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  • Pluripotent stem cells such as embryonic stem cells

    2018-11-08

    Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have special abilities to differentiate into cells of all three germ layers (pluripotency) and to undergo unlimited proliferation while retaining their identities (self-renewal) (Nichols and Smith, 2012). In addition, they are known to be able to maintain genetic integrity, which is an essential requirement for their utilization in genetic studies or medical applications. Maintaining chromosome number is particularly important in pluripotent stem cells because Phenformin can lead not only to oncogenic transformation but also to differentiation dysregulation (Peterson and Loring, 2014; Ben-David et al., 2014; Lamm et al., 2016; Zhang et al., 2016). Nevertheless, aneuploidy is often observed in some human ESC (hESC) and iPSC lines (Spits et al., 2008; Mayshar et al., 2010; Taapken et al., 2011). A screening study of a large number of hESC/iPSC lines documented a progressive tendency to acquire karyotypic abnormality during long-term culture, indicating a culture-associated susceptibility to aneuploidy (International Stem Cell Initiative et al., 2011). Although previous reports describe several putative risks contributing to chromosome instability, including excessive replication stresses and DNA damage responses (Zhao et al., 2015; Lamm et al., 2016; Jacobs et al., 2016), safeguarding mechanisms to counteract these threats remain to be elucidated. We previously reported that the aberrant activation of the RHO-ROCK pathway was responsible for dissociation-induced hESC apoptosis (Watanabe et al., 2007; Ohgushi et al., 2010). We also identified ABR, a modulator of RHO family small GTPase activities, as an upstream factor controlling the survival-or-death decision of dissociated hESCs. The ROCK activation is thought to affect cellular motility (Li et al., 2010), but whether this phenomenon represents any biological implications has remained a mystery. To tackle this question, we sought to explore ABR function. We found that ABR did not have direct effects on cell survival unless cell-cell contact was impaired. Instead, we obtained unexpected data indicating that ABR depletion increased the frequency of chromosome missegregation. These findings shed light on the safeguarding mechanism that prevents chromosomal instability in hESCs.
    Results
    Discussion In this study, we explored ABR function in clump-cultured hESCs. We first noticed that ABR depletion impeded G2-to-M-to-G1 transitions. Deeper investigations at a single-cell level revealed that ABR-depleted cells struggled to complete a couple of mitotic steps, including centrosome separation at prophase and chromosome alignment at metaphase. These observations indicated that ABR has a crucial role in mitosis progression. Important information lacking now is subcellular localization of ABR. Our attempts to determine its localization in hESCs did not work well, but a large-scale proteomics analysis demonstrated ABR as a putative interactor of some centrosomal proteins (e.g., CEP25, Fogeron et al., 2013), supporting our conclusion. ABR seems to play a safeguarding role in mitotic fidelity, in addition to being an apoptosis promoter in dissociated cells (Figure S3D), and these different outcomes upon ABR activation are dictated by the cellular adhesive state, dissociation versus clumping. A previous report demonstrated that the mitotic activation of actomyosin sometimes stimulated cell death, mirroring the dissociation-induced phenotype (Barbaric et al., 2014). Considering that cellular adhesiveness is dynamically rearranged during mitosis, spontaneous failures in the adhesion-mediated control of ABR activity could occur upon mitosis. An intriguing possibility is that mitotic cells in which ABR is inappropriately regulated might be intrinsically programmed to be eliminated, representing a mechanism restraining expansion of genetically abnormal cells. Consistently, it seems that ABR is not absolutely required for mitosis completion, but mitosis without ABR is an error-prone process leading to frequent chromosome missegregation. These results indicate that ABR sets a robust way for chromosome segregation in hESCs. This might be favorable, particularly to the self-renewing pluripotent stem cells in which the postmitotic checkpoint signaling is likely uncoupled to apoptosis-mediated elimination of genetically abnormal cells (Mantel et al., 2007).