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

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Nature Biotechnology (Impact Factor: 41.51). 08/2008; 26(8):916-24. DOI: 10.1038/nbt1483
Source: PubMed


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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    • "Cell Culture and Mice Mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) were isolated as previously described (Wernig et al., 2008). MEFs and TTFs were grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics . "
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    ABSTRACT: Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency.
    Cell stem cell 09/2015; DOI:10.1016/j.stem.2015.08.006 · 22.27 Impact Factor
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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), 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 · 5.37 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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