title: Temporal repression of endogenous pluripotency genes during reprogramming of porcine induced pluripotent stem cells.
abstract: Porcine induced pluripotent stem cells (piPSCs) have the capacity to differentiate in vitro and in vivo and form chimeras. However, the lack of transgene silencing of exogenous DNA integrated into the genome and the inability of cells to proliferate in the absence of transgene expression are underlying reported problems, suggesting that reprogramming is not complete. The aim of the present study was to evaluate reprogramming events using a partially reprogrammed piPSC-like line expressing hOCT4, hNANOG, and hcMYC under tetracycline-regulated control to investigate the effects of these particular transgenes on the expression of the porcine endogenous pluripotency machinery. Endogenous and exogenous gene expression of OCT4, NANOG, SOX2, KLF4, and cMYC was determined at passages 5, 10, 15, and 20, both in cells cultured at 1 μg/mL doxycycline or 4 μg/mL doxycycline. Our results revealed that endogenous genes are repressed by their transgene counterparts in culture and that lack of expression of the transgenes, SOX2 and KLF4 allows for expression of endogenous SOX2 and KLF4. Furthermore, we report that alternate endogenous transcripts for pNANOG, pSOX2, and pKLF4 can also be detected in the pig. Despite the ability for some endogenous genes to be expressed in these lines, the piPSC-like cells still cannot be maintained without doxycycline, indicating that the culture system of piPSCs may not be optimal or that the reprogramming factor combination used may not currently be optimal for maintaining pluripotency in the pig. This may help to explain the difficulties in producing stable piPSCs and bona fide embryonic stem cell lines in this species.
authors.affiliation: University of Copenhagen, Faculty of Life Sciences, Department of Basic Animal and Veterinary Sciences, Frederiksberg C., DK-1870, Denmark. vha@life.ku.dk
authors.forename: Vanessa J
authors.initials: VJ
authors.lastname: Hall
authors.forename: Marianne
authors.initials: M
authors.lastname: Kristensen
authors.forename: Mikkel A
authors.initials: MA
authors.lastname: Rasmussen
authors.forename: Olga
authors.initials: O
authors.lastname: Ujhelly
authors.forename: András
authors.initials: A
authors.lastname: Dinnyés
authors.forename: Poul
authors.initials: P
authors.lastname: Hyttel
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000502:physiology*; D015536:Down-Regulation / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology; D018507:Gene Expression Regulation, Developmental; D050437:Genes, Developmental / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism* / Q000502:physiology; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D030781:Organisms, Genetically Modified; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D013552:Swine / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D019076:Transgenes
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors