title: Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia.
abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research.
authors.affiliation: Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Shimada
authors.forename: Yoshiya
authors.initials: Y
authors.lastname: Hashimoto
authors.forename: Akira
authors.initials: A
authors.lastname: Nakada
authors.forename: Keiji
authors.initials: K
authors.lastname: Shigeno
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Nakamura
mesh_terms: D000577:Amides / Q000494:pharmacology; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D002087:Butyrates / Q000494:pharmacology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015687:Cell Hypoxia; D002460:Cell Line; D019175:DNA Methylation; D004159:Diphenylamine / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020868:Gene Silencing; D016259:Genes, myc; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012190:Retroviridae; D055748:SOXB1 Transcription Factors / Q000235:genetics; D013859:Thiocarbamates / Q000494:pharmacology; D013882:Thiosemicarbazones; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: C507011:A-83-01; D000577:Amides; D001549:Benzamides; D002087:Butyrates; C473711:Chir 99021; D004791:Enzyme Inhibitors; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; D011720:Pyrazoles; D011725:Pyridines; D011743:Pyrimidines; D055748:SOXB1 Transcription Factors; D013859:Thiocarbamates; D013882:Thiosemicarbazones; C108830:Y 27632; C506614:mirdametinib; D004159:Diphenylamine