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.

Download full-text

Full-text

Available from: Keiko Muguruma
  • Source
    • "Since Bedzhov and Zernicka-Goetz (2014) recently showed that murine ESC can form cysts with dominant lumens by 36–48 hr in a 3D culture system, we tested whether H9 hESC (NIH code, WA09) can also undergo lumenogenesis. H9 cells were grown in standard medium containing Y-27632 (ROCK inhibitor) to inhibit apoptosis (Ohgushi et al., 2010). Three days after plating dispersed H9 hESC in Geltrex, the vast majority of cells had formed multicell cysts, 86.7% ± 1.8% of which had a single dominant lumen (Figure 1A). "
    [Show abstract] [Hide abstract]
    ABSTRACT: We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCζ, is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly, actin polymerization, regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA), promotes lumen formation, whereas actin contraction, mediated by MYOSIN-II, inhibits this process. Finally, we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development, this system can provide a powerful model for investigation of this process in a controlled environment. Overall, our data establish that lumenogenesis is a fundamental cell biological property of human PSCs.
    Full-text · Article · Nov 2015 · Stem Cell Reports
  • Source
    • "allowed cells to spontaneously slip into a telencephalic progenitor fate (Watanabe et al., 2005). Another major innovation was the discovery of an efficient method to culture human ESCs (Watanabe et al., 2007; Ohgushi et al., 2010). Until the mid-2000s, advances using human ESCs had been hampered by the fact that, unlike mouse ESCs, human ESCs are vulnerable to dissociation, and thus are lost through passaging. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Stefano Piccolo looks back at the life and research of his friend and colleague Yoshiki Sasai.
    Preview · Article · Aug 2014 · Development
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Aug 2014 · Stem Cell Reports
Show more