A chemical platform for improved induction of human iPSCs

Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA.
Nature Methods (Impact Factor: 32.07). 11/2009; 6(11):805-8. DOI: 10.1038/nmeth.1393
Source: PubMed


The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (200-fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, nonviral methods for reprogramming human somatic cells.

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    • "We used the combination of LDN193189, SB431542, and TTNPB to initiate the reprogramming process by inhibiting glial signaling pathways and activating neuronal signaling pathways simultaneously. Tzv, an inhibitor of Rho-associated kinase (ROCK), promotes cell survival and improves reprogramming efficiency (Lin et al., 2009; Watanabe et al., 2007). Tzv was included throughout the 8 days of the reprogramming period. "
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    ABSTRACT: We have recently demonstrated that reactive glial cells can be directly reprogrammed into functional neurons by a single neural transcription factor, NeuroD1. Here we report that a combination of small molecules can also reprogram human astrocytes in culture into fully functional neurons. We demonstrate that sequential exposure of human astrocytes to a cocktail of nine small molecules that inhibit glial but activate neuronal signaling pathways can successfully reprogram astrocytes into neurons in 8-10 days. This chemical reprogramming is mediated through epigenetic regulation and involves transcriptional activation of NEUROD1 and NEUROGENIN2. The human astrocyte-converted neurons can survive for >5 months in culture and form functional synaptic networks with synchronous burst activities. The chemically reprogrammed human neurons can also survive for >1 month in the mouse brain in vivo and integrate into local circuits. Our study opens a new avenue using chemical compounds to reprogram reactive glial cells into functional neurons.
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    • "Butyrate appears to be particularly effective when combined with the TGF-b and MAPK/ ERK pathway inhibitors (SB431542 and PD0325901, or others), which have previously been reported to aid iPSC reprogramming. Increased hiPSC reprogramming efficiency has also been achieved with the HDAC inhibitor trichostatin A (TSA) but with lower efficiency and greater toxicity than other HDAC inhibitors (Lin et al., 2009; Mali et al., 2010; Zhang and Wu, 2013). "
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    ABSTRACT: Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a comprehensive epigenetic process involving genome-wide modifications of histones and DNA methylation. This process is often incomplete, which subsequently affects iPSC reprograming, pluripotency, and differentiation capacity. Here, we review the epigenetic changes with a focus on histone modification (methylation and acetylation) and DNA modification (methylation) during iPSC induction. We look at changes in specific epigenetic signatures, aberrations and epigenetic memory during reprogramming and small molecules influencing the epigenetic reprogramming of somatic cells. Finally, we discuss how to improve iPSC generation and pluripotency through epigenetic manipulations.
    No preview · Article · Oct 2015 · Journal of Genetics and Genomics
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    • "PD and CH are used to convert human pluripotent stem cells to the naive state [4], [17]. Combination of SB and PD, or SB, PD, and sodium butyrate (NAB) can convert partially reprogrammed colonies to a fully reprogrammed state, thereby improving the efficiency of reprogramming [18], [19]. Moreover, epigenetic modifier NAB is more reliable and efficient than VPA in generation of human iPS cells and contributes to more efficient reprogramming [20], [21]. "
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    ABSTRACT: Induced pluripotent stem (iPS) cells from somatic cells have great potential for regenerative medicine. The efficiency in generation of iPS cells has been significantly improved in recent years. However, the generation of high-quality iPS cells remains of high interest. Consistently, we demonstrate that knockout serum replacement (KSR)-based medium accelerates iPS cell induction and improves the quality of iPS cells, as confirmed by generation of chimeras and all iPS cell-derived offspring with germline transmission competency. Both alkaline phosphatase (AP) activity assay and expression of Nanog have been used to evaluate the efficiency of iPS cell induction and formation of ES/iPS cell colonies; however, appropriate expression of Nanog frequently indicates the quality of ES/iPS cells. Interestingly, whereas foetal bovine serum (FBS)-based media increase iPS cell colony formation, as revealed by AP activity, KSR-based media increase the frequency of iPS cell colony formation with Nanog expression. Furthermore, inhibition of MAPK/ERK by a specific inhibitor, PD0325901, in KSR- but not in FBS-based media significantly increases Nanog-GFP+ iPS cells. In contrast, addition of bFGF in KSR-based media decreases proportion of Nanog-GFP+ iPS cells. Remarkably, PD can rescue Nanog-GFP+ deficiency caused by bFGF. These data suggest that MAPK/ERK pathway influences high quality mouse iPS cells and that KSR- and PD-based media could enrich homogeneous authentic pluripotent stem cells.
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