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Wernig, M, Lengner, CJ, Hanna, J, Lodato, MA, Steine, E, Foreman, R et al.. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat Biotechnol 26: 916-924

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Nature Biotechnology (Impact Factor: 39.08). 08/2008; 26(8):916-24. DOI: 10.1038/nbt1483
Source: PubMed

ABSTRACT The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming.

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Available from: Jacob H. Hanna, May 24, 2015
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    • "Please cite this article in press as: Kim et al., KLF4 N-Terminal Variance Modulates Induced Reprogramming to Pluripotency, Stem Cell Reports (2015), http://dx.doi.org/10.1016/j.stemcr.2015.02.004 et al., 2013; Polo et al., 2012; Samavarchi-Tehrani et al., 2010; Wernig et al., 2008). "
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    ABSTRACT: As the quintessential reprogramming model, OCT3/4, SOX2, KLF4, and c-MYC re-wire somatic cells to achieve induced pluripotency. Yet, subtle differences in methodology confound comparative studies of reprogramming mechanisms. Employing transposons, we systematically assessed cellular and molecular hallmarks of mouse somatic cell reprogramming by various polycistronic cassettes. Reprogramming responses varied in the extent of initiation and stabilization of transgene-independent pluripotency. Notably, the cassettes employed one of two KLF4 variants, differing only by nine N-terminal amino acids, which generated dissimilar protein stoichiometry. Extending the shorter variant by nine N-terminal amino acids or augmenting stoichiometry by KLF4 supplementation rescued both protein levels and phenotypic disparities, implicating a threshold in determining reprogramming outcomes. Strikingly, global gene expression patterns elicited by published polycistronic cassettes diverged according to each KLF4 variant. Our data expose a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, predicts reprogramming hallmarks, and guides comparison of compatible public data sets. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Stem Cell Reports 03/2015; 26(4). DOI:10.1016/j.stemcr.2015.02.004 · 5.64 Impact Factor
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    • "Somatic cells can be reprogrammed into pluripotency by expression of defined transcription factors (Lowry et al., 2008; Park et al., 2008; Takahashi et al., 2007; Takahashi and Yamanaka , 2006; Wernig et al., 2007). Although most cell types can be reprogrammed, this dramatic cell fate conversion occurs only at low frequency following long latency, even when all cells are engineered to express the reprogramming factors (Carey et al., 2010; Stadtfeld and Hochedlinger, 2010; Wernig et al., 2008). The prevailing theory for this low efficiency and long latency is a stochastic model, which calls upon stochastic changes to help subvert the various barriers limiting the fate transitions (reviewed in Hanna et al., 2010; Stadtfeld and Hochedlinger , 2010; Yamanaka, 2009). "
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    • "To assess the reproducibility of iNSC formation, we developed a ''secondary'' system (Wernig et al., 2008b) in which dox-inducible vectors were carried in all cells of chimeras and allowed transdifferentiation in the absence of virus transduction (Figure 3A). For this, iNSC-14F cells were reprogrammed to pluripotency using retroviral vectors (Figure S3A). "
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    ABSTRACT: Overexpression of transcription factors has been used to directly reprogram somatic cells into a range of other differentiated cell types, including multipotent neural stem cells (NSCs), that can be used to generate neurons and glia. However, the ability to maintain the NSC state independent of the inducing factors and the identity of the somatic donor cells remain two important unresolved issues in transdifferentiation. Here we used transduction of doxycycline-inducible transcription factors to generate stable tripotent NSCs. The induced NSCs (iNSCs) maintained their characteristics in the absence of exogenous factor expression and were transcriptionally, epigenetically, and functionally similar to primary brain-derived NSCs. Importantly, we also generated tripotent iNSCs from multiple adult cell types, including mature liver and B cells. Our results show that self-maintaining proliferative neural cells can be induced from nonectodermal cells by expressing specific combinations of transcription factors. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    Stem Cell Reports 11/2014; 3(6). DOI:10.1016/j.stemcr.2014.10.001 · 5.64 Impact Factor
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