao L, Gregorich ZR, Zhu W, Mattapally S, Oduk Y, Lou X, Kannappan R, Borovjagin AV, Walcott GP, Pollard AE, Fast VG, Hu X, Lloyd SG, Ge Y, Zhang J (2018) Large cardiac muscle patches engineered from human induced-pluripotent stem cell-derived cardiac cells improve recovery from myocardial infarction in swine. Circulation 137(16):1712–1730. https://doi.org/10.1161/CIRCULATIONAHA.117.030785
Ezashi T, Yuan Y, Roberts RM (2016) Pluripotent stem cells from domesticated mammals. Annu Rev Anim Biosci 4:223. https://doi.org/10.1146/annurev-animal-021815-111202
Shiue YL, Yang JR, Liao YJ, Kuo TY, Liao CH, Kang CH, Tai C, Anderson GB, Chen LR (2016) Derivation of porcine pluripotent stem cells for biomedical research. Theriogenology 86(1):176–181. https://doi.org/10.1016/j.theriogenology.2016.04.030
Brevini TA, Pennarossa G, Attanasio L, Vanelli A, Gasparrini B, Gandolfi F (2010) Culture conditions and signalling networks promoting the establishment of cell lines from parthenogenetic and biparental pig embryos. Stem Cell Rev Rep 6(3):484–495. https://doi.org/10.1007/s12015-010-9153-2
Vassiliev I, Vassilieva S, Beebe LF, Harrison SJ, McIlfatrick SM, Nottle MB (2010) In vitro and in vivo characterization of putative porcine embryonic stem cells. Cell Reprogram 12(2):223–230. https://doi.org/10.1089/cell.2009.0053
Haraguchi S, Kikuchi K, Nakai M, Tokunaga T (2012) Establishment of self-renewing porcine embryonic stem cell-like cells by signal inhibition. J Reprod Dev 58(6):707–716. https://doi.org/10.1262/jrd.2012-008
Hou DR, Jin Y, Nie XW, Zhang ML, Ta N, Zhao LH, Yang N, Chen Y, Wu ZQ, Jiang HB, Li YR, Sun QY, Dai YF, Li RF (2016) Derivation of porcine embryonic stem-like cells from in vitro-produced blastocyst-stage embryos. Sci Rep 6:25838. https://doi.org/10.1038/srep25838
Xue B, Li Y, He Y, Wei R, Sun R, Yin Z, Bou G, Liu Z (2016) Porcine pluripotent stem cells derived from IVF embryos contribute to chimeric development in vivo. PLoS One 11(3):e0151737. https://doi.org/10.1371/journal.pone.0151737
Ma Y, Yu T, Cai Y, Wang H (2018) Preserving self-renewal of porcine pluripotent stem cells in serum-free 3i culture condition and independent of LIF and b-FGF cytokines. Cell Death Discov 4:21. https://doi.org/10.1038/s41420-017-0015-4
Gao X, Nowak-Imialek M, Chen X, Chen D, Herrmann D, Ruan D, Chen ACH, Eckersley-Maslin MA, Ahmad S, Lee YL, Kobayashi T, Ryan D, Zhong J, Zhu J, Wu J, Lan G, Petkov S, Yang J, Antunes L, Campos LS, Fu B, Wang S, Yong Y, Wang X, Xue SG, Ge L, Liu Z, Huang Y, Nie T, Li P, Wu D, Pei D, Zhang Y, Lu L, Yang F, Kimber SJ, Reik W, Zou X, Shang Z, Lai L, Surani A, Tam PPL, Ahmed A, Yeung WSB, Teichmann SA, Niemann H, Liu P (2019) Establishment of porcine and human expanded potential stem cells. Nat Cell Biol 21(6):687–699. https://doi.org/10.1038/s41556-019-0333-2
Acknowledgments
The study was supported by Seed fund from Southern Medical University to X.G., and P.L. is supported by GRF 17127219.
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Gao, X., Ruan, D., Liu, P. (2021). Reprogramming Porcine Fibroblast to EPSCs. In: Hu, K. (eds) Nuclear Reprogramming. Methods in Molecular Biology, vol 2239. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1084-8_13
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DOI: https://doi.org/10.1007/978-1-0716-1084-8_13
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