Excision of Reprogramming Transgenes Improves the Differentiation Potential of iPS Cells Generated with a Single Excisable Vector

Sommer, Cesar; Sommer, Andreia Gianotti; Longmire, Tyler; Christodoulou, Constantina; Thomas, Dylan C.; Gostissa, Monica; Alt, F W; Murphy, George J.; Kotton, Darrell N.; Mostoslavsky, Gustavo

doi:10.1002/stem.255

Cited by 249 publications

(252 citation statements)

References 36 publications

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“…We excised integrated and epigenetically silenced lentiviral reprogramming vectors to exclude their potential reactivation, thus reducing the risks of inhibited differentiation and teratoma formation (51,52). It may be interesting to explore whether Gag-mediated protein transduction allows the induction of reprogramming or differentiation by the transient delivery of transcription factors.…”

Section: Discussionmentioning

confidence: 99%

Protein transduction from retroviral Gag precursors

Voelkel¹,

Galla²,

Maetzig³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

125

118

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Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes.Flp recombinase | murine leukemia virus | pluripotent cells | protein transfer

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Section: Discussionmentioning

confidence: 99%

Protein transduction from retroviral Gag precursors

Voelkel¹,

Galla²,

Maetzig³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

125

118

View full text Add to dashboard Cite

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“…Immortalized Mll3 −/− Mll4 f/f and DKO brown preadipocytes were described (8). MEFs and preadipocytes were reprogrammed with lentiviral infection of OSKM as described (21). Animal experiments in this study were approved by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Animal Care and Use Committee.…”

Section: Methodsmentioning

confidence: 99%

Enhancer priming by H3K4 methyltransferase MLL4 controls cell fate transition

Wang

Lee

Lai

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

189

188

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Transcriptional enhancers control cell-type-specific gene expression. Primed enhancers are marked by histone H3 lysine 4 (H3K4) mono/di-methylation (H3K4me1/2). Active enhancers are further marked by H3K27 acetylation (H3K27ac). Mixed-lineage leukemia 4 (MLL4/KMT2D) is a major enhancer H3K4me1/2 methyltransferase with functional redundancy with MLL3 (KMT2C). However, its role in cell fate maintenance and transition is poorly understood. Here, we show in mouse embryonic stem cells (ESCs) that MLL4 associates with, but is surprisingly dispensable for the maintenance of, active enhancers of cell-identity genes. As a result, MLL4 is dispensable for cell-identity gene expression and self-renewal in ESCs. In contrast, MLL4 is required for enhancer-binding of H3K27 acetyltransferase p300, enhancer activation, and induction of cell-identity genes during ESC differentiation. MLL4 protein, rather than MLL4-mediated H3K4 methylation, controls p300 recruitment to enhancers. We also show that, in somatic cells, MLL4 is dispensable for maintaining cell identity but essential for reprogramming into induced pluripotent stem cells. These results indicate that, although enhancer priming by MLL4 is dispensable for cell-identity maintenance, it controls cell fate transition by orchestrating p300-mediated enhancer activation.enhancer | MLL4/KMT2D | H3K4 methyltransferase | cell fate transition | p300 I n mammalian cells, enhancers coordinate with promoters to precisely control cell-type-specific gene transcription, which determines the cell identity (1, 2). Comprehensive genome-wide studies have provided insights into the chromatin signatures of enhancers. Primed enhancers are marked by H3K4me1/2. Active enhancers (AEs) are further marked by the histone acetyltransferases CBP/p300-mediated H3K27ac (3). Recent studies classify AEs into typical enhancers and superenhancers. Superenhancers are clusters of AEs bound by lineage-determining transcription factors (TFs). Compared with typical enhancers, superenhancers are more cell-lineage-specific and control cell identity (4). Enhancer activation is orchestrated through a regulatory network involving lineage-determining TFs and chromatinmodifying complexes (2). However, how chromatin-modifying complexes regulate enhancer activation and cell fate transition is poorly understood.Embryonic stem cells (ESCs) derived from blastocysts are capable of unlimited replication in vitro, a property known as ESC self-renewal. ESC identity is maintained during self-renewal. ESC self-renewal is controlled by a core circuitry of TFs including Oct4, Sox2, and Nanog (4). ESCs can rapidly respond to environmental cues and differentiate into three germ layers-ectoderm, endoderm, and mesoderm-which consist of all cell lineages within days (5). Differentiated somatic cells can also be converted back to the pluripotent stage by ectopic expression of Oct4 and Sox2 together with Klf4 and c-Myc, a process known as somatic cell reprogramming into induced pluripotent stem cells (iPSCs) (6). The dramatic changes of ce...

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“…Liver cells are highly permissive for adenoviral infections and, therefore, less adenovirus is required for the efficient reprogramming of liver cells compared with fibroblasts. However, the continuous expression of transgenes in iPSCs can limit their differentiation potential (17). Many studies have explored the removal of viral vectors after reprogramming.…”

Section: Induced Pluripotent Stem Cellsmentioning

confidence: 99%

Pluripotent stem cells to hepatocytes, the journey so far

2017

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Abstract. Over the past several years, there has been substantial progress in the field of regenerative medicine, which has enabled new possibilities for research and clinical application. For example, there are ongoing efforts directed at generating functional hepatocytes from adult-derived pluripotent cells for toxicity screening, generating disease models or, in the longer term, for the treatment of liver failure. In the present review, the authors summarise recent developments in regenerative medicine and pluripotent stem cells, the methods and tissues used for reprogramming and the differentiation of induced pluripotent stem cells (iPSCs) into hepatocyte-like cells. In addition, the hepatic disease models developed using iPSC technologies are discussed, as well as the potential for gene editing.

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Excision of Reprogramming Transgenes Improves the Differentiation Potential of iPS Cells Generated with a Single Excisable Vector

Cited by 249 publications

References 36 publications

Protein transduction from retroviral Gag precursors

Protein transduction from retroviral Gag precursors

Enhancer priming by H3K4 methyltransferase MLL4 controls cell fate transition

Pluripotent stem cells to hepatocytes, the journey so far

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