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
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
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8
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hr after dissociation
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cyto c
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control
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mESC
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control
Y-27632
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hESC
20
TMRM+ cells (%)
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Bcl-X L
20
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6hr
dnFADD
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mock
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% Annexin V+ cells
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A
hESC
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226 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.
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low
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caspase-3
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P
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min after dissociation
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04:02:00
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SCAT3
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no rupture
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Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
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.
Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 227
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
A
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min after hESC dissociation
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GTP-RhoA
pMLC2
pMLC2
total RhoA
MLC2
MLC2
3
1
control
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3
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E
Y-27632
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6
shLacZ
pMLC2
pMLC2
F-actin
F-actin
merge/DAPI
merge/GFP
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shROCK-I/ROCK-II
hESC
hESC
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% apoptosis
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Blebbistatin
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cyto c
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Hsc70
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control
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Blebbistatin
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AP-positive colonies
+Blebbistatin
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Blebbistatin
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228 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.
Blebbistatin
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Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
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.
Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 229
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
mock
Figure 3. Actomyosin Hyperactivation,
Rather than Blebbing Movement per se,
Primarily Causes Apoptosis of Dissociated
hESCs
Ezrin(T567D)
hESC
A
GFP/pMLC2/DAPI
hESC(mock)
B
00h:00m:00s
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caspase-3
FRET
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(A) No obvious effect of overexpression of a constitutively-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 expressing SCAT3 and Ezrin(T567D). The scale bar represents 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 deviations. See also Movie S2.
03:52:00 high
+Ezrin(T567D)
C
Ezrin(T567D)
Bcl-X L
mock
% apoptosis
venus/CFP ratio
caspase-3
FRET
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
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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
Cell Stem Cell
hESC colony
Mechanism of Dissociated-Induced Apoptosis in hESCs
A
B
C
G
+
+
+
Y-27632
EGTA
pMLC2
a-Catenin
D
E
MLC2
merge/DAPI
E-Cadherin
F
1
+EGTA
J
2
3
70
0hr
8hr
% apoptosis
60
a-Catenin
H
-
merge/DAPI
E-Cadherin
hESC
50
40
***
30
20
00:10:00
00h:00m:00s
00:30:00
01:00:00
Y-27632
0
control
+EGTA
10
1
2
+EGTA
+Y-27632
I
00h:00m:00s
K
++
Ca (-)
0hr
00:10:00
01:00:00
00:30:00
++
Ca addition
Ca++(+)
00h:00m:00s
00:30:00
00h:00m:00s
5hr
1.5hr
hESC
+EGTA
N mEpiSC
01:30:00
03:00:00
00:10:00
05:00:00
+EGTA
O min after dissociation
0 30
0 30
GTP-RhoA
total RhoA
L
++
Ca (-)
0hr
hESC
1
5hr
1.5hr
00:30:00
mESC
Ca ++(+), a-E-cad nAb
01:30:00
03:00:00
P
mEpiSC
2
3
4
epiblast cell (E6.25)
05:00:00
+EGTA
00h:00m:00s
Ca++ addition
Y-27632
vehicle
Q
M
blebbing (-)
blebbing (+)
hESC
YFP
+EGTA
100
00h:00m:00s
00:12:00
00:16:00
00:19:00
01:00:00
Rho-FRET
high
% cells
80
60
40
20
0
00:00:00
00:12:00
00:16:00
00:19:00
01:00:00 low
+
Y-27632
1
232 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.
2
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
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.
Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 233
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
GAP
PH
C2
GAP
80
**
60
F
shAbr(A)
shROCK-I/II
shAbr(A)
shAbr(B)
shAbr(C)
0
1
2
3
4
5
4
G
80
60
shLacZ
1
shAbr(A)
shLacZ
0
shROCK-I/II
**
20
**
2
3
% apoptosis
80
% blebbing
100
***
80
60
40
20
1
2
0hr
6hr
**
60
100
0
H
100
40
**
shLacZ
3
** **
40
Hsc70
2
E
shLacZ
20
Abr
1
D
0hr
6hr
shAbr(A)
C2
100
shLacZ
PH
Abr
AbrDDH
Abr(RA/NA)
shAbr(C)
GAP
shAbr(B)
shLacZ
B
C2
shAbr(A)
DH
PH
AP-positive colonies
C
DH
% apoptosis
A
n.s.
40
20
0
+
shAbr(A)
+
+
+
2
3
GFP/pMLC2/DAPI
+
+
1
shLacZ
shAbr(A)
Abr(WT)
Abr*
4
Flag
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
B
hESC dissociation
0 10 30 time (min)
GTP-Rac1
hESC
YFP
+EGTA
A
00h:00m:00s
high
00:12:00
D
dissociated hESC
E
venus/CFP ratio
+caRac
10:00:00
02:00:00
caspase-3
FRET
low
6
no rupture
4
2
60
**
40
20
0
0
200
400
0
600
min after dissociarion
02:00:00
00:00:00
F
high
10:00:00
H
dissociated hESC
I
n.s.
n.s.
100
**
caRac
% apoptosis
mock
GFP/pMLC2/DAPI
shLacZ
0
30
0
0
30
0
30
GTP-RhoA
60
total RhoA
40
GTP-Rac1
total Rac1
0
+
30
+
GTP-RhoA
total RhoA
+
+
2
3
+
+
1
2
3
caRac
1
2
shAbr(A)
20
min after dissociation
mock
caRac
1
min after dissociation
0hr
6hr
80
G
0hr
6hr
80
SCAT3
8
00h:00m:00s
01:00:00 low
00:30:00
00:15:00
caRac
3
00:00:00
C
01:00:00
mock
2
00:30:00
% apoptosis
1
00:15:00
00:12:00
Rac-FRET
total Rac1
4
shLacZ
+ shAbr(A)
Abr*
Abr*DDH
+ Abr*(RA/NA)
1
2
3
4
5
Flag
4
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.
236 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
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.
Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 237
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
A
0hr
6hr
***
***
** n.s.
100
B
C
shAbr(A)+mock
D
0hr
6hr
n.s.
*
shAbr(A)+mock
80
**
n.s.
60
caRho
shAbr(A)+ dnRac
40
% apoptosis
80
% apoptosis
***
**
100
*
n.s.
60
caRock
shAbr(A)+ dnRac
40
20
20
0
0
+
+ + +
+
+
1
E
2
3
4
+
shLacZ
+ + shAbr(A)
+
caRho
+
dnRac
+ Abr*
V5-Abr*
Myc-caRho
Flag-dnRac
5
shLacZ
+ + shAbr(A)
+
caROCK
+
dnRac
+ Abr*
+ + +
+
+
V5-Abr*
Myc-caROCK
Flag-dnRac
1
6
adhesion culture
2
3
4
5
6
cell dissociation
apex
cell-cell
adhesion
epiblast state-linked
regulator (?)
GEF
GAP
Abr
epiblast state-linked
regulator (?)
Abr
E-cadherin
Rac
Rac
Rho
ROCK
1
2
shAbr
3
Rho
Loss of cell-cell
adhesion
ROCK
Y-27632
myosin
myosin
myosin
hyperactivation
salvage
integrin?
Ras?
Blebbistatin
caEzrin
4
blebbing
PI3K/Akt
growth factors?
mitochondria
caspase
Bcl-XL
zVAD
5
terminal surge Annexin V(+)
apoptotic body
BM
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.
238 Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc.
Cell Stem Cell
Mechanism of Dissociated-Induced Apoptosis in hESCs
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.
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Cell Stem Cell 7, 225–239, August 6, 2010 ª2010 Elsevier Inc. 239
Cell Stem Cell, Volume 7
Supplemental Information
Molecular Pathway and Cell State Responsible
for Dissociation-Induced Apoptosis
in Human Pluripotent Stem Cells
Masatoshi Ohgushi, Michiru Matsumura, Mototsugu Eiraku, Kazuhiro Murakami, Toshihiro Aramaki, Ayaka Nishiyama, Keiko
Muguruma, Tokushige Nakano, Hidetaka Suga, Morio Ueno, Toshimasa Ishizaki, Hirofumi Suemori, Shuh Narumiya, Hitoshi Niwa,
and Yoshiki Sasai
Inventory of Supplemental Information
Figure S1 related to Figure 1.
