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

Cell Stem Cell

Article Molecular Pathway and Cell State Responsible for Dissociation-Induced Apoptosis in Human Pluripotent Stem Cells Masatoshi Ohgushi,1,2 Michiru Matsumura,1,2 Mototsugu Eiraku,1 Kazuhiro Murakami,3 Toshihiro Aramaki,1 Ayaka Nishiyama,1 Keiko Muguruma,1 Tokushige Nakano,1 Hidetaka Suga,1 Morio Ueno,1 Toshimasa Ishizaki,4 Hirofumi Suemori,5 Shuh Narumiya,4 Hitoshi Niwa,3 and Yoshiki Sasai1,2,* 1Organogenesis

and Neurogenesis Group of Human Stem Cell Technology 3Laboratory for Pluripotent Cell Studies RIKEN Center for Developmental Biology, Kobe 650-0047, Japan 4Department of Pharmacology, Graduate School of Medicine 5Institute for Frontier Medical Sciences Kyoto University, Kyoto 606-8315, Japan *Correspondence: [email protected] DOI 10.1016/j.stem.2010.06.018 2Division

SUMMARY

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.

INTRODUCTION Although hESCs are pluripotent cells derivatized from the blastocyst embryo like mESCs, there are in fact several substantial differences between them (Thomson et al., 1998; Sato et al., 2003). Among the interspecies differences, a particularly intriguing one is the requirement of hESCs to be cultured as cell clumps because they undergo apoptosis when dissociated (Watanabe et al., 2007 and references therein). Their apoptotic response is remarkably extensive and their fragility upon dissociation has

been a large obstacle to the development of techniques for manipulating hESCs. We recently reported that the application of Y-27632, a specific inhibitor for Rho-dependent protein kinase (ROCK), permit the survival of hESCs in clonal culture by efficiently blocking the dissociation-induced cell death (Watanabe et al., 2007; also see an example of greatly improved plating efficiency in Figure S1A available online). The addition of the ROCK inhibitor to dissociated hESCs has already greatly improved a number of practical procedures. However, several fundamental questions about the ROCKdependent hESC apoptosis have remained unsolved to date. For instance, it is not known how Y-27632 protects dissociated hESCs from massive cell death, what the upstream signals are that trigger the hESC apoptosis after dissociation, why only hESCs, but not mESCs, are vulnerable to dissociation-induced cell death, or what the biological relevance of the dissociationinduced hESC death is. ROCK, the target of Y-27632, is one of the major downstream mediators of Rho (Riento and Ridley, 2003; Harb et al., 2008; Krawetz et al., 2009). The GTP-bound form of Rho interacts with ROCK, inducing a conformational change in ROCK that elevates its kinase activity. Rho signaling plays crucial regulatory roles in cellular proliferation, differentiation, cytokinesis, motility, adhesion, and cytoskeletal arrangement (Jaffe and Hall, 2005). At the molecular level, Rho subfamily members, such as Rho and Rac (Burridge and Wennerberg, 2004), function as molecular switches that cycle between GDP-bound inactive and GTPbound active forms. This transition is strictly controlled by the cooperation of positive and negative regulators. The positive regulator molecules, termed GEFs (guanine nucleotide exchange factors), can activate specific Rho subfamily molecules. The negative regulators, called GAPs (GTPase activating proteins), can reverse this reaction by facilitating the hydrolysis of the bound GTP. Rho/ROCK activation induces various outcomes depending on the cellular context (Riento and Ridley, 2003). Although a number of substrates for ROCK have been already identified as potential downstream effectors (Riento and Ridley, 2003; Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 225

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Jaffe and Hall, 2005), the responsible effecter for a variety of cases is still uncertain. In particular, the downstream effectors in the survival-or-death regulation of hESCs have been unknown. In this study, we first demonstrate that the ROCK-dependent hyperactivation of myosin is the direct cause of dissociationinduced apoptosis in hESCs. Disruption of E-cadherin-mediated cell-cell adhesion is sufficient to trigger an immediate activation of the Rho/ROCK/MLC2 signaling cascade. This dissociationinduced myosin hyperactivation is specific to the epiblast-equivalent cell state rather than to their species of origin. We also show the involvement of a unique Rho-GEF family factor, Abr, as an essential role in this regulation and the biased activity of Rho versus Rac as a critical factor for this phenomenon. RESULTS Dissociation of hESCs Causes a ROCK-Dependent Early Apoptotic Response via the Mitochondrial Pathway We first sought to clarify how early after dissociation the apoptotic reaction begins and which apoptotic pathway is involved there. FACS analysis showed an obvious increase in the Annexin V+ population of hESCs (cells undergoing apoptosis or dead ones) even a few hours after dissociation (Figure 1A; control, black; also see Figures S1B and S1C). At the 6 hr time point, the majority (about three quarters) of the dissociated hESC population was already positive for this early apoptosis marker (Figure 1B, lane 1). Such an apoptotic response was not observed in dissociated mESCs (lane 2). The rapid induction of the apoptosis marker in hESCs was significantly inhibited by the addition of the ROCK inhibitor Y-27632 or the pan-Caspase inhibitor zVAD (Figure 1A; red and blue). Strong suppression of apoptosis was also seen when both ROCK-I and -II were knocked down by RNAi (lane 4 in Figure S1Dc; Figures S1Da and S1Db for RNAi controls). The overexpression of dominant-negative FADD (dnFADD), which efficiently blocks the FAS-FADD pathway, a typical nonmitochondrial cascade, did not have a substantial effect on the apoptosis marker (Figure 1C, lane 2; see Figure S1E for controls).

In contrast, the overexpression of Bcl-XL, which antagonizes the upstream trigger of the mitochondrial pathway (Youle and Strasser, 2008), markedly decreased the Annexin V+ population (lane 3). Furthermore, the mitochondrial potential (indicated by the TMRM dye) decreased after cell dissociation (Figure 1D, red) and this reduction was inhibited by Y-27632 (Figure 1D, blue). In addition, the cytoplasmic release of cytochrome C, a major messenger of the mitochondrial pathway (Youle and Strasser, 2008), was observed with a similar time course as the decrease in TMRM+ staining (Figure 1E). These findings showed that the mitochondrial pathway plays a major role in the early apoptotic response downstream of ROCK activity. The ROCK-Dependent Apoptosis of Dissociated hESCs Is Associated with an Atypical, Extensive Early-Onset Blebbing We next performed live-cell imaging during the early phase of dissociation culture (Figures 1F–1L). Unlike dissociated mESCs, which spread normally on the plate bottom and were not particularly mobile (Figure 1F and Movie S1, part A), the dissociated hESC exhibited a high motility and formed a number of blebs on their cell surface (Figures 1G and 1H; also see Figure S1G). The blebbing began immediately upon the start of the dissociation culture and continued until the cells burst and formed apoptotic bodies (Movie S1, part B). This blebbing in the dissociated hESCs was strongly suppressed by Y-27632 (Figures 1I and 1J and Movie S1, part C). The suppression of blebbing appeared to require the continuous (or concurrent) presence of Y-27632 at least during the first 6–12 hr. The cells that had once calmly spread on the plate bottom in the presence of Y-27632 started blebbing when the Y-27632 was removed afterwards (Figure S1H). Conversely, cells that had started blebbing (but were not yet dead) in the absence of Y-27632 stopped blebbing upon the later addition of Y-27632 (Figure S1I). Similar earlyonset blebbing was also observed in dissociated human iPS cells (see Experimental Procedures). Blebbing is an indication of unregulated hyperactivation of the actomyosin system (Charras and Paluch, 2008), which leads to

Figure 1. Unusual Early-Onset Blebbing during ROCK-Dependent Apoptosis of Dissociated hESCs (A) Time course FACS analysis of apoptosis in dissociated hESCs (black line, no inhibitor; red line, 10 mM Y-27632; blue line, 20 mM zVAD). (B) Apoptosis induction of hESCs and mESCs 6 hr after dissociation. (C) hESCs were transfected with expression plasmids for an inhibitor with H2B-Venus expression plasmid. Dunnett’s test (n = 3) versus lane 1. n.s., not significant; **p < 0.01. (D) FACS measurement of mitochondrial potentials by the uptake of mitochondrial dye TMRM. (E) Cytosolic release of cytochrome c from mitochondria in dissociated hESC. Bottom, loading control. (F and G) Live imaging of dissociated mESCs and hESCs on Matrigel (F, mESCs; G, hESCs). (H) Percentages of blebbing cells in dissociated hESCs and mESCs 15–30 min after dissociation. (I) Live imaging of dissociated hESCs in the presence of 10 mM Y-27632. (J) Effects of inhibitors on blebbing occurrence. (K) A Snapshot of confocal live imaging for dynamics of blebbing movement in dissociated hESCs. Plasma membrane (red, Lyn-mCherry) and nuclei (green, H2Bvenus). (L) Snapshots of live imaging of dissociated hESC in the presence of 20 mM zVAD. Images were obtained every 5 min for 12 hr after cell seeding. Scale bars represent 20 mm in (F), (G), (I), and (L). (M) Snapshots of dissociated hESCs expressing the FRET probe SCAT3. Upper panels show bright field; the bottom panel show Venus/CFP ratio image. Pseudocolors are used to represent the Venus/CFP ratio with blue and red indicated high and low activities, respectively. (N) Time course for Caspase-3 activation. (O and P) Analysis of dissociated hESCs expressing the SCAT3 probe together with Bcl-XL. Images were obtained every 2 min for 10 hr after cell seeding. Time course of the mean Venus/CFP ratios over the whole cell was shown. Scale bars represent 10 mm in (M) and (O). (G and L–O) White arrowheads indicate blebbing cells; the red arrow indicates membrane rupture. The bars in the graphs represent standard deviations. See also Figures S1 and S2 and Movie S1.

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the excessive contraction of the cortical actin network and dramatically increases the intracellular pressure. This repeatedly causes multiple local detachments of the plasma membrane (evagination) from the cytoskeleton (Charras et al., 2005; Charras et al., 2006). Some transient blebbing is generally observed in dying cells, but occurs only for a short period during the terminal phase of apoptosis when cells are close to cell bursting (Coleman et al., 2001; Sebbagh et al., 2001). In contrast, the blebbing of dissociated hESCs started soon after dissociation, long before the cell burst, and continued for a substantial length of time until the cells burst from a few hours to a day (Figure 1G). By careful observation with multicolor fluorescence live imaging, the plasma membrane movement of the dissociated hESC (shown by Lyn-mCherry) per se was indistinguishable from that of conventional blebbing: a rapid outward protrusion of saclike blebs (within a few seconds; Figure 1K) and their subsequent slow retraction (a few minute later) (Figures S2A–S2C and Movie S1, parts D–F). The conventional blebbing at the terminal phase of apoptosis is typically caused by the Caspase-3-induced cleavage of ROCK-I, which then becomes constitutively active (Coleman et al., 2001; Sebbagh et al., 2001). In dissociated hESCs, however, the caspase inhibitor zVAD did not substantially inhibit the blebbing (Figures 1J and 1L), although it suppressed the appearance of the apoptosis marker Annexin V (Figure 1A). In addition, we did not detect the cleaved form of ROCK-I in a western blot (data not shown). These findings indicated that the blebbing of hESCs is also atypical in its molecular regulation and does not occur downstream of caspases. We next carried out a time-course study, in which the Caspase activity was measured in real-time FRET assay (Figures 1M–1P and Figures S2D–S2G). The level of Caspase-3 activity remained low until 10 min before cell rupture, when an abrupt all-ornone-type activation was observed (Figures 1M and 1N; Movie S2, part A; the SCAT3 FRET probe gives a high Venus/CFP fluorescence ratio when the Caspase-3 activity is low; Takemoto et al., 2003). A similar observation was made in the temporal profile of Caspase-9 activation (the SCAT9 probe; Takemoto et al., 2003), which occurs upstream of Caspase-3 in the mitochondrial pathway, except that the Caspase-9 activation started earlier (30 min before cell rupture) and became elevated more gradually than the Caspase-3 activation (Figures S2D and S2E). Thus, the robust terminal activation of both Caspases occurred much later than the onset of blebbing.

Both the blebbing and the Caspase-3 activation were inhibited by Y-27632 treatment (not shown) and by ROCK-I/II-knockdown (Figures S2F and S2G; Movie S2, part B). Consistent with the zVAD data, the blebbing was not substantially inhibited when the Caspase-3 activation was suppressed by overexpressing Bcl-XL, which prevents cells from undergoing apoptosis (Figures 1O and 1P; Movie S2, part C). These observations at the single-cell level demonstrated that the blebbing seen in dissociated hESCs is not the consequence of a strong precocious activation of Caspases but instead is directly associated with ROCK activity. Rho/ROCK-Mediated Hyperactivation of Myosin Is a Primary Cause of Rapid Apoptosis of Dissociated hESCs We next analyzed how the ROCK activity was regulated after dissociation. In pull-down assays, an elevated level of active Rho (GTP-bound) was observed upon dissociation (Figure 2A). Similarly, substantial augmentation of ROCK activity was observed upon dissociation in the in vitro kinase assay with ROCK proteins immunoprecipitated from hESC lysates (Figure S3A; MYPT1 was used as a substrate). Western blot analysis showed that the phosphorylation level of the nonmuscle myosin light chain 2 (MLC2), a known ROCK substrate (Riento and Ridley, 2003), was significantly and continuously elevated after dissociation (Figure 2B). This elevation of phosphorylated MLC2 (pMLC2) in dissociated hESCs was inhibited by both Y-27632 and the Rho inhibitor C3 (Figure 2C, lanes 4 and 6), consistent with previous reports on the Rho-ROCK-myosin axis functioning in a variety of cells including pluripotent cells (Harb et al., 2008). In addition, three other ROCK inhibitors (HA1077, H-1152P, and GSK269962A) attenuated MLC2 phosphorylation at the concentrations effective for apoptosis inhibition (Figure S3B). Consistent with these Western blot results, a strong immunostaining signal for phosphorylated MLC2 was observed in dissociated hESCs and was diminished by Y-27632 (Figure 2D, top) and by RNAi knockdown of ROCK-I/II (Figure 2E). These observations indicate that the dissociation of hESC induces a quick and substantial increase in pMLC2 in a Rho/ROCKdependent manner. The phosphorylation of MLC2 is known to activate myosin and to create intracellular contractive forces via the actomyosin network (Charras and Paluch, 2008). Treatment with the myosin inhibitor Blebbistatin rescued dissociated hESCs from not only

Figure 2. ROCK-Dependent Actomyosin Hyperactivation Is a Primary Cause for Dissociation-Induced Apoptosis in hESCs (A) Detection of active Rho in pull-down assay from the lysates of dissociated hESCs. (B and C) MLC2 phosphorylation in dissociated hESCs. Time course analysis (B) and effects of inhibitors (C, 10 mM Y-27632 and 2 mg/ml C3). (D) Immunohistochemistry for p-MLC (green) in dissociated hESCs without (left) or with (right) 10 mM Y-27632. Cells were counter-stained with F-actin (red) and DAPI (blue). (E) Immunohistochemistry for p-MLC (red) in ROCK-depleted hESCs (left panels, control shRNA; right panels, shRNAs for ROCK-I/II). Cells were counter-stained with F-actin (blue). Arrowheads indicate shRNA-expressing cells (positive for the tracer GFP, green). (F) Live imaging of dissociated hESCs on Matrigel without (upper) or with (bottom) 10 mM Blebbistatin. Images were obtained every 5 min for 12 hr after cell seeding. The scale bar represents 20 mm in (D)–(F). (G and H) Effects of Blebbistatin on apoptosis in dissociated hESCs. Apoptosis assay (G) and cytosolic cytochrome c release (H) were shown. Dunnett’s test (n = 3) versus lane 1. **p < 0.01. (I and J) Effects of Blebbistatin on colony formation of dissociated hESCs. Formed hESC colonies were visualized by AP staining (I, scale bar represents 500 mm) and counted (J). Student’s t test (n = 3). ***p < 0.001. The bars in the graphs represent standard deviations. See also Figure S3.

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Figure 3. Actomyosin Hyperactivation, Rather than Blebbing Movement per se, Primarily Causes Apoptosis of Dissociated hESCs

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stream of ROCK plays an essential role in the dissociation-induced cell death of hESCs. Consistent with this idea, similar sup00h:00m:00s 02:30:00 01:00:00 02:18:00 02:16:00 pression of both blebbing and apoptosis low was observed in dissociated hESCs in which the myosin function was inhibited by overexpressing a dominant-negative MLC2 (MLC2-AA: nonphosphorylatable 02:30:00 high 01:00:00 02:18:00 02:16:00 00:00:00 form; Figures S3G–S3I) or by shRNAs for nonmuscle myosin heavy chain IIA/ D E 100 0hr n.s. IIC (or myh9/14; Figures S3J–S3L). In 6hr rupture 7 80 contrast to Blebbistatin treatment, the 6 MLCK inhibitor ML-7 did not show sub60 5 stantial suppressing effects on dissocia4 tion-induced apoptosis even at high con40 ** 3 centrations (e.g., 20 mM; Figure S3M), 20 2 suggesting that MLCK is not an essential 1 downstream mediator of ROCK in this 0 0 particular context (multiple ROCK targets 60 120 180 240 300 0 are discussed in Riento and Ridley, 2003). min after dissociation To examine the role of blebbing in apoptosis, we next overexpressed a constitutively active Ezrin (EzrinT567D), 1 2 3 which physically strengthens the link between the plasma membrane and the actomyosin cortex and thereby specifithe blebbing (Figure 2F) but also the apoptosis (Figure 2G), and cally reduces blebbing (Charras et al., 2006). The overexpression decreased the dissociation-induced release of cytochrome C of EzrinT567D did not affect the high level of pMLC2 accumula(Figure 2H, lanes 2 and 6). As a result, the Blebbistatin treatment tion in the dissociated hESC (Figure 3A; the overexpressing cells significantly increased the colony formation efficiency in the are indicated by coexpressing GFP). However, EzrinT567D hESC dissociation culture (Figures 2I and 2J). In addition, treat- strongly inhibited the blebbing, which then occurred only during ment with cytochalasin D (an inhibitor of actin polymerization) the terminal stage of apoptosis when Caspase-3 was strongly also attenuated both blebbing and apoptosis in dissociated activated (Figures 3B–3D, Movie S2, part D). In contrast to the hESCs (Figures S3C–S3F; MLC2 phosphorylation was unaf- efficient suppression of blebbing, EzrinT567D did not substanfected, Figure S3F), suggesting that the actin-myosin interaction tially inhibit the dissociation-induced apoptosis (Figure 3E, is indispensable for the early-onset cell death. These inhibitor lane 3; lane 2 positive control with Bcl-XL). These findings of studies suggested that the actomyosin hyperactivation down- uncoupling between apoptosis and blebbing indicated that the 230 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.

Cell Stem Cell Mechanism of Dissociated-Induced Apoptosis in hESCs

early-onset blebbing is not a direct cause of cell death but rather a parallel phenomenon induced by myosin hyperactivation. ROCK/Myosin Hyperactivation as well as Rho Activation Is Induced by the Loss of Ca2+-Dependent Intercellular Adhesion between hESCs Unlike ICM-like mESCs, hESCs exhibit an epithelial character with a clear apico-basal polarity (Krtolica et al., 2007; e.g., apical junctions and a basement membrane) as does the epiblast. A typical form of dissociation-induced apoptosis of epithelial cells is anoikis, which is caused specifically by the loss of cellular adhesion (anchorage) to a substrate or the basement membrane (Frisch and Screaton, 2001). We and others previously guessed that the dissociation-induced hESC apoptosis was probably also anoikis (Watanabe et al., 2007; Krawetz et al., 2009). However, we noticed in our live-imaging analysis that, although the dissociated hESCs successfully attached to the substrate matrix, the cells still underwent massive blebbing and apoptosis, arguing the hypothesis that this cell death is anoikis. The nonanoikis nature was further supported by our immunostaining data of dissociated hESC that clearly showed the formation of paxillin+ focal adhesions onto the culture substrate, demonstrating the presence of the cell-substrate anchorage (Figure S4A; also see the accumulation of phospho-tyronsine and phospho-FAK, indicative of local integrin-related signaling). Therefore, we next examined the role of cell-cell adhesion in the control of the hESC apoptosis. In this case, we first cultured the hESC in colonies so that the cells formed tight intercellular adhesion via the cadherin/catenin system involving E-cadherin (Figures 4A–4C). We then added the Ca2+ chelator EGTA, which disrupts the cadherin-mediated cell attachment, and caused hESC to detach from one another but not from the plate bottom (Figures 4D–4F). Importantly, this dissociation without substrate detachment was sufficient to increase the level of pMLC2 (Figure 4G, lane 2) in a ROCK-dependent manner (lane 3). Live imaging showed that the EGTA-induced mild dissociation was sufficient to induce blebbing in a major population of hESCs, particularly at the periphery of the colonies, where the cellular dissociation by EGTA was most evident (Figure 4H and Movie S3, part A; under this mild dissociation condition, the blebbing typically started 20–40 min after dissociation). Y-27632 treatment reduced both the blebbing (Figure 4I and Movie S3, part B) and also the apoptosis induced by EGTA (Figure 4J, lane 2). The Ca2+ depletion-induced blebbing was suppressed when cell adhesion was restored by adding Ca2+ back to the medium (Figure 4K). In contrast, cells pretreated with the E-cadherinblocking antibody remained separated even after Ca2+ was added back. These cells continued to show blebbing even in the presence of Ca2+ (Figure 4L) and exhibited a higher rate of apoptosis (Figure S4B, lane 3). These findings indicated that the continuous loss of E-Cadherin-dependent intercellular adhesion (not of Ca2+) is responsible for the blebbing and the cell death. Consistent with this idea, RNAi knockdown of E-cadherin sufficiently induced blebbing and apoptosis even in hESCs present in colonies (Figures S4C–S4F and Movie S3, part C). Taken together, these findings show that, although some minor contribution of anoikis is not totally excluded, the loss of E-cadherin-dependent intercellular adhesion plays a major decisive role in the apoptosis in dissociated hESCs.

We next performed a FRET analysis (using Rho-Raichu; Yoshizaki et al., 2003) to analyze the early response of Rho to the cellular dissociation by EGTA treatment. Consistent with the western blot study of the dissociation culture (Figure 2A, lane 1), the level of Rho activation was low before EGTA treatment (Figure 4M; blue) but substantially increased soon after the addition of the chelator to the medium (red in the cell periphery; this FRET probe has a membrane-anchor motif). This activation clearly preceded the onset of blebbing (Movie S4), indicating that Rho activation was not induced by blebbing. These observations showed that Rho activation is an early event occurring prior to the myosin hyperactivation in hESC. The ROCK/Myosin Hyperactivation Occurs in an Epiblast State-Specific Manner One fundamental question about the dissociation-induced hESC apoptosis is why it occurs only in hESCs and not in mESCs. Recently, it was reported that mouse epiblast-derived pluripotent stem cells (mEpiSCs) behave more like hESCs than ICMderived mESCs do (Brons et al., 2007; Tesar et al., 2007). We therefore examined the effect of the EGTA treatment on mEpiSC. Interestingly, in contrast to mESCs (Figure 1F), mEpiSCs started blebbing soon after dissociation (Figure 4N, Figure S5A and Movie S5, part A), and the blebbing was suppressed by Y-27632 (Figure S5B and Movie S5, part B). In addition, the dissociation of mEpiSC causes a higher rate of Y-27632-sensitive apoptosis than did that of mESCs (Figure S5C, lane 3). These observations suggested that the difference in the vulnerability of pluripotent cells is more dependent on the cell state (epiblast-like versus ICM-like), rather than on the species of origin (human versus mouse). We next compared the activation state of Rho in different pluripotent cells. The dissociation of mEpiSCs, but not of mESCs, increased the level of active Rho (Figure 4O, lane 4). In addition, FRET analysis showed a rapid activation of Rho in mEpiSCs after their cellular disaggregation by EGTA (data not shown). Thus, the mEpiSCs also resembled hESCs in the control of the Rho/ROCK pathway after dissociation, suggesting that the epiblast-like nature of hESCs is relevant to the dissociationinduced Rho/ROCK activation. In accordance with this idea, the dissociation culture of mouse epiblast cells (primary culture from E6.25 embryos) showed extensive blebbing that was sensitive to Y-27632 (Figures 4P and 4Q; Figure S5D and Movie S5, part C), indicating that a strong tendency for ROCK-myosin hyperactivation is common to epiblast-related cells, both primary culture cells and stem cell lines. Similar dissociation-induced blebbing is also seen in the epiblastlike cells (E-cadherin+, Fgf5+, Crypto+, and Klf4 ) generated from mESCs cultured for 2 days under differentiating conditions of SFEBq culture (Eiraku et al., 2008) (unpublished data). Interestingly, the similar Y-27632-sensitive blebbing was seen in dissociated ectodermal cells of Xenopus gastrulae (Movie S6), suggesting that the dissociation-induced ROCK/Myosin hypreactivation is a common phenomenon in vertebrate early ectodermal cells. Dissociation-Induced ROCK/Myosin Hyperactivation in hESCs Is Dependent on the Rho-GEF Family Factor Abr Previous studies have shown that distinct Rho-GEF family proteins (which have several dozen members) function as the Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 231

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Mechanism of Dissociated-Induced Apoptosis in hESCs

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upstream activator of Rho in various cellular events (Bos et al., 2007; Rossman et al., 2005). RT-PCR analysis revealed that at least 26 Rho-GEF family genes (with a DH domain encoding, Rossman et al., 2005) were expressed in hESC (data not shown). We performed an shRNA-mediated knockdown screen focusing on genes with nonredundant structures and found that the knockdown of one Rho-GEF member (Figures 5A and 5B, efficient knockdown was also confirmed by quantitative PCR, data not shown) strongly rescued hESCs from the dissociation-induced apoptosis (Figure 5C, lanes 3–5; confirmed with three independent shRNAs) as efficiently as the knockdown of ROCK-I/II (positive control; lane 2), and promoted colony formation from dissociated hESCs (Figures 5D and 5E). This Rho-GEF family molecule was Abr, which is structurally related to Bcr. In contrast to the well-known Bcr, whose fusion to Abl causes chronic myelogenous leukemia, relatively little is known about the role and function of Abr. Importantly, in addition to suppressing apoptosis, the knockdown of Abr markedly inhibited both the blebbing (Figure 5F) and the pMLC accumulation (Figure 5G; Abr-shRNA-expressing cells were marked by GFP) after dissociation. These effects of Abr-shRNA were reversed by the coexpression of an shRNA-resistant codonswapped Abr (Figure 5H, lanes 2 and 4; lane 3 is a negative control using shRNA-sensitive wild-type Abr, which did not generate the gene product), supporting the specificity of the knockdown phenotype. These finding demonstrated that Abrdependent ROCK/myosin activation plays a key role in triggering the downstream myosin hyperactivation and apoptosis of hESCs. Combinatory Rac Inhibition with Rho/ROCK Activation Plays a Crucial Role in Myosin Hyperactivation upon Dissociation of hESCs Given that two small G proteins, Rho and Rac, interact in the regulation of various cellular events (positively and negatively; Jaffe and Hall, 2005), we next examined the control of Rac in hESCs. In a pull-down assay, the cell dissociation decreased the level of active Rac (Figure 6A), in contrast to increased Rho activity (Figure 2A). Consistent with this finding, in live FRET analysis (Rac-Raichu; Itoh et al., 2002), whereas a substantial Rac

activity was observed in hESCs before and immediately after the EGTA treatment, the Rac activity subsequently decreased down to a basal level (Figure 6B). Similar Rac suppression upon dissociation was also observed in mEpiSCs (data not shown). Thus, the regulation of Rac activity makes a clear difference from that of Rho activity, which is low before dissociation and increases upon dissociation (Figures 2A and 4M). These findings raised the possibility that Rho and Rac act in opposite directions in the dissociation-induced ROCK/myosin hyperactivation in hESCs. To test this idea, we introduced a constitutively active Rac (caRac, Rac1V12), which makes Rac activity persistently high, into hESC (Figures 6C–6F). It suppressed the blebbing movement (Figure 6C, top) and inhibited the activation of Caspase-3 and the apoptosis (Figures 6C–6E; Movie S7). It also lowered the accumulation of pMLC in dissociated hESCs (Figure 6F). Thus, the persistent activation of Rac signaling has a clear inhibitory effect on the dissociation-induced ROCK-myosin hyperactivation. Interestingly, we found that this dissociation-induced Rhohigh/Rac-low state was greatly altered in cells of a dissociation-resistant hESC subclone reported previously (subline 1; Hasegawa et al., 2006). Although these cells expressed hESCspecific markers and formed teratoma (Figure S6A), they did not undergo blebbing or apoptosis in dissociation culture (Figures S6B–S6D). Consistent with these findings, no substantial elevation of pMLC2 upon dissociation was observed (Figure S6E, lanes 5 and 6). In pull-down assays, no substantial elevation of Rho activity was observed upon dissociation, whereas Rac activity was increased (Figure S6F, lane 4). These findings provide additional circumstantial evidence for the strong correlation between the Rho/Rac control and the dissociationinduced myosin hyperactivation. The involvement of the reciprocal Rho/Rac control was particularly intriguing because one special structural feature of Abr is to contain a GAP (inhibitor) domain for Rho-class GTPases in addition to the typical GEF (activator; DH) domain (Figure 5A, top; Heisterkamp et al., 1993; Chuang et al., 1995). Previous studies have shown that the GAP domain of Abr preferentially binds to Rac (and Cdc42), but not to Rho, and has an inhibitory GAP activity for Rac, whereas the amino-terminal portion

Figure 4. Disrupted E-Cadherin-Mediated Intercellular Contact Plays a Causal Role in the Dissociation-Induced Apoptosis of hESCs (A–F) Immunostaining of adherens junction proteins in an intact hESC colony (A–C) and their collapse in hESC dissociated by EGTA (3 mM) treatment. Adherens junction proteins were stained with a-Catenin (A and D, green) and E-cadherin (B and E, red) antibodies. The scale bar represents 50 mm. (G) MLC2 phosphorylation in EGTA-treated hESC clumps in the absence or presence of 10 mM Y-27632. (H and I) Snapshots of live imaging of EGTA-dissociated hESCs on Matrigel in the absence (H) or presence (I) of 10 mM Y-27632. Images were obtained every 1 min for 2 hr after EGTA addition. (J) Apoptosis assay before or 8 hr after EGTA addition. ***p < 0.001 in t test (n = 3). (K–L) Ca2+ switching experiments. Ca2+ was added back 1.5 hr after the initial EGTA treatment. Snapshots of live imaging of EGTA-dissociated hESCs on the Matrigel substrate in the absence (K) or presence (L) of E-cadherin neutralizing antibody are shown. Images were obtained every 2 min for 5 hr after EGTA addition. Scale bars represent 20 mm. (M) Snapshots of EGTA-treated hESC expressing the Rho-Raichu FRET probes (identified as YFP-positive cells). EGTA was added at 10 min after the starting point of recording. Images were obtained every 30 s for 1 hr. In this case, red and blue indicated high and low Rho activities, respectively. The scale bar represents 20 mm. (N) Live imaging of EGTA-treated mEpiSC colonies on MEF feeder cells. (O) Measurement of Rho activation by a pull-down assay in the lysates of dissociated mESC (lanes 1 and 2) and mEpiSC (lanes 3 and 4). (H, K, L, N, and O) White arrowheads indicate blebbing cells. (P and Q) Snapshots of dissociated epiblast cells in the absence (left) or presence (right) of 10 mM Y-27632 (P). The scale bar represents 10 mm. Percentages of blebbing cells in dissociated epiblast cells upon dissociation (Q). For each condition, four epiblasts were subjected to dissociation and blebbing cells were counted. The contingency table analysis (Fisher’s exact test) showed a high statistical significance (p < 0.001, two-sided). Arrowheads indicate blebbing cells. The bars in the graphs represent standard deviations. See also Figures S4 and S5 and Movies S3–S6.

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Figure 5. Rho-GEF Abr Mediates Rho Activation Induced by Loss of Intercellular Adhesion (A) Abr domain structure and Abr mutants. (B) Efficiency of three independent shRNAs-mediated Abr-knockdown was measured by western blotting. (C–F) Effects of Abr-knockdown on hESC apoptosis and blebbing. Apoptosis assay (C), colony formation (D and E) and blebbing occurrence (F) were assayed in Abr-depleted hESC. (C) and (F) show a Dunnett’s test (n = 3) versus lane 1; **p < 0.01. (E) shows a Student’s t test (n = 3); ***p < 0.001. (G) Attenuated MLC2 phosphorylation in Abr-shRNA-expressing hESCs. Arrowheads mark GFP-positive shRNAs-expressing cells. The scale bar represents 10 mm. (H) Dissociation-induced apoptosis was restored in Abr-knockdown hESC by transfection with RNAi-resistant Abr mutants (Abr*). Expression was confirmed by western blotting against the animo-terminal Flag tag (bottom). Dunnett’s test (n = 3) versus lane 2 (among Abr-depleted cells) is shown. n.s., not significant; **p < 0.01. The bars in the graphs represent standard deviations.

possesses a Rho-GEF activity (Chuang et al., 1995). A mutant Abr in which two essential residues in the GAP domain were mutated (Cho et al., 2007; Figure 5A, bottom) failed to rescue the dissociation-induced phenotypes of the Abr-shRNA hESC (Figure 6H, lane 5, bottom panel for control). Likewise, an Abr mutant lacking a Rho-GEF domain (DH; Figure 5A, middle row) was unable to replace the wild-type Abr (Figure 6H, lane 4). These findings indicated that both the Rho-GEF and Rac-GAP domains are essential for Abr to induce the ROCK-dependent downstream events in dissociated hESCs. Consistent with this idea, neither Rho activation nor Rac inhibition was observed in Abr-depleted hESCs upon dissociation (Figure 6I). Furthermore, the vulnerability to dissociation-induced apoptosis was almost fully restored in Abr-depleted hESCs when both caRho and dnRac (or caROCK and dnRac) were introduced, although the overexpression of each of them 234 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.

alone caused a substantial but only partial recovery (Figures 7A–7D). Taken together, these findings indicate that a biased Rho/Rac regulation (high Rho and low Rac activity) plays a crucial role for the induction of the ROCK/myosin hyperactivation in dissociated mammalian pluripotent cells. Figure 7E illustrates the summary of the apoptosis-inducing pathway elucidated in the present study. In nondissociated hESCs, the Rho/ROCK/pMLC system is kept low by an inhibitory mechanism dependent on the E-cadherin-mediated intercellular adhesion. Upon cell dissociation, this system becomes desuppressed (activated) in an Abr-dependent manner. Rac is an antagonistic factor for the dissociation-induced activation of the Rho/ROCK/myosin system. However, in dissociated hESCs, the Rac-GAP function of Abr attenuates the antagonistic Rac function and facilitates a unilateral augmentation of the

Cell Stem Cell Mechanism of Dissociated-Induced Apoptosis in hESCs

‘‘Rho-high/Rac-low’’ state, leading to myosin hyperactivation, which is the main cause of the hESC-specific apoptosis executed via the conventional mitochondrial pathway. Interestingly, we observed in our preliminary study that Rac inhibition (by dnRac) combined with Rho activation (by caRho) induced Y-27632-sensitive blebbing movements even in dissociated mESC (Figures S5E and S5F), suggesting that the reciprocal control of Rho/Rac can induce ROCK-Myosin hyperactivation at least to some extent in non-epiblast-like cells. So far, we do not have experimental evidence showing Abr’s direct interaction with the E-cadherin-catenin complex. Unlike catenins, Abr did not substantially bind E-cadherin (Figure S7A). In addition, simple activation of E-cadherin alone (by culturing hESCs on E-cadherin-protein-coated dishes) appeared to be insufficient to attenuate blebbing or apoptosis (Figures S7B– S7E). These observations imply that Abr is controlled by a third factor that acts downstream of E-cadherin-dependent intercellular adhesion. We deduce that the dissociation-induced apoptosis may be antagonized to some extent by the PI3K-Akt pathway (Downward, 2004), which can be activated by various extrinsic signals including Fgf and extracellular matrix/integrin, both are essential for hESC culture. The inhibitors of the PI3K-Akt pathway (e.g., LY-294002) facilitated the apoptosis in dissociated hESCs (Figure S7F). Conversely, the overexpression of a constitutively active Akt (caAkt) at least partially reduced the dissociationinduced apoptosis without interfering with blebbing (Figure S7G), suggesting that PI3K-Akt signaling can negatively modulate the apoptotic signal downstream of the myosin hyperactivation. Western blot analysis showed that dissociation induces a gradual accumulation of phosphorylated (active) Akt in hESCs (Figure S7H). This accumulation of active Akt may help delay the onset of cell death. The contribution of the active Akt to the strong resistance of mESCs is worth analyzing in-depth, given that both Eras (strongly expressed in mouse pluripotent stem cells, but not in hESCs; Takahashi et al., 2003; Kameda and Thomson, 2005) and LIF can activate the PI3K pathway. DISCUSSION

apoptotic pathways. Two typical apoptosis-inducing factors related to cellular stress responses are MAPKs (e.g., JNK; Chang and Karin, 2001) and p53 (Vousden and Lane, 2007), which are known to act upstream of the Bcl/Bax family. However, we have so far obtained no evidence for the involvement of these two typical apoptosis inducers in the hESC apoptosis (unpublished data). For instance, the dissociation of hESCs did not increase the level of phosphorylated JNK, and the MAPK inhibitors (SP600125 and SB203580) had little effects on the dissociation-induced apoptosis. Similarly, no significant increase in p53 was induced by hESC dissociation, and the overexpression of Mdm2, an inhibitor (ubiquitin ligase) of p53, failed to inhibit the apoptosis. A second possibility is that the apoptosis is caused by the excessive energy consumption by the actomyosin hyperactivation. With this in mind, we measured the ATP content in dissociated hESCs. We found no substantial decrease of the ATP level in the Annexin V cell population even 5 hr after dissociation (although most cells started blebbing soon after dissociation; Figure 1G), arguing against the idea that the apoptosis of dissociated hESCs is due to an excessive consumption of intracellular energy (data not shown). A third interpretation is that the augmented physical tension within the actomyosin cytoskeleton itself triggers the activation of the mitochondrial pathway, for instance, by controlling the subcellular localization of apoptosis-inducing proteins in a tension-dependent fashion. This is certainly an attractive hypothesis to be tested systematically in future investigation. The present study focused on the molecular mechanism of the early culture period (<2 days) of hESC apoptosis. During this time, in addition to Y-27632, the Caspase inhibitor zVAD also effectively suppressed the early-onset apoptotic reaction (Figure 1A). Interestingly, however, although Y-27632 and Blebbistatin (Figure S1A and Figure 2I) fully enable the survival of dissociated hESCs for a long time (>2 days), continuous zVAD treatment only partially supports the long-term survival (Watanabe et al., 2007), suggesting the precence of a minor Caspase-independent pathway that also lies downstream of the ROCK-myosin hyperactivation for efficient long-term survival.

Myosin Hyperactivation Is the Direct Cause of the Early-Onset Apoptosis of hESCs In this study, we first revealed that an unregulated activation of the actin-myosin system is the cause for the cell death of isolated hESCs. The myosin hyperactivation per se, not the blebbing, directly leads to the apoptosis. In other words, although the blebbing is like a ‘‘death dance’’ preceding the suicide of a solitary hESC, it is an epiphenomenon and not the actual trigger for death. An important question to be addressed in future investigations is how the information of myosin hyperactivation is transduced to the mitochondria. The present study also showed that cytochalasin D treatment prevented dissociated hESCs from undergoing early-onset apoptosis without inhibiting the pMLC2 accumulation, indicating that the hyperactivation of the actomyosin system, rather than elevated activity of myosin per se, is essential for the apoptosis induction. One model for the induction mechanism is that the actomyosin hyperactivation nonspecifically augments intracellular stress, which is shown to trigger apoptosis via stress-responsive

Upstream Regulation of the Dissociation-Induced ROCK/Myosin Hyperactivation in hESCs In the context of dissociation-induced ROCK/myosin hyperactivation, the two small GTPases Rho and Rac seem mutually antagonistic in function. For instance, the overexpression of caRac effectively inhibited the blebbing of hESCs. The data in the present study suggest at least two aspects of Rac’s antagonistic functions in dissociated hESCs. First, overexpression of caRac attenuated the elevation of Rho activity in dissociated hESC (Figure S6G, top row), consistent with previous reports on mutual inhibition of Rho and Rac (Burridge and Wennerberg, 2004). Second, Rac may also play an inhibitory role at more downstream levels. When coexpressed with caRho or caROCK, dnRac further increased apoptosis in dissociation culture of Abrdepleted hESCs (Figures 7A–7D). These findings suggest that Rac inhibition plays an effective role even under the condition of forced Rho-ROCK activation. The effect of the reciprocal Rho/Rac activity is in accordance with a dual regulatory role of Abr as a Rho-GEF and Rac-GAP, Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 235

Cell Stem Cell Mechanism of Dissociated-Induced Apoptosis in hESCs

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which potentially contributes to the Rho-high/Rac-low state. Abr is required for both the dissociation-induced myosin hyperactivation and the apoptosis in hESCs. However, given that Abr is expressed both in hESCs and mESCs (data not shown), just the presence or absence of Abr in the cell is unlikely to determine the differential vulnerability of the mammalian ESCs, suggesting that the upstream regulation of Abr’s function, rather than its expression, plays a key role. How the function of Abr is differentially regulated in attached and dissociated cells at the molecular level is an open question for future investigation. Possible Biological Roles of the ROCK/Myosin Hyperactivation and Apoptosis In vivo, the cells in the ICM of the preimplantation embryo are not polarized and form a simple cell mass with no epithelialization (Rossant and Tam, 2009). After implantation, the pluripotent cells derived from the ICM become epithelialized and form an epiblast tissue with a clear apico-basal polarity, including the appearance of a basement membrane and tight junctions (Krtolica et al., 2007). During this process, cells that do not contribute to the epiblast undergo apoptosis, forming a cavity in the center. One possible role of the dissociation-induced apoptosis is the quality control of the epiblast tissue formation in the postimplantation embryo, in other words, elimination of those ICM-derived cells that fail to be incorporated into the epiblast cell sheet. Another intriguing possibility is that the hyperactive state of the ROCK/myosin system in vivo is related more to morphogenesis rather than to cell survival. The process by which the ICM (a simple cell mass) is reshaped into the epiblast (a cell sheet) involves a dramatic 3D rearrangement of cells that requires high cell motility. The hyperactivity of the ROCK/myosin system may enable the epiblast-stage cells to prepare to undergo rapid cell movement. Consistent with this idea, recent studies have identified a novel type of Rho-ROCK-dependent blebbing (or myosin hyperactivation) that is used as a driving tool for directed migration of cells in 3D culture (Sanz-Moreno et al., 2008; Charras and Paluch, 2008). Finally, another stimulating open question regarding the cellstate-specific ROCK/myosin hyperactivation is whether this phenomenon is limited to the early embryonic cells of mammalian species. In the Xenopus embryo, the inner layer cells of the blastula animal cap are equivalent to mouse ICM cells. They are pluripotent and form the animal pole roof lining the blastocoel, but do not have evident apico-basal polarity (e.g., no basement membrane). These cells become epithelialized during early gastrulation upon their fate specification into the ectoderm lineage. Importantly, the dissociated Xenopus gastrulae ectodermal cells also exhibit blebbing (Johnson, 1976) in a ROCKdependent manner (Movie S6), implying the possibility that the ‘‘hyperactivation’’-ready nature of epiblast/early-ectoderm cells has a profound biological role across species in the reproducible formation of the first-born epithelial structure in vertebrate ontogeny.

EXPERIMENTAL PROCEDURES Cell Culture The hESCs (KhES-1, KhES-3) were used in accordance with the hESC guidelines of the Japanese government. Five hiPSCs (gift from Y. Nakamura and S. Yamanaka) were also tested and similar observations were made. Undifferentiated hESCs and its subline 1 were maintained as described previously (Hasegawa et al., 2006: Watanabe et al., 2007). Additional details are in the Supplemental Experimental Procedures. Plasmids and Transfection PCR-amplified cDNAs was sequenced and subcloned into the pCAG-IP or pCAG-IG expression vector. FRET probes for Caspases (SCAT3 and SCAT9) and Rho proteins (Rho-Raichu and Rac-Raichu) were kindly provided by M. Miura (University of Tokyo) and M. Matsuda (Kyoto University), respectively. The pSIREN RNAi system (Clontech) was used for knocking down the expression of specific genes. The transfection of hESC with cDNA- or shRNA-expression plasmids was performed with the FuGENE HD transfection reagent (Roche). Additional information was in Supplemental Experimental Procedures. Biochemical Analyses For the evaluation of apoptosis, cells were stained with fluorescence-conjugated Annexin-V/Propidium Iodide (Biovision) and flow cytometric analysis was performed with FACSAria (BD Biosciences). Immunostaining and colony formation assay was performed as described previously (Watanabe et al., 2007; Eiraku et al., 2008). The Rho/Rac activity was evaluated with a GST pull-down assay with MLB solution, Rhotekin-RBD, and Pak1-RBD (Upstate). For the detection of protein expression, cell lysates were made in RIPA lysis buffer. For dissection of protein-protein interaction, cell lysates were made in NT lysis buffer and immunoprecipitation was performed as described before. The cell lysates and immunoprecipitates were analyzed by SDSPAGE and sequential western blotting. Antibodies used in this study were shown in Supplemental Experimental Procedures. Live Imaging For the live single-cell imaging, dissociated cells were seeded onto a Matrigelcoated 35 mm glass-bottom dish. The recording was started at 15 min after the first contact with the dissociation reagent. For the EGTA experiments, cell clumps maintained on a feeder layer or a Matrigel-coated 35 mm glass-bottom dish for a few days were used for imaging. In this case, the recording was started when EGTA was added to the culture medium. For confocal observation, the images were collected with a CSU-X1 unit (Yokogawa) configured with an IX81-ZDC microscope (Olympus). The ratiometric analyses were performed with MetaMorph 7.5 (Molecular Devices). Statistical Analysis Error bars shown in the figures represent standard deviations and n in the legends is the number of experiments. Statistical significance (two-sided) was tested by a Student’s t test for two-group comparison, and by the oneway ANOVA for multiple-group comparison (for Annexin-V staining analysis, samples at the 6 hr point were analyzed unless otherwise mentioned) with a post-hoc Tukey’s (among all groups) or Dunnett’s test (versus control) with the Prism 4 program (GraphPad).

SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures and seven movies and can be found with this article online at doi:10.1016/j.stem.2010.06.018.

(H) No significant restoration of dissociation-induced apoptosis by expression of RNAi-resistant Abr mutants (Abr*) lacking the GEF domain (DDH) or GAP activity (RA/NA) in Abr-depleted hESC. Abr* was used as a positive control. The bottom panel shows a western blot against the amino-terminal Flag tag. Dunnett’s test (n = 3) versus lane 2 (among Abr-depleted cell groups) is shown. n.s., not significant; **p < 0.01. (I) Pull-down assay for Rho and Rac activity in Abr-depleted hESC before or after dissociation (lanes 3 and 4). The bars in the graphs represent standard deviations. See also Figure S6 and Movie S7.

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Figure 7. Rho-High/Rac-Low State Responsible for the Dissociation-Induced hESC Apoptosis (A–D) Rac inhibition contributes to ROCK-dependent blebbing and apoptosis induction. The sensitivity to dissociation-induced blebbing and apoptosis were fully restored in Abr-depleted hESCs when dominant negative forms of Rac1 (dnRac and Rac1N17) were coexpressed with constitutive active forms of RhoA (caRho, RhoAV12) (A) or caROCK (ROCK1-D3) (C) (compare lanes 5 and 6; Annexin-V staining). Snapshots of dissociation culture of mock- or caROCK/dnRac or caRho/ dnRac-transfected hESCs from which Abr was depleted are shown (C and D). Arrowheads indicate blebbing cells. The scale bar represents 20 mm. The bottom panel shows a western blot with an antibody against the amino-terminal tags. Tukey’s test (n = 3) among all groups is shown. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001. (E) The molecular pathway of dissociation-induced hESC apoptosis contains at least five regulatory steps in the cascade: (1) Desuppression of epiblast statelinked regulator by dissociation; (2) Rho-GEF/Rac-GAP function of Abr; (3) generation of Rho-high/Rac-low state; (4) ROCK-dependent myosin hyperactivation; and (5) actomyosin-dependent apoptosis induction via mitochondria. The bars in the graphs represent standard deviations. See also Figure S7.

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ACKNOWLEDGMENTS We are grateful to Drs. M. Matsuda and E. Kiyokawa for the Rho and Rac FRET probes and stimulating discussion, to Drs. G. Sheng, H. Enomoto, and N. Takata for invaluable comments, to Drs. M. Takeichi, M. Wenxiang, and T. Nishimura for advice on cell-adhesion and Abr analyses, to Dr. P. Tesar for mEpiSCs, to Drs. M. Miura and E. Kuranaga for FRET probes of Capases, to Dr. Y. Gotoh for caAkt, and to members of the Sasai lab for discussion and advice. This work was supported by grants-in-aid from MEXT, the Kobe Cluster Project, and the Leading Project for Realization of Regenerative Medicine (Y.S.).

Heisterkamp, N., Kaartinen, V., van Soest, S., Bokoch, G.M., and Groffen, J. (1993). Human ABR encodes a protein with GAPrac activity and homology to the DBL nucleotide exchange factor domain. J. Biol. Chem. 268, 16903–16906. Itoh, R.E., Kurokawa, K., Ohba, Y., Yoshizaki, H., Mochizuki, N., and Matsuda, M. (2002). Activation of rac and cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells. Mol. Cell. Biol. 22, 6582–6591. Jaffe, A.B., and Hall, A. (2005). Rho GTPases: Biochemistry and biology. Annu. Rev. Cell Dev. Biol. 21, 247–269. Johnson, K.E. (1976). Circus movements and blebbing locomotion in dissociated embryonic cells of an amphibian, Xenopus laevis. J. Cell Sci. 22, 575–583.

Received: December 14, 2009 Revised: April 18, 2010 Accepted: June 4, 2010 Published: August 5, 2010

Kameda, T., and Thomson, J.A. (2005). Human ERas gene has an upstream premature polyadenylation signal that results in a truncated, noncoding transcript. Stem Cells 23, 1535–1540.

REFERENCES

Krtolica, A., Genbacev, O., Escobedo, C., Zdravkovic, T., Nordstrom, A., Vabuena, D., Nath, A., Simon, C., Mostov, K., and Fisher, S.J. (2007). Disruption of apical-basal polarity of human embryonic stem cells enhances hematoendothelial differentiation. Stem Cells 25, 2215–2223.

Bos, J.L., Rehmann, H., and Wittinghofer, A. (2007). GEFs and GAPs: Critical elements in the control of small G proteins. Cell 129, 865–877. Brons, I.G., Smithers, L.E., Trotter, M.W., Rugg-Gunn, P., Sun, B., Chuva de Sousa Lopes, S.M., Howlett, S.K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R.A., and Vallier, L. (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195. Burridge, K., and Wennerberg, K. (2004). Rho and Rac take center stage. Cell 116, 167–179. Chang, L., and Karin, M. (2001). Mammalian MAP kinase signalling cascades. Nature 410, 37–40. Charras, G., and Paluch, E. (2008). Blebs lead the way: How to migrate without lamellipodia. Nat. Rev. Mol. Cell Biol. 9, 730–736. Charras, G.T., Yarrow, J.C., Horton, M.A., Mahadevan, L., and Mitchison, T.J. (2005). Non-equilibration of hydrostatic pressure in blebbing cells. Nature 435, 365–369. Charras, G.T., Hu, C.K., Coughlin, M., and Mitchison, T.J. (2006). Reassembly of contractile actin cortex in cell blebs. J. Cell Biol. 175, 477–490. Cho, Y.J., Cunnick, J.M., Yi, S.J., Kaartinen, V., Groffen, J., and Heisterkamp, N. (2007). Abr and Bcr, two homologous Rac GTPase-activating proteins, control multiple cellular functions of murine macrophages. Mol. Cell. Biol. 27, 899–911.

Krawetz, R.J., Li, X., and Rancourt, D.E. (2009). Human embryonic stem cells: Caught between a ROCK inhibitor and a hard place. Bioessays 31, 336–343.

Riento, K., and Ridley, A.J. (2003). Rocks: Multifunctional kinases in cell behaviour. Nat. Rev. Mol. Cell Biol. 4, 446–456. Rossant, J., and Tam, P.P. (2009). Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse. Development 136, 701–713. Rossman, K.L., Der, C.J., and Sondek, J. (2005). GEF means go: Turning on RHO GTPases with guanine nucleotide-exchange factors. Nat. Rev. Mol. Cell Biol. 6, 167–180. Sanz-Moreno, V., Gadea, G., Ahn, J., Paterson, H., Marra, P., Pinner, S., Sahai, E., and Marshall, C.J. (2008). Rac activation and inactivation control plasticity of tumor cell movement. Cell 135, 510–523. Sato, N., Sanjuan, I.M., Heke, M., Uchida, M., Naef, F., and Brivanlou, A.H. (2003). Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev. Biol. 260, 404–413. Sebbagh, M., Renvoize´, C., Hamelin, J., Riche´, N., Bertoglio, J., and Bre´ard, J. (2001). Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat. Cell Biol. 3, 346–352. Takahashi, K., Mitsui, K., and Yamanaka, S. (2003). Role of ERas in promoting tumour-like properties in mouse embryonic stem cells. Nature 423, 541–545.

Chuang, T.H., Xu, X., Kaartinen, V., Heisterkamp, N., Groffen, J., and Bokoch, G.M. (1995). Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family. Proc. Natl. Acad. Sci. USA 92, 10282–10286.

Takemoto, K., Nagai, T., Miyawaki, A., and Miura, M. (2003). Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects. J. Cell Biol. 160, 235–243.

Coleman, M.L., Sahai, E.A., Yeo, M., Bosch, M., Dewar, A., and Olson, M.F. (2001). Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat. Cell Biol. 3, 339–345.

Tesar, P.J., Chenoweth, J.G., Brook, F.A., Davies, T.J., Evans, E.P., Mack, D.L., Gardner, R.L., and McKay, R.D. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199.

Downward, J. (2004). PI 3-kinase, Akt and cell survival. Semin. Cell Dev. Biol. 15, 177–182. Eiraku, M., Watanabe, K., Matsuo-Takasaki, M., Kawada, M., Yonemura, S., Matsumura, M., Wataya, T., Nishiyama, A., Muguruma, K., and Sasai, Y. (2008). Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3, 519–532. Frisch, S.M., and Screaton, R.A. (2001). Anoikis mechanisms. Curr. Opin. Cell Biol. 13, 555–562. Harb, N., Archer, T.K., and Sato, N. (2008). The Rho-Rock-Myosin signaling axis determines cell-cell integrity of self-renewing pluripotent stem cells. PLoS ONE 3, e3001. Hasegawa, K., Fujioka, T., Nakamura, Y., Nakatsuji, N., and Suemori, H. (2006). A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation. Stem Cells 24, 2649–2660.

Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147. Vousden, K.H., and Lane, D.P. (2007). p53 in health and disease. Nat. Rev. Mol. Cell Biol. 8, 275–283. Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J.B., Nishikawa, S., Nishikawa, S., Muguruma, K., and Sasai, Y. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686. Yoshizaki, H., Ohba, Y., Kurokawa, K., Itoh, R.E., Nakamura, T., Mochizuki, N., Nagashima, K., and Matsuda, M. (2003). Activity of Rho-family GTPases during cell division as visualized with FRET-based probes. J. Cell Biol. 162, 223–232. Youle, R.J., and Strasser, A. (2008). The BCL-2 protein family: Opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 9, 47–59.

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