title: Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig.
abstract: Cellular reprogramming of committed cells into a pluripotent state can be induced by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. Reprogrammed cells can be maintained by activating endogenous pluripotent networks without transgene expression. Although various research groups have attempted to generate pig induced pluripotent stem cells (iPSCs), authentic iPSCs have not be obtained, instead showing dependence on transgene expression. In this study, iPSCs were derived from porcine fetal fibroblasts via drug-inducible vectors carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study investigated characteristics of iPSCs and reprogramming mechanisms in pig. The iPSCs were stably maintained over an extended period with potential in vitro differentiation into three germ layers. In addition, the pluripotent state of iPSCs was regulated by modulating culture conditions. They showed naive- or primed-like pluripotent states in LIF or bFGF supplemented culture conditions, respectively. However, iPSCs could not be maintained without ectopic expression of transgenes. The cultured iPSCs expressed endogenous transcription factors such as OCT4 and SOX2, but not NANOG (a known gateway to complete reprogramming). Endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated, as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occur in female iPSCs. Based on our results, expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu to achieve faithful pluripotency in pig. Accordingly, investigating iPSCs could help us improve and develop reprogramming methods by understanding reprogramming mechanisms in pig.
authors.affiliation: Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Korea.
authors.forename: Kwang-Hwan
authors.initials: KH
authors.lastname: Choi
authors.affiliation: Division of Animal Sciences and Bond Life Sciences Center, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Jin-Kyu
authors.initials: JK
authors.lastname: Park
authors.affiliation: Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Korea.
authors.forename: Dongchan
authors.initials: D
authors.lastname: Son
authors.affiliation: Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Korea.
authors.forename: Jae Yeon
authors.initials: JY
authors.lastname: Hwang
authors.affiliation: Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Korea.
authors.forename: Dong-Kyung
authors.initials: DK
authors.lastname: Lee
authors.affiliation: Department of Biological Resources and Technology, Yonsei University, Wonju, Korea.
authors.forename: Hakhyun
authors.initials: H
authors.lastname: Ka
authors.affiliation: LG Life Sciences, R&D Park, Daejeon, Republic of Korea.
authors.forename: Joonghoon
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Korea.|Institute of Green Bio Science and Technology, Seoul National University, Pyeong Chang, Kangwon do, Korea.
authors.forename: Chang-Kyu
authors.initials: CK
authors.lastname: Lee
mesh_terms: D000818:Animals; D015415:Biomarkers; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000235:genetics; D044127:Epigenesis, Genetic; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D013552:Swine; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation; D019076:Transgenes
chemical_list: D015415:Biomarkers; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors