Molecular Pathway and Cell State Responsible for Dissociation-Induced Apoptosis in Human Pluripotent Stem Cells

Organogenesis and Neurogenesis Group, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
Cell stem cell (Impact Factor: 22.27). 08/2010; 7(2):225-39. DOI: 10.1016/j.stem.2010.06.018
Source: PubMed

ABSTRACT Human embryonic stem cells (hESCs), unlike mouse ones (mESCs), are vulnerable to apoptosis upon dissociation. Here, we show that the apoptosis, which is of a nonanoikis type, is caused by ROCK-dependent hyperactivation of actomyosin and efficiently suppressed by the myosin inhibitor Blebbistatin. The actomyosin hyperactivation is triggered by the loss of E-cadherin-dependent intercellular contact and also observed in dissociated mouse epiblast-derived pluripotent cells but not in mESCs. We reveal that Abr, a unique Rho-GEF family factor containing a functional Rac-GAP domain, is an indispensable upstream regulator of the apoptosis and ROCK/myosin hyperactivation. Rho activation coupled with Rac inhibition is induced in hESCs upon dissociation, but not in Abr-depleted hESCs or mESCs. Furthermore, artificial Rho or ROCK activation with Rac inhibition restores the vulnerability of Abr-depleted hESCs to dissociation-induced apoptosis. Thus, the Abr-dependent "Rho-high/Rac-low" state plays a decisive role in initiating the dissociation-induced actomyosin hyperactivation and apoptosis in hESCs.

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Available from: Keiko Muguruma, Sep 26, 2015
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    • "These new techniques, however, are expensive, have limited scalability, and may have high batch-to-batch variability. Y-27632, a ROCK inhibitor , is used to prevent cell apoptosis after cell dissociation and to promote cell viability after plating (Ohgushi et al., 2010; Watanabe et al., 2007), but the benefit of this chemical is limited to a brief period after cell dissociation and its continued effects on cell survival and proliferation are questionable (Couture, 2010). Thus, culture methods that are low cost, robust, scalable, easy to use, and consistent remain to be further developed to allow widespread applications of hESC/iPSCs in basic research and clinical. "
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    ABSTRACT: We here report that doxycycline, an antibacterial agent, exerts dramatic effects on human embryonic stem and induced pluripotent stem cells (hESC/iPSCs) survival and self-renewal. The survival-promoting effect was also manifest in cultures of neural stem cells (NSCs) derived from hESC/iPSCs. These doxycycline effects are not associated with its antibacterial action, but mediated by direct activation of a PI3K-AKT intracellular signal. These findings indicate doxycycline as a useful supplement for stem cell cultures, facilitating their growth and maintenance.
    Stem Cell Reports 08/2014; 3(2). DOI:10.1016/j.stemcr.2014.06.013 · 5.37 Impact Factor
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    • "Indeed, although the cell-cycle time of hESCs is relatively short (less than 24 hr) (Becker et al., 2006), hESCs are commonly passaged only every 4–5 days at low split ratios (1:3 or even 1:2), implying a loss of up to 90% of cells from cultures (Olariu et al., 2010). The extensive hESC death is even further exacerbated upon passaging of cells by enzymatic methods that entail dissociation of cell colonies to single cells (Chen et al., 2010; Ohgushi et al., 2010; Watanabe et al., 2007), resulting in a very low single-cell cloning efficiency (typically <1%) (Enver et al., 2005; Harrison et al., 2007). The low cloning efficiency is at least partly due to an excessive apoptosis of cells upon dissociation (Chen et al., 2010; Ohgushi et al., 2010), but the discrepancy in the number of cells surviving the initial plating and the overall cloning efficiency suggests that critical restriction points exist between initial plating and when robust colony formation is established. "
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    ABSTRACT: Summary Using time-lapse imaging, we have identified a series of bottlenecks that restrict growth of early-passage human embryonic stem cells (hESCs) and that are relieved by karyotypically abnormal variants that are selected by prolonged culture. Only a minority of karyotypically normal cells divided after plating, and these were mainly cells in the later stages of cell cycle at the time of plating. Furthermore, the daughter cells showed a continued pattern of cell death after division, so that few formed long-term proliferating colonies. These colony-forming cells showed distinct patterns of cell movement. Increasing cell density enhanced cell movement facilitating cell:cell contact, which resulted in increased proportion of dividing cells and improved survival postplating of normal hESCs. In contrast, most of the karyotypically abnormal cells reentered the cell cycle on plating and gave rise to healthy progeny, without the need for cell:cell contacts and independent of their motility patterns.
    Stem Cell Reports 07/2014; 3(1):142-155. DOI:10.1016/j.stemcr.2014.05.006 · 5.37 Impact Factor
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    • "The inherent sensitivity of hPS cells arises from the relatively poor understanding of cell–cell and cell–substrate interactions underlying the maintenance of pluripotency. It is now known that hPS cells undergo dissociation-associated apoptosis and inhibiting RhoA/ROCK mediated, NMMII-dependent cytoskeletal tension enhances hPS cell survival (Ohgushi et al., 2010). As RhoA/ROCK mediated cytoskeletal tension is an important factor in mechanotransduction (McBeath et al., 2004 "
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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.
    Frontiers in Pharmacology 07/2014; 5:150. DOI:10.3389/fphar.2014.00150 · 3.80 Impact Factor
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