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A kinase inhibitor screen identifies small-molecule enhancers of reprogramming and iPS cell generation

1] Program for RNA Biology, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, USA. [2] Chemical Biology Program, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Nature Communications (Impact Factor: 11.47). 09/2012; 3:1085. DOI: 10.1038/ncomms2059
Source: PubMed

ABSTRACT

Somatic cells can be reprogrammed to form embryonic stem cell-like induced pluripotent stem cells (iPSCs), but the process suffers from low efficiency and the underlying molecular mechanisms that control reprogramming remain poorly understood. Here we perform an inhibitor screen to identify kinases that enhance, or present a barrier to, reprogramming. In particular, inhibitors of p38, inositol trisphosphate 3-kinase, and Aurora A kinase potently enhance iPSC generation, and iPSCs derived from inhibitor-treated somatic cells are capable of reaching a fully reprogrammed state. Knockdown of target kinases by short interfering RNAs confirms that they function as barrier genes. We show that Aurora A kinase, which functions in centrosome activity and spindle assembly, is highly induced during reprogramming and inhibits Akt-mediated inactivation of GSK3β, resulting in compromised reprogramming efficiency. Together, our results not only identify new compounds that enhance iPSC generation but also shed new light on the function of Aurora A kinase in the reprogramming process.

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    • "Small molecules used to generate iPSCs are comprised of epigenetic modifiers, WNT signal modulators, cell senescence attenuators, metabolism modulators, and regulators of cell apoptosis/senescence pathways. Small molecules inducing iPSCs can be classified into three types: (1) small molecules that improve reprogramming efficiency[55]; (2) compounds replacing one or more reprogramming factors565758; and (3) compound cocktails alone that are sufficient to induce iPSCs[59,60]. Small molecule methods have been successfully applied to reprogram mouse fibroblasts directly into functional neurons using only a combination of small molecules[59,60]. "

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    • "We will not discuss further studies aimed at isolating compounds that improve reprogramming as these are discussed elsewhere (Chen et al., 2010b; Yang et al., 2011; Li and Rana, 2012). In reviewing HCA screens using hPS cells with a reasonable throughput we will start by focusing on survival and self-renewal (Table 1). "
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    ABSTRACT: Self-renewing stem cell populations are increasingly considered as resources for cell therapy and tools for drug discovery. Human pluripotent stem (hPS) cells in particular offer a virtually unlimited reservoir of homogeneous cells and can be differentiated toward diverse lineages. Many diseases show impairment in self-renewal or differentiation, abnormal lineage choice or other aberrant cell behavior in response to chemical or physical cues. To investigate these responses, there is a growing interest in the development of specific assays using hPS cells, artificial microenvironments and high content analysis. Several hurdles need to be overcome that can be grouped into three areas: (i) availability of robust, homogeneous, and consistent cell populations as a starting point; (ii) appropriate understanding and use of chemical and physical microenvironments; (iii) development of assays that dissect the complexity of cell populations in tissues while mirroring specific aspects of their behavior. Here we review recent progress in the culture of hPS cells and we detail the importance of the environment surrounding the cells with a focus on synthetic material and suitable high content analysis approaches. The technologies described, if properly combined, have the potential to create a paradigm shift in the way diseases are modeled and drug discovery is performed.
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    • "However, the inhibitor did not affect RHAMM+/+ mouse ES cells, which suggests that inhibition of AURKA affects self-renewal in a genotype-dependent manner. In fact, a recent double blind screen of 244 inhibitors identified AURKA as one of three barrier kinases to reprogramming and pluripotency [39]; a small-molecule AURKA inhibitor potently enhanced induced pluripotent stem (iPS) cell generation [39], which is similar to our finding that the same AURKA inhibitor restored pluripotency of RHAMM+/- mouse ES cells. However, an alternate screen of 104 ES cell-associated phosphoregulators found that the same AURKA inhibitor caused ES cells and iPS cells to differentiate [40]. "
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    ABSTRACT: Receptor for hyaluronan mediated motility (RHAMM, encoded by HMMR) may be a cell-surface receptor for hyaluronan that regulates embryonic stem cell pluripotency and differentiation, however, a precise mechanism for its action is not known. We examined murine embryonic stem cells with and without hemizygous genomic mutation of Hmmr/RHAMM, but we were not able to find RHAMM on the cell-surface. Rather, RHAMM localized to the microtubule cytoskeleton and along mitotic spindles. Genomic loss of Hmmr/RHAMM did not alter cell cycle progression but augmented differentiation and attenuated pluripotency in murine embryonic stem cells. Through a candidate screen of small-molecule kinase inhibitors, we identified ERK1/2 and aurora kinase A as barrier kinases whose inhibition was sufficient to rescue pluripotency in RHAMM(+/-) murine embryonic stem cells. Thus, RHAMM is not found on the cell-surface of embryonic stem cells, but it is required to maintain pluripotency and its dominant mechanism of action is through the modulation of signal transduction pathways at microtubules.
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