A Small-Molecule Inhibitor of Tgf-β Signaling Replaces Sox2 in Reprogramming by Inducing Nanog

Harvard Stem Cell Institute, Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
Cell stem cell (Impact Factor: 22.27). 10/2009; 5(5):491-503. DOI: 10.1016/j.stem.2009.09.012
Source: PubMed


The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.

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Available from: Kyle M Loh, Jun 20, 2014
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    • "The sustained expression of somatic cell pathways and suppression of mesenchymal-to-epithelial transition (MET) are two major barriers in the initial stages of reprogramming (Li et al., 2010; Samavarchi-Tehrani et al., 2010). Suppression of the transforming growth factorb (TGF-b) pathway or activation of the bone morphogenetic protein (BMP) pathway promote MET and increase reprogramming efficiency (Ichida et al., 2009; Samavarchi- Tehrani et al., 2010). The class of partially reprogrammed cells that successfully pass through the initial stages and fail to activate the endogenous pluripotency genes are referred to as pre-iPSCs (Theunissen et al., 2011). "
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    ABSTRACT: Reprogramming of somatic cells to generate induced pluripotent stem cells (iPSCs) has considerable latency and generates epigenetically distinct partially and fully reprogrammed clones. To understand the molecular basis of reprogramming and to distinguish the partially reprogrammed iPSC clones (pre-iPSCs), we analyzed several of these clones for their molecular signatures. Using a combination of markers that are expressed at different stages of reprogramming, we found that the partially reprogrammed stable clones have significant morphological and molecular heterogeneity in their response to transition to the fully pluripotent state. The pre-iPSCs had significant levels of OCT4 expression but exhibited variable levels of mesenchymal-to-epithelial transition. These novel molecular signatures that we identified would help in using these cells to understand the molecular mechanisms in the late of stages of reprogramming. Although morphologically similar mouse iPSC clones showed significant heterogeneity, the human iPSC clones isolated initially on the basis of morphology were highly homogeneous with respect to the levels of pluripotency.
    Full-text · Article · Nov 2015
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    • "In addition, many epigenetic regulators have been showed to play critical roles during the reprogramming process such as enzymes that modulate histone modification and DNA (de) methylation (Apostolou and Hochedlinger, 2013; Buganim et al., 2013; Liang and Zhang, 2013; Theunissen and Jaenisch, 2014). Furthermore, previous studies have uncovered the important roles of multiple signaling pathways during reprogramming such as TGFb (Ichida et al., 2009; Maherali and Hochedlinger, 2009; Liu et al., 2013), BMP (Samavarchi-Tehrani et al., 2010; Chen et al., 2013), Wnt/b-catenin (Marson et al., 2008; Ho et al., 2013), p53-p21 (Hong et al., 2009; Kawamura et al., 2009; Marion et al., 2009), NF-kB (Lee et al., 2012), MAPK/ERK (Silva et al., 2008), mTOR (Wang et al., 2013) and Eras-Akt pathways (Tang et al., 2014; Yu et al., 2014). However, there might be additional factors and signals associated with the reprogramming process. "
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    ABSTRACT: Calcineurin-NFAT signaling is critical for early lineage specification of mouse embryonic stem cells and early embryos. However, its roles in somatic cell reprogramming remain unknown. Here, we report that calcineurin-NFAT signaling has a dynamic activity and plays diverse roles at different stages of reprogramming. At the early stage, calcineurin-NFAT signaling is transiently activated and its activation is required for successful reprogramming. However, at the late stage of reprogramming, activation of calcineurin-NFAT signaling becomes a barrier for reprogramming and its inactivation is critical for successful induction of pluripotency. Mechanistically, calcineurin-NFAT signaling contributes to the reprogramming through regulating multiple early events during reprogramming, including mesenchymal to epithelial transition (MET), cell adhesion and emergence of SSEA1(+) intermediate cells. Collectively, this study reveals for the first time the important roles of calcineurin-NFAT signaling during somatic cell reprogramming and provides new insights into the molecular regulation of reprogramming. This article is protected by copyright. All rights reserved.
    Full-text · Article · Oct 2015 · Journal of Cellular Physiology
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    • "However, we observed a significantly lower reprogramming efficiency compared with reprogramming using a feeder system. Therefore, further modifications were incorporated in the reprogramming process, including the use of hypoxic culture conditions (3%–5% O 2 ) and a transforming growth factor b (TGF-b) inhibitor (A83-01), a selective inhibitor of the TGF-b type I receptor ALK5, which has been shown to enhance reprogramming efficiency (Ichida et al., 2009). We also incorporated Alhydrogel (i.e., an aluminum hydroxide wet gel suspension), which enhanced integration-free reprogramming under defined and feeder-free conditions (Figure S1). "
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    ABSTRACT: The discovery of induced pluripotent stem cells (iPSCs) and the concurrent development of protocols for their cell-type-specific differentiation have revolutionized our approach to cell therapy. It has now become critical to address the challenges related to the generation of iPSCs under current good manufacturing practice (cGMP) compliant conditions, including tissue sourcing, manufacturing, testing, and storage. Furthermore, regarding the technical challenges, it is very important to keep the costs of manufacturing and testing reasonable and solve logistic hurdles that permit the global distribution of these products. Here we describe our efforts to develop a process for the manufacturing of iPSC master cell banks (MCBs) under cGMPs and announce the availability of such banks.
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