Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells.

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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Cell Stem Cell
Article
Molecular Pathway and Cell State Responsible
for Dissociation-Induced Apoptosis
in Human Pluripotent Stem Cells
Masatoshi Ohgushi,1,2Michiru Matsumura,1,2Mototsugu Eiraku,1Kazuhiro Murakami,3Toshihiro Aramaki,1
Ayaka Nishiyama,1Keiko Muguruma,1Tokushige Nakano,1Hidetaka Suga,1Morio Ueno,1Toshimasa Ishizaki,4
Hirofumi Suemori,5Shuh Narumiya,4Hitoshi Niwa,3and Yoshiki Sasai1,2,*
1Organogenesis and Neurogenesis Group
2Division 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: yoshikisasai@cdb.riken.jp
DOI 10.1016/j.stem.2010.06.018
SUMMARY
Human embryonic stem cells (hESCs), unlike mouse
ones (mESCs), are vulnerable to apoptosis upon dis-
sociation. Here, we show that the apoptosis, which is
of a nonanoikis type, is caused by ROCK-dependent
hyperactivation of actomyosin and efficiently sup-
pressed by the myosin inhibitor Blebbistatin. The
actomyosin hyperactivation is triggered by the loss
of E-cadherin-dependent intercellular contact and
alsoobservedindissociatedmouseepiblast-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
Racinhibition isinduced inhESCsupondissociation,
but not in Abr-depleted hESCs or mESCs. Further-
more, 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
AlthoughhESCsarepluripotentcellsderivatizedfromtheblasto-
cyst 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 intrigu-
ingone istherequirement ofhESCsto becultured ascellclumps
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
ofY-27632,aspecificinhibitorforRho-dependentproteinkinase
(ROCK), permit the survival of hESCs in clonal culture by effi-
ciently blocking the dissociation-induced cell death (Watanabe
et al., 2007; also see an example of greatly improved plating effi-
ciency 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 ROCK-
dependent 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 dissociation-
induced 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
andRac(BurridgeandWennerberg,2004),functionasmolecular
switches that cycle between GDP-bound inactive and GTP-
bound active forms. This transition is strictly controlled by the
cooperationofpositiveandnegativeregulators.Thepositivereg-
ulator 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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0
20
40
60
80
100
024681012
% Annexin V+ cells
hr after dissociation
control
Y-27632
zVAD
0
80
60
40
20
100
hESC
mESC
0hr
6hr
0
60
80
40
20
mock
dnFADD
2
Bcl-XL
0hr
6hr
0
20
40
60
80
100
02468
control
Y-27632
TMRM+ cells (%)
hr after dissociation
cyto c
Hsc70
0 1 2 4 6
% blebbing
0
40
80
20
60
100
control
Y-27632
2
zVAD
0
40
80
20
60
100
hESC
mESC
0
2
4
1
3
5
060120180240300
venus/CFP ratio
min after dissociation
0120
min after dissociation
600240480
360
0
2
4
1
3
venus/CFP ratio
5
P
04:06:00
04:02:00
03:54:00
03:52:00
04:06:00
04:02:00
03:54:00
03:52:00
03:50:00
10:00:00
05:00:00
00:03:00
01:00:00
00:00:00
10:00:0005:00:0000:03:00
01:00:00
12:00:0006:00:0003:00:0001:00:00
12:00:0006:00:0003:00:0001:00:00
12:00:0006:00:0003:00:0001:00:00
01:00:00
% Annexin V+ cells
1
2
13
hr after dissociation
12
13
% blebbing
% Annexin V+ cells
ABCD
E
F
mESC
hESC
G
I
+Y-27632
+zVAD
L
M
hESC
caspase-3
HJ
K
H2B/Lyn
FRET
O
+Bcl-X
caspase-3
FRET
L
N
00h:00m:00s
00h:00m:00s
00h:00m:00s
00h:00m:00s
00h:00m:00s
03h:50m:00s
SCAT3
rupture
no rupture
hESC
SCAT3
12:00:00
06:00:00
03:00:00
100
**
n.s.
high
low
high
low
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
226 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.

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Jaffe and Hall, 2005), the responsible effecter for a variety of
cases is still uncertain. In particular, the downstream effectors
inthesurvival-or-deathregulationofhESCshavebeenunknown.
In this study, we first demonstrate that the ROCK-dependent
hyperactivation of myosin is the direct cause of dissociation-
induced 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 dissociation-
induced myosin hyperactivation is specific to the epiblast-equiv-
alentcellstateratherthantotheirspeciesoforigin.Wealsoshow
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 non-
mitochondrial cascade, did not have a substantial effect on the
apoptosismarker(Figure1C,lane2;seeFigureS1Eforcontrols).
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 partic-
ularly 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 dissocia-
tion culture and continued until the cells burst and formed
apoptotic bodies (Movie S1, part B). This blebbing in the disso-
ciated 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 after-
wards (Figure S1H). Conversely, cells that had started blebbing
(but were not yet dead) in the absence of Y-27632 stopped bleb-
binguponthelateradditionofY-27632(FigureS1I).Similarearly-
onset 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)hESCsweretransfected withexpressionplasmidsforaninhibitorwithH2B-Venusexpressionplasmid. Dunnett’stest(n=3)versuslane1.n.s.,notsignificant;
**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)ASnapshot ofconfocal liveimagingfordynamicsofblebbing movement indissociated hESCs.Plasmamembrane(red, Lyn-mCherry) andnuclei(green,H2B-
venus).
(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)SnapshotsofdissociatedhESCsexpressingtheFRETprobeSCAT3.Upperpanelsshowbrightfield;thebottompanelshowVenus/CFPratioimage.Pseudo-
colors 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.
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 227

Page 4

GTP-RhoA
total RhoA
010 30
pMLC2
MLC2
60 180 360010 30
B
pMLC2
MLC2
+-+dissociation-+-
C
+-- Y-27632--+
-++ C3---
control Y-27632
D
12:00:00
06:00:00
03:00:0001:00:00
F
0
60
50
30
10
80
% apoptosis
70
40
20
control
Y-27632
Blebbistatin
0hr
6hr
90
dissociation
cyto c
Hsc70
+
+
-
-
-
+
Y-27632-
-
-
+
+
-
-++Blebbistatin---
G H
I
control Blebbistatin
0
60
AP-positive colonies
40
20
control
J
Blebbistatin
pMLC2
F-actin
merge/DAPI
A
213
213
546
213546
hESC

hESC(vehicle)
213546
213
21
min after hESC dissociation
min after hESC dissociation
+Blebbistatin
70
50
30
10
00h:00m:00s
00:00:00
01:00:00 03:00:00
04:00:00
06:00:00
shLacZ
pMLC2
shROCK-I/ROCK-II
F-actin
merge/GFP
E
hESC
**
**
***
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
228 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.

Page 5

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)fromthecytoskeleton(Charrasetal.,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 (Cole-
man 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 imag-
ing, 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 sac-
like 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.
Wenextcarriedoutatime-course study,inwhichtheCaspase
activity was measured in real-time FRET assay (Figures 1M–1P
and Figures S2D–S2G). The levelof Caspase-3 activity remained
low until ?10 min before cell rupture, when an abrupt all-or-
none-type activation was observed (Figures 1M and 1N; Movie
S2, part A; the SCAT3 FRET probe gives a high Venus/CFP fluo-
rescence 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 mito-
chondrial 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 theblebbingand theCaspase-3activationwere 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 (Fig-
ure 2A). Similarly, substantial augmentation of ROCK activity
was observed upon dissociation in the in vitro kinase assay
withROCKproteinsimmunoprecipitatedfromhESClysates(Fig-
ure 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 andtheRhoinhibitor C3(Figure2C,lanes4and6),con-
sistent 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 (Fig-
ureS3B).ConsistentwiththeseWesternblotresults,astrongim-
munostaining 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/ROCK-
dependent 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 seed-
ing. 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.
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 229

Page 6

the blebbing (Figure 2F) but also the apoptosis (Figure 2G), and
decreased the dissociation-induced release of cytochrome C
(Figure 2H, lanes 2 and 6). As a result, the Blebbistatin treatment
significantly increased the colony formation efficiency in the
hESC dissociation culture (Figures 2I and 2J). In addition, treat-
ment with cytochalasin D (an inhibitor of actin polymerization)
also attenuated both blebbing and apoptosis in dissociated
hESCs (Figures S3C–S3F; MLC2 phosphorylation was unaf-
fected, Figure S3F), suggesting that the actin-myosin interaction
is indispensable for the early-onset cell death. These inhibitor
studies suggested that the actomyosin hyperactivation down-
02:30:00
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02:16:00
01:00:00
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02:16:00
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00:00:00
0
60
min after dissociation
300120
240
180
0
2
4
1
3
venus/CFP ratio
5
6
7
% apoptosis
mock
Ezrin(T567D)
0hr
6hr
Bcl-XL
mock
Ezrin(T567D)
B
A
DE
GFP/pMLC2/DAPI
hESC(mock)
+Ezrin(T567D)
caspase-3
FRET
hESC
123
00h:00m:00s
rupture
high
low
C
0
20
40
60
80
100
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01:00:00
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00h:00m:00s
high
low
caspase-3
FRET
**
n.s.
Figure
Rather than Blebbing Movement per se,
Primarily Causes Apoptosis of Dissociated
hESCs
(A) No obvious effect of overexpression of a con-
stitutively-active form of Ezrin, Ezrin(T567D), on
MLC2 phosphorylation in dissociated hESCs.
Arrowheads indicate GFP-positive transfected
cells. The scale bar represents 10 mm.
(B–E) Effects of the overexpressing Ezrin(T567D)
on blebbing and apoptosis in dissociated hESCs.
(B) Mock-transfected cell.
(C) FRET imaging of dissociated hESCs express-
ing SCAT3 and Ezrin(T567D). The scale bar repre-
sents 10 mm.
(D) Time course of the mean Venus/CFP ratios.
(E) Apoptosis assay with Annexin V staining before
or 6 hr after dissociation. Dunnett’s test (n = 3)
versus lane 1. n.s., not significant; **p < 0.01.
The bars in the graphs represent standard devia-
tions. See also Movie S2.
3. ActomyosinHyperactivation,
stream of ROCK plays an essential role in
the dissociation-induced cell death of
hESCs.
Consistent with this idea, similar sup-
pression of both blebbing and apoptosis
was observed in dissociated hESCs in
which the myosin function was inhibited
by overexpressing a dominant-negative
MLC2 (MLC2-AA: nonphosphorylatable
form; Figures S3G–S3I) or by shRNAs
for nonmuscle myosin heavy chain IIA/
IIC (or myh9/14; Figures S3J–S3L). In
contrast to Blebbistatin treatment, the
MLCK inhibitor ML-7 did not show sub-
stantial suppressing effects on dissocia-
tion-induced apoptosis even at high con-
centrations (e.g., 20 mM; Figure S3M),
suggesting that MLCK is not an essential
downstream mediator of ROCK in this
particular context (multiple ROCK targets
arediscussedinRientoandRidley,2003).
To examine the role of blebbing in
apoptosis, we next overexpressed a
constitutively active Ezrin (EzrinT567D),
which physically strengthens the link
between the plasma membrane and the
actomyosin cortex and thereby specifi-
callyreducesblebbing(Charrasetal.,2006).Theoverexpression
of EzrinT567D did not affect the high level of pMLC2 accumula-
tion in the dissociated hESC (Figure 3A; the overexpressing cells
are indicated by coexpressing GFP). However, EzrinT567D
strongly inhibited the blebbing, which then occurred only during
the terminal stage of apoptosis when Caspase-3 was strongly
activated (Figures 3B–3D, Movie S2, part D). In contrast to the
efficient suppression of blebbing, EzrinT567D did not substan-
tially inhibit the dissociation-induced apoptosis (Figure 3E,
lane 3; lane 2 positive control with Bcl-XL). These findings of
uncoupling between apoptosis and blebbing indicated that the
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
230 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.

Page 7

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 isanoikis, which iscaused specifically bythe loss of cellular
adhesion (anchorage) to a substrate or the basement membrane
(Frisch and Screaton, 2001). We and others previously guessed
thatthedissociation-inducedhESCapoptosiswasprobablyalso
anoikis (Watanabe et al., 2007; Krawetz et al., 2009). However,
we noticed in our live-imaging analysis that, although the disso-
ciated 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 (Fig-
ure 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 treat-
ment 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-cadherin-
blocking 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 adhe-
sion (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-cad-
herin-dependent intercellular adhesion plays a major decisive
role in the apoptosis in dissociated hESCs.
We next performed a FRET analysis (using Rho-Raichu; Yosh-
izaki 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 treat-
ment (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
Onefundamentalquestionaboutthedissociation-induced hESC
apoptosis is why it occurs only in hESCs and not in mESCs.
Recently, it was reported that mouse epiblast-derived pluripo-
tent stem cells (mEpiSCs) behave more like hESCs than ICM-
derived mESCs do (Brons et al., 2007; Tesar et al., 2007). We
thereforeexaminedtheeffectoftheEGTAtreatmentonmEpiSC.
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-sensi-
tiveapoptosisthandidthatofmESCs(FigureS5C,lane3).These
observations suggested that the difference in the vulnerability of
pluripotentcellsismoredependentonthecellstate(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
pluripotentcells.ThedissociationofmEpiSCs,butnotofmESCs,
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 dissociation-
induced 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-relatedcells,bothprimaryculturecellsandstemcelllines.
Similar dissociation-induced blebbing is also seen in the epiblast-
like 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). Interest-
ingly, the similar Y-27632-sensitive blebbing was seen in dissoci-
atedectodermalcellsofXenopusgastrulae(MovieS6),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
Mechanism of Dissociated-Induced Apoptosis in hESCs
Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 231

Page 8

a-Catenin
E-Cadherin
a-Catenin
E-Cadherin
merge/DAPI
merge/DAPI
pMLC2
MLC2
-++ EGTA
--+Y-27632
A B C
D E F
G
01:00:00
00:30:00
00:10:00
00h:00m:00s
01:00:00
00:30:00
00:10:00
H
I
0
60
50
30
10
% apoptosis
70
40
20
control
Y-27632
2
0hr
8hr
05:00:00
03:00:00
01:30:00
00:30:00
Ca
1.5hr
0hr5hr
++(-)
Ca++(+)
Ca
++addition
Ca
1.5hr
0hr
5hr
++(-)
Ca++(+), a-E-cad nAb
Ca++addition
05:00:00
03:00:00
01:30:00
00:30:00
J
K
L
01:00:00
00:19:00
00:16:00
00:12:00
01:00:00
00:19:00
00:16:00
00:12:00
00:00:00
M
hESC colony
+EGTA
123
hESC
+EGTA
+EGTA
+Y-27632
1
hESC
00h:00m:00s
hESC
00h:00m:00s
+EGTA
+EGTA
00h:00m:00s
00h:00m:00s
hESC
YFP
+EGTA
Rho-FRET
high
low
00:10:00
00h:00m:00s
mEpiSC
+EGTA
GTP-RhoA
total RhoA
0 30
min after dissociation
0 30
mESC
1
mEpiSC
3
O
24
N
***
Y-27632
epiblast cell (E6.25)
vehicle
Y-27632
1
-
+
2
% cells
0
100
60
20
80
40
blebbing (+)
blebbing (-)
Q
P
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
232 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.

Page 9

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, effi-
cient knockdown was also confirmed by quantitative PCR,
data not shown) strongly rescued hESCs from the dissocia-
tion-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 forma-
tion 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
knownabouttheroleandfunctionofAbr.Importantly,inaddition
to suppressing apoptosis, the knockdown of Abr markedly
inhibited both the blebbing (Figure 5F) and the pMLC accumula-
tion (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 codon-
swapped 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 Abr-
dependent ROCK/myosin activation plays a key role in trig-
gering 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(Figure2A).Consistentwiththisfinding,inliveFRETanal-
ysis (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 differ-
ence 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 sup-
pressed 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 dissoci-
ated hESCs (Figure 6F). Thus, the persistent activation of Rac
signalinghasaclearinhibitoryeffectonthedissociation-induced
ROCK-myosin hyperactivation.
Interestingly, we found that this dissociation-induced Rho-
high/Rac-low state was greatly altered in cells of a dissocia-
tion-resistant hESC subclone reported previously (subline 1;
Hasegawa et al., 2006). Although these cells expressed hESC-
specific markers and formed teratoma (Figure S6A), they did
not undergo blebbing or apoptosis in dissociation culture
(Figures S6B–S6D). Consistent with these findings, no substan-
tial elevation of pMLC2 upon dissociation was observed (Fig-
ure 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
findingsprovideadditionalcircumstantialevidenceforthestrong
correlation between the Rho/Rac control and the dissociation-
induced myosin hyperactivation.
The involvement of the reciprocal Rho/Rac control was partic-
ularly 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.
(HandI)SnapshotsofliveimagingofEGTA-dissociatedhESCsonMatrigelintheabsence(H)orpresence(I)of10mMY-27632.Imageswereobtainedevery1min
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 addi-
tion. 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
pointofrecording.Imageswereobtainedevery30sfor1hr.Inthiscase,redandblueindicated highandlowRhoactivities, respectively.Thescalebarrepresents
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.
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 233

Page 10

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 inhi-
bition was observed in Abr-depleted hESCs upon dissociation
(Figure6I).Furthermore,thevulnerabilitytodissociation-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
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
theinduction oftheROCK/myosinhyperactivationindissociated
mammalian pluripotent cells.
Figure 7E illustrates the summary of the apoptosis-inducing
pathway elucidated in the present study. In nondissociated
hESCs,theRho/ROCK/pMLC systemiskeptlowbyaninhibitory
mechanism dependent on the E-cadherin-mediated intercellular
adhesion. Upon cell dissociation, this system becomes desup-
pressed (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
shLacZ
shAbr(A) shAbr(B)
shAbr(C)
shROCK-I/II
2
0hr
6hr
% apoptosis
shLacZ
shAbr(A)
% blebbing
0
80
20
60
100
40
80
20
60
100
40
0
0hr
6hr
% apoptosis
0
80
20
60
100
40
GFP/pMLC2/DAPI
ACE
F
shLacZ
shAbr(A)
Abr(WT)
Abr*
+
+ +
+
+
+
4
shROCK-I/II
1
345
123
123
Flag
Hsc70
Abr
shLacZ
shAbr(A)
shAbr(B)
shAbr(C)
1234
D
DH
PH
C2 GAP
Abr
AbrDDH
Abr(RA/NA)
PH C2GAP
DH
PH
C2
GAP
B
shLacZ
shAbr(A)
AP-positive colonies
0
80
20
60
100
40
shLacZ
shAbr(A)
H
12
**
****
**
***
**
**
**
n.s.
shLacZ
shAbr(A)
G
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.
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
234 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.

Page 11

‘‘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. Interest-
ingly, 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 intercel-
lular adhesion.
We deduce that the dissociation-induced apoptosis may be
antagonized to some extent by the PI3K-Akt pathway (Down-
ward, 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 (Fig-
ure S7F). Conversely, the overexpression of a constitutively
active Akt (caAkt) at least partially reduced the dissociation-
inducedapoptosiswithoutinterferingwithblebbing(FigureS7G),
suggesting that PI3K-Akt signaling can negatively modulate the
apoptotic signal downstream of the myosin hyperactivation.
Westernblotanalysisshowedthatdissociationinducesagradual
accumulation of phosphorylated (active) Akt in hESCs (Fig-
ure 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
Myosin Hyperactivation Is the Direct Cause
of the Early-Onset Apoptosis of hESCs
In this study, we first revealed that an unregulated activation of
theactin-myosinsystemisthecauseforthecelldeathofisolated
hESCs. The myosin hyperactivation per se, not the blebbing,
directlyleadstotheapoptosis.Inotherwords,althoughthebleb-
bing 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 investi-
gationsishowtheinformationofmyosinhyperactivationistrans-
duced 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 actomy-
osin system, rather than elevated activity of myosin per se, is
essential for the apoptosis induction.
Onemodelfortheinductionmechanismisthattheactomyosin
hyperactivation nonspecifically augments intracellular stress,
which is shown to trigger apoptosis via stress-responsive
apoptotic pathways. Two typical apoptosis-inducing factors
relatedtocellularstressresponsesareMAPKs(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 (unpub-
lished data). For instance, the dissociation of hESCs did not
increase the level of phosphorylated JNK, and the MAPK inhibi-
tors (SP600125 and SB203580) had little effects on the dissoci-
ation-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 hyperactiva-
tion. With this in mind, we measured the ATP content in dissoci-
ated 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 disso-
ciated 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 activa-
tion 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 Bleb-
bistatin (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 (Wata-
nabe et al., 2007), suggesting the precence of a minor Cas-
pase-independent pathway that also lies downstream of the
ROCK-myosin hyperactivation for efficient long-term survival.
Upstream Regulation of the Dissociation-Induced
ROCK/Myosin Hyperactivation in hESCs
In the context of dissociation-induced ROCK/myosin hyperacti-
vation, 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 antago-
nistic 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 Abr-
depleted 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
Mechanism of Dissociated-Induced Apoptosis in hESCs
Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 235

Page 12

GTP-Rac1
total Rac1
010 30 time (min)
A
01:00:00
00:30:00
00:15:00
00:12:00
00h:00m:00s
01:00:00
00:30:00
00:15:00
00:12:00
00:00:00
B
02:00:00
10:00:00
02:00:00
C
0
2
4
6
8
0200400600
min after dissociarion
venus/CFP ratio
0
20
40
60
80
% apoptosis
mock
caRac
6hr
DE
caRac
GFP/pMLC2/DAPI
mock
F
0
20
40
60
80
% apoptosis
100
shLacZ
shAbr(A)
Abr*
Abr*DDH
Abr*(RA/NA)
+
+ +
+
+
+
H
+
5
123
hESC dissociation
hESC
YFP
+EGTA
Rac-FRET
dissociated hESC
00h:00m:00s
+caRac
caspase-3
FRET
dissociated hESC
3 4 2 1
Flag
SCAT3
no rupture
00:00:00
10:00:00
high
low
high
low
+
total RhoA
GTP-RhoA
total Rac1
GTP-Rac1
300300
min after dissociation
shLacZ shAbr(A)
3214
I
12
0hr
6hr
0hr
**
**
n.s.
n.s.
total RhoA
GTP-RhoA
caRac
300
300
min after dissociation
mock caRac
3214
G
Figure 6. Rho-High/Rac-Low State Plays a Key Role for Induction of Myosin Hyperactivation
(A) Measurement of Rac activity in dissociated hESCs by pull-down assay.
(B) Snapshots of EGTA-treated hESC expressing the Rac-Raichu FRET probes (identified as a YFP-positive cell). White arrowheads indicate blebbing cells. The
scale bar represents 10 mm.
(C and D) Effects of a constitutive active form of Rac1 (caRac, Rac1V12) on blebbing and apoptosis in dissociated hESCs. Snapshots of dissociated hESCs
expressing the SCAT3 probes together with caRac (C) The scale bar represents 20 mm. The time course of the mean Venus/CFP ratios over the whole cell
was shown (D).
(E) Apoptosis assay in caRac-expressing hESCs. **p < 0.01 in t test (n = 3).
(F) Decreased MLC2 phosphorylation in dissociated hESC expressing caRac (white arrowheads indicate as a GFP-positive cell, green). The scale bar represents
10 mm.
(G) Rho activity in mock- or caRac-transfected hESC before and after dissociation.
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
236 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.

Page 13

which potentially contributes to the Rho-high/Rac-low state. Abr
is required for both the dissociation-induced myosin hyperacti-
vation 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 differen-
tially 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
Invivo, the cells in the ICM of the preimplantation embryo arenot
polarized and form a simple cell mass with no epithelialization
(RossantandTam,2009).Afterimplantation,thepluripotentcells
derived from the ICMbecome epithelialized and form anepiblast
tissuewithaclearapico-basalpolarity,includingtheappearance
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 postimplan-
tation 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; Char-
ras and Paluch, 2008).
Finally, another stimulating open question regarding the cell-
state-specific ROCK/myosin hyperactivation is whether this
phenomenon is limited to the early embryonic cells of mamma-
lian 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 blasto-
coel, but do not have evident apico-basal polarity (e.g., no base-
ment membrane). These cells become epithelialized during early
gastrulation upon their fate specification into the ectoderm
lineage. Importantly, the dissociated Xenopus gastrulae ecto-
dermal cells also exhibit blebbing (Johnson, 1976) in a ROCK-
dependent 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 guide-
lines of the Japanese government. Five hiPSCs (gift from Y. Nakamura and
S. Yamanaka) were also tested and similar observations were made. Undiffer-
entiated 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), respec-
tively. 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-conju-
gated 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 SDS-
PAGE 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 Matrigel-
coated35mmglass-bottomdish.Therecordingwasstartedat15minafterthe
first contact with the dissociation reagent. For the EGTA experiments, cell
clumpsmaintainedonafeeder layeror aMatrigel-coated35mmglass-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 observa-
tion, the images were collected with a CSU-X1 unit (Yokogawa) configured
with an IX81-ZDC microscope (Olympus). The ratiometric analyses were per-
formed 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 one-
way 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 restorationof dissociation-induced apoptosis by expression of RNAi-resistant Abr mutants (Abr*) lacking theGEF 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.
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 237

Page 14

cell-cell
adhesion
epiblast state-linked
regulator (?)
myosin
hyperactivation
mitochondria
caspase
Annexin V(+)
blebbing
Y-27632
Blebbistatin
caEzrin
Rho
Bcl-XL
zVAD
shAbr
apoptotic body
PI3K/Akt
growth factors?
integrin?
Ras?
terminal surge
Loss of cell-cell
adhesion
E-cadherin
Rac
Rho
ROCK
myosin
Rac
Abr
GEF
GAP
Abr
epiblast state-linked
regulator (?)
salvage
1
2
3
4
5
apex
BM
adhesion culture
cell dissociation
ROCK
myosin
0
20
40
80
60
100
% apoptosis
shLacZ
shAbr(A)
caROCK
Abr*
V5-Abr*
Myc-caROCK
dnRac
+
+ + +
+
+
+
++
+
+
Flag-dnRac
AB
321456
6hr
0hr
n.s.
n.s.
*
**
*
***
shLacZ
shAbr(A)
caRho
Abr*
V5-Abr*
Myc-caRho
dnRac
+
+ + +
+
+
+
++
+
+
0
20
40
80
60
100
% apoptosis
shAbr(A)+mock
dnRac
caRho
shAbr(A)+
n.s.
n.s.
**
***
**
***
6hr
0hr
CD
dnRac
caRock
shAbr(A)+
shAbr(A)+mock
Flag-dnRac
321456
E
Figure 7. Rho-High/Rac-Low State Responsible for the Dissociation-Induced hESC Apoptosis
(A–D)RacinhibitioncontributestoROCK-dependent blebbing and apoptosis induction. Thesensitivity todissociation-induced blebbing and apoptosis werefully
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 state-
linked 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.
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
238 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.

Page 15

ACKNOWLEDGMENTS
Weare gratefultoDrs. M. Matsuda and E.Kiyokawafor 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 Medi-
cine (Y.S.).
Received: December 14, 2009
Revised: April 18, 2010
Accepted: June 4, 2010
Published: August 5, 2010
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