Figure S2 related to Figure 1.
Figure S3 related to Figure 2.
Figure S4 related to Figure 4.
Figure S5 related to Figure 4.
Figure S6 related to Figure 6.
Figure S7 related to Figure 7.
Movie S1 relates to Figure 1.
Movie S2 relates to Figure 1 and 3.
Movie S3 relates to Figure 4.
Movie S4 relates to Figure 4.
Movie S5 relates to Figure 4.
Movie S6 relates to Figure 4.
Movie S7 relates to Figure 6.
-1-
-2-
Figure S1. ROCK-dependent induction of blebbing and apoptosis in dissociated hESC, related to Figure 1.
(A) Low-density culture of dissociated hESC in the absence (left) and presence (right) of 10 μM Y-27632 on MEF
cells for 7 days. Colonies were visualized with AP staining. (B-C) FACS analysis of dissociated hES cells with
Annexin-V and propidium iodide staining (B). Left panel, no inhibitor; right panel, 10 µM Y-27632. Percentages of
+/- cells were presented as a graph (C). Double-negative (white), live cells; Annexin V-positive (brown), early
apoptotic cells; double-positive (light brown), dead cells. (D) shRNA-mediated ROCK knockdown experiments.
(a-b) Efficiency of shRNAs-mediated knockdown of ROCK-I (rock1) and -II (rock2) was measured by quantitative
PCR (a) and western blot (b). (c) Annexin-V staining of ROCK-depleted hESC. Prior to the experiment,
non-transfected cells had been eliminated by the puromycin treatment. Dunnett’s test (n = 3) vs. lane 1 (control). n.s.,
not significant; **, p < 0.01. (E) Control experiment of dnFADD activity and expression. HeLa cells were
transfected with mock- or dnFADD-expression plasmids together with a H2B-venus-expression plasmid, and their
apoptosis was induced by treating with 1 µg/ml Fas-activating antibody + 1 µg/ml cycloheximide (gray) or 2 µg/ml
etoposide (black) for 12 hours. The expression levels of dnFADD in HeLa and hESC were comparable (right;
co-expressed GFP as a loading control). (F) Effects of zVAD with Y-27632 on colony formation from dissociated
hESC. Formed hESC colonies were visualized by AP staining and counted. Tukey’s test (n = 3) among all groups.
n.s., not significant. (G) Snapshots of dissociated hESC cultured on MEF feeder cells (top), a Collagen IV-coated
(row) or a non-adhesive dish (bottom). (H-I) Snapshots of live imaging of dissociated hESC. (H) Dissociated hESC
were cultured without the inhibitor for 1 hour, and 10 μM Y-27632 was then added to culture medium. (I)
Conversely, dissociated hESC were cultured in the presence of 10 μM Y-27632 for 1 hour, and the inhibitor was
then washed out. (G-I) Arrowheads, blebbing cells. Bar, 20 μm. The bars in the graphs represent standard
deviations.
-3-
-4-
Figure S2. Snapshots of confocal imaging and FRET imaging of blebbing hESC, related to Figure 1.
(A) The plasma membrane and the nucleus were visualized with mCherry-fused Lyn and venus-fused H2B,
respectively. (B) Disassociation of the plasma membrane and cortical actin during the bleb evagination and their
following reassembly in the blebs during the retraction phase. Actin movement was visualized with venus-fused
actin. (C) Myosin accumulation at the bleb surface during the retraction phase. Myosin movement was visualized
with eGFP-fused MLC2. Images were obtained every 10 seconds for 15 minutes. Red arrows, newly formed blebs.
Yellow arrowheads, relocalization of the actin rim or myosin in the bleb membrane. (D-E) Snapshots of dissociated
hESC expressing the FRET probe for Caspase-9 proteolytic activity (SCAT9) (D). Time course for Caspase-9
activation (E). (F-G) Analysis of dissociated hESC expressing the SCAT3 probe and shRNAs for
ROCK-I/ROCK-II. Images were obtained every 2 minutes for 10 hours after cell seeding. White arrowheads,
blebbing cells; red arrows, membrane rupture. Bar, 20 μm.
-5-
-6-
Figure S3. Attenuation of the ROCK/actomyosin system abolished the dissociation-induced blebbing and apoptosis,
related to Figure 2.
(A) In vitro kinase assay using recombinant MYPT1 as a substarate (bottom, CBB staining). ROCK-II was
immunoprecipitated from cell lysates of undissociated (lane 1) or dissociated (lanes 2, 3) hESC. ROCK-dependent
activity was confirmed by adding 10 µM Y-27632 to the kinase reaction mixture (lane 4). (B-C). Titration of four
ROCK inhibitors on the MLC2 phosphorylation (upper) and apoptosis (bottom) in dissociated hESC. The strong
correlation was observed between the concentrations effective for the inhibition of MLC2 phospholyration and that
of apoptosis. (C) Snapshots of live imaging of dissociated hESC in the absence (upper) and presence (bottom) of 2
µM Cytochalasin D. Images were obtained every 5 minutes for 12 hours after cell seeding. Bars, 20 μm. No
pretreatment with Cytochalasin D was given (see Supplemental Experimental procedures). Although some blebs
were seen at the 0 time point in both samples, later, Cytochalasin D-treated hESC showed no substantial blebbing
movements, unlike the control cells. (D-E) Annexin V staining of dissociated hESC cultured in the absence (upper)
or presence (bottom) of 2 µM Cytochalasin D (D). FITC-positive cells were counted and the percentages were
shown as a graph (E). Bar, 50 µm. (F) The MLC2 phosphorylation level was elevated in hESC upon dissociation
regardless of Cytochalasin D treatment. (G-H) Effects of a dominant-negative MLC2 (MLC2-AA) on
dissociation-induced blebbing and apoptosis. Snapshots of mock- or MLC2-AA-transfected hESC during
dissociation culture. MLC2-AA-expressing cells (right, marked by GFP) showed no blebbing (G). Bar, 20 µm.
Percentages of blebbing cells are shown as a graph (H). Annexin V staining (I). ***, p < 0.01 in t-test between 6-hr
groups (n = 3). (J-L) Effects of shRNA-mediated knockdown of non-muscle myosin heavy chains (NMMHCs) on
dissociation-induced blebbing and apoptosis. Effective MHCs knockdown (NMMHC-IIA and -IIC) was
confirmed by western blot analyses using specific antibodies (J). Annexin V staining (K). Dunnett’s test (n = 3) vs.
lane 1 (control). **, p < 0.001. (L) Snapshots of control and MHCs-shRNA-expressing hESC in dissociation culture.
Cells co-expressing shRNAs for Myh9 and Myh14 (bottom) showed no blebbing (bottom), unlike the control cells
(top). Bar, 20 µm. In contrast, knockdown of NMMHC-IIB (MYH10) has little effect on dissociation-induced
blebbing and apoptosis in hESC (data not shown). (M) Snapshots of dissociated hESC in the presence of 20 µM
ML-7. Bars, 20 μm. (C, G, L, M) White arrowheads, blebbing cells; red arrows, membrane rupture. The bars in the
graphs represent standard deviations.
-7-
-8-
Figure S4. E-Cadherin knockdown induces blebbing and cell death in non-dissociated hESC, related to Figure 4.
(A) Focal adhesion formation in dissociated hESC cultured on the Matrigel substrate was visualized by
immunostaining for Paxillin (a, red and f, green), a component of focal adhesion. The accumulation of
phospholyrated FAK (g, red) and phospho-tyrosin (c, green) in focal adhesion indicates the clustering and activation
of the local integrin system. The cells were fixed at 3 hours after cell seeding and subjected to immunostaining. Bar,
10 µm. (B) Apoptosis assay was performed in the indicated time in the procedure of the Figure 4K-L experiment.
***, p < 0.001 in t-test (n = 3). (C-F) E-Cadherin knockdown experiments. (C) Efficiency of shRNA-mediated
E-Cadherin knockdown was measured by western blot (a) and immunostaining (b). shRNA-expressing cells was
marked by GFP (asterisks). (D) Time table of live imaging. (E-F) Snapshots of live imaging of hESC expressing
control shRNAs (E) or E-Cadherin shRNAs within an intact colony (F). Recording was started 48 hours after the
plasmid transfection. Transfected cells were identified as GFP-positive cells. Images were obtained every 10
minutes for 48 hours. Bar, 10 µm. White arrowheads, blebbing cells; red arrows, membrane rupture. The bars in the
graphs represent standard deviations.
-9-
Figure S5. Dissociation-induced ROCK/myosin hyperactivation is related to the epiblast-like pluripotent state,
related to Figure 4.
(A-B) Snapshots of live imaging of EGTA-treated mEpiSC colonies on MEF feeder cells in the absence (A) or
presence (B) of 10 μM Y-27632. Images were obtained every 1 minute for 2 hours after EGTA addition. Bars, 10
μm. (C) Apoptosis frequency in dissociated mESC (lanes 1-2) and mEpiSC (lanes 3-4) with or without Y-27632.
n.s., not significant; **, p < 0.01 in t-test (n = 3) for 6-hour data of each line. (D) Experiments using primary-culture
epiblast cells (E6.25). Snapshots of live imaging of dissociated epiblast cells on the Matrigel substrate in the absence
(upper) or presence (bottom) of 10 μM Y-27632. Images were obtained every 30 seconds for 15 minutes after cell
seeding. Bar, 10 μm. (E) Snapshots of dissociated mESC in which the artificial Rho-high/Rac-low state was
generated by the combinatory overexpression of constitutively-active RhoA (RhoAV14, caRho) and
dominant-negative Rac1 (Rac1N17, dnRac). Transfected cells were selected with puromycin and blebbing mESC
in dissociation culture were counted in three independent experiments (F). Tukey’s test showed a significant
difference between the lane-4 values and any of the rest (p < 0.001, n = 3).
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Figure S6. Comparison between apoptosis-resistant subline 1 hESC and their parental KhES-1 cells, related to
Figure 6.
(A) Immunostaining images of pluripotency markers (Oct3/4, Nanog, TRA-1-60 and E-cadherin). (B) Snapshots of
live imaging of dissociated each line. top, parental line; bottom, subline 1. Images were obtained every 5 minutes for
12 hours after cell seeding. Bars, 20 μm. White arrowheads, blebbing cells; red arrows, membrane rupture. (C)
Comparison of blebbing occurance. (D) Apoptosis induction (Annexin-V staining). (E-F) MLC2 phosphorylation
(E) and Rho/Rac activity (F) before and after dissociation. (G) mRNA expression of the components of the
Abr-ROCK-myosin pathway (Q-PCR). The bars in the graphs represent standard deviations.
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Figure S7. Roles of E-Cadherin activation and PI3K/Akt signaling in controlling apoptosis of dissociated hESC,
related to Figure 7.
(A) Immunoprecipitation assay of the E-Cadherin/catenin complex. Lane 1, total lysate; lane 2, immunoprecipitates
from undissociated hESC by an E-cadherin antibody. Abr was not co-precipitated with this complex (top panel). (B)
Immobilization of E-Cadherin-Fc chimera protein onto a culture dishes. (C) Images of mESC seeded as single cells
on the E-cadherin-coated dish. Cells were visualized by AP staining 2 day after seeding. Bar, 500 µm. a, non-coated
dish; b-c, E-Cadherin (5 or 10 µg/ml)-coated dish; d, an enlarged image of the square region in c. mESC attached
efficiently onto the coated dish and grew well as dispersed cells without forming colonies, as reported previously.
(D) PlasDIC images of hESC seeded as single cells on the E-cadherin-coated dish in the absence (top) or presence
(bottom) of 10 µM Y-27632. Images were obtained at the indicated time points in dissociation culture. Arrowheads,
blebbing cells; red arrows, dead cells. Bar; 20 µm. (E) Apoptosis induction in dissociated hESC on the
E-Cadherin-coated dishe 8h after dissociation (gray bars). White bar, before dissociation. The coating concentrations
of E-cad protein are indicated below. (F) The PI3K inhibitor LY-294002 enhanced hESC’s apoptosis in dissociation
culture. Blue and red lines, LY294002-treated cells; black line, control. (G) Overexpression of constitutive active Akt
(caAkt) delayed the onset of apoptosis in dissociated hESC (green line, caAkt-expressing cells; black line,
mock-transfected cells). Expression of caAkt is shown in the inset. (H) Accumulation of phosphorylated Akt during
dissociation culture of hESC. top, phosphorylated (active) Akt; bottom, total Akt.
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Supplemental Experimental Procedures
Cell culture.
The hESC (KhES-1) were a gift from N. Nakatsuji (Kyoto University) and were used following the hES cell
guidelines of the Japanese government. Another hESC line (KhES-3 from N. Nakatsuji) and five hiPS cells (gift
from Y. Nakamura and S. Yamanaka) were also tested and similar observations were made. Undifferentiated hESC
were maintained as described previously (Hasegawa et al., 2006: Watanabe et al., 2007). The cells were cultured on
a feeder layer of mouse embryonic fibroblasts (MEF; purchased from Kitayama Labes; inactivated with 10 µg/ml
mitomycin C and seeded at 1.2x106 per 10 cm plate) in D-MEM/F12 (Sigma) supplemented with 20% KSR
additive, 2 mM glutamine, 0.1 mM non-essential amino acids (Invitrogen), 5 ng/ml recombinant human bFGF
(Wako) and 0.1 μM 2-ME under 2% CO2. For passage, hES cell colonies were detached and recovered en bloc
from the feeder layer by treating them with CTK dissociation solution at 37°C for 5-7 minutes, followed by tapping
the cultures and flushing them with a pipette. The detached ES clumps were broken into smaller pieces by gently
pipetting them several times and then these small clumps were transferred onto a MEF-seeded dish. hESC were also
maintained on Matrigel substrate (BD Biosciences) in MEF-conditioned medium. The culture medium was
exchanged to fresh one daily until the next passage. The dissociation-resistant subclone of KhES-1 (subline1;
Hasegawa et al, 2006) was also maintained as decribed above. The cells expressed hES cell-specific markers such as
Oct3/4, Nanog and Tra-1-60 (Figure S6; also expressed genes involved in the Abr-ROCK-myosin pathway) and
had the ability of forming teratomas when injected SCID mouse testes (100%, n = 6, 9 weeks after injection 10,000
cells/testis) as their parental line did and as reported previously (Hasegawa et al, 2006).
The mESC (EB5) were maintained as described previously (Eiraku et al., 2008). The mEpiSC (kindly
provided by P. Tesar; Tesar et al., 2007) were maintained on a feeder layer of MEF in hES medium supplemented
with recombinant human bFGF (10 ng/ml) and activin A (20 ng/ml, R&D). The mEpiSC were passaged using the
same procedure as described for the hESC.
Dissociation of hESC clumps.
The hESC were dissociated as described previously (Watanabe et al., 2007). First, they were detached from the
feeder layer as described above for the passage procedure. Next, contaminating MEF cells were removed by
incubating the cell suspension on a gelatin-coated plate at 37°C for 2 hours in the maintenance culture medium. In
this procedure, MEF cells adhere to the dish bottom, but hESC do not. The floating hES cell clumps were recovered
from the suspension by centrifugation, washed with PBS, incubated in TrypLE select (Invitrogen) at 37°C for 5
minutes, dissociated into single cells by pipetting, and then passed through a Cell Strainer (BD Falcon). The
dissociated cells (2 x 105 cell / 35 mm dish) were seeded onto an MEF feeder layer or Matrigel. For inhibitor studies,
unless mentioned otherwise, the inhibitors were added to the culture medium 1 hour prior to detaching the cells from
the feeder layer and again upon seeding the cells.
Reagents were purchased from Calbiochem (Y-27632, HA-1077, H-1152P, LY-294002, ML-7,
cytochalasin D, (-)-Blebbistatin), Sigma (puromycine, Collagen-IV, actinomysin D, cycloheximide), PEPTIDE
Institute (zVAD-fmk), Cytoskeleton (C3 transferase), TMRM (Molecular Probes) and Axon Medchem
(GSK269962). In the case of cytochalasin D experiment, pre-treatment prior to dissociation was omitted because
cytochalasin-D-treated hES cells are extremely fragile and vulnerable to physical stress that yielded by pipetