A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types

Wernig, Marius; Lengner, Christopher J.; Hanna, Jacob; Lodato, Michael A.; Steine, Eveline J.; Foreman, Ruth K.; Staerk, Judith; Markoulaki, Styliani; Jaenisch, Rudolf

doi:10.1038/nbt1483

Cited by 404 publications

(417 citation statements)

References 28 publications

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“…We found that 8 days of dox-induced Oct4 expression was sufficient for iPSC generation observed at day 24 after transduction ( Figure 4A). Efficiency increased when Oct4 expression was induced for 12 days (Figure 4A), which is in consistent with previous reports that a greater number of iPSCs are generated when exogenous reprogramming factors were expressed for longer period of time [19,20]. However, iPSC colony numbers decreased when Oct4 was induced for more than 12 days, and most of the iPSC colonies emerged 4-8 days after dox withdrawal ( Figure 4A).…”

Section: Reprogramming Kinetics Of Mouse Fibroblasts By Doxinducible supporting

confidence: 81%

Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules

et al. 2010

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The introduction of four transcription factors Oct4, Klf4, Sox2 and c-Myc by viral transduction can induce reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), but the use of iPSCs is hindered by the use of viral delivery systems. Chemical-induced reprogramming offers a novel approach to generating iPSCs without any viral vector-based genetic modification. Previous reports showed that several small molecules could replace some of the reprogramming factors although at least two transcription factors, Oct4 and Klf4, are still required to generate iPSCs from mouse embryonic fibroblasts. Here, we identify a specific chemical combination, which is sufficient to permit reprogramming from mouse embryonic and adult fibroblasts in the presence of a single transcription factor, Oct4, within 20 days, replacing Sox2, Klf4 and c-Myc. The iPSCs generated using this treatment resembled mouse embryonic stem cells in terms of global gene expression profile, epigenetic status and pluripotency both in vitro and in vivo. We also found that 8 days of Oct4 induction was sufficient to enable Oct4-induced reprogramming in the presence of the small molecules, which suggests that reprogramming was initiated within the first 8 days and was independent of continuous exogenous Oct4 expression. These discoveries will aid in the future generation of iPSCs without genetic modification, as well as elucidating the molecular mechanisms that underlie the reprogramming process.

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Section: Reprogramming Kinetics Of Mouse Fibroblasts By Doxinducible supporting

confidence: 81%

Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules

et al. 2010

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“…The Brachyury-green fluorescent protein (Brachyury-GFP) ES cell line was provided by Dr. Gordon Keller (McEwen Center for Regenerative Medicine, Toronto, ON, Canada). The murine iPS cell line, NANOG-GFP2 was gifted from Prof. Rudolf Jaenisch (Whitehead Institute, Cambridge, MA) and has been described previously [21]. All chemicals, unless otherwise stated, were purchased from Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com).…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Reactive Oxygen Species Mediate Cardiomyocyte Formation from Embryonic Stem Cells in High Glucose

et al. 2010

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Accumulating evidence points to reactive oxygen species (ROS) as important signaling molecules for cardiomyocyte differentiation in embryonic stem (ES) cells. Given that ES cells are normally maintained and differentiated in medium containing supraphysiological levels of glucose (25 mM), a condition which is known to result in enhanced cellular ROS formation, we questioned whether this high glucose concentration was necessary for cardiomyocyte lineage potential. We show here that ES cells cultured in physiological glucose (5 mM), maintained their general stemness qualities but displayed an altered mitochondrial metabolism, which resulted in decreased ROS production. Furthermore, ES and induced pluripotent stem (iPS) cells differentiated in lower glucose concentrations failed to generate cardiomyocyte structures; an effect mimicked with antioxidant treatments using catalase, N-acetyl cysteine and mitoubiquinone, under high glucose conditions in ES cells. Molecular analysis revealed that ES cells differentiated in 5 mM glucose had reduced expression of the pro-cardiac NOX4 gene and diminished phosphorylation of p38 mitogen-activated protein kinase (MAPK), together with specific changes in the cardiac transcriptional network. These outcomes could be reversed by supplementation of low glucose cultures with ascorbic acid, paradoxically acting as a pro-oxidant. Furthermore, forced expression of an upstream p38 MAPK kinase (MKK6) could bypass the requirement for ROS during differentiation to cardiomyocytes under low glucose conditions, illustrating a key role for p38 in the cardiac differentiation program. Together these data demonstrate that endogenous ROS control is important for cardiomyocyte formation from ES cells, and furthermore that supraphysiological glucose, by supplying ROS, is absolutely required.

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“…Secondary mouse embryonic fibroblasts (2° MEFs) were isolated from chimeric embryos as previously described 20 , providing doxycycline inducible Oct4, Sox2, Klf4, and c-Myc expression; Neomycin selection at the Nanog locus; and a fluorescent transcriptional reporter for Oct4. The parental pluripotent stem cells used to generate the secondary line were derived by replacing an Oct4 allele with an Oct4-IRES-GFP sequence in Nanog-Neo iPS cells 20 .…”

Section: Secondary Somatic Cell Generation and Reprogrammingmentioning

confidence: 99%

“…The parental pluripotent stem cells used to generate the secondary line were derived by replacing an Oct4 allele with an Oct4-IRES-GFP sequence in Nanog-Neo iPS cells 20 . Secondary MEFs were plated at optimal density 20 and passaged after 48 hours. Doxycycline (2μg/ml, Stemgent) was added 24 hours after replating, marking the start of reprogramming.…”

Section: Secondary Somatic Cell Generation and Reprogrammingmentioning

confidence: 99%

Transcriptional profiling of cells sorted by RNA abundance

et al. 2014

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We have developed a quantitative technique for sorting cells based on endogenous RNA abundance with a molecular resolution of 10-20 transcripts. We demonstrate efficient and unbiased RNA extraction from transcriptionally sorted cells and report a high fidelity transcriptome measurement of (mouse) induced pluripotent stem cells (iPSCs) isolated from a heterogeneous reprogramming culture. This method is broadly applicable to profiling transcriptionally distinct cellular states without requiring antibodies or transgenic fluorescent proteins.

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A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types

Cited by 404 publications

References 28 publications

Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules

Generation of iPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules

Mitochondrial Reactive Oxygen Species Mediate Cardiomyocyte Formation from Embryonic Stem Cells in High Glucose

Transcriptional profiling of cells sorted by RNA abundance

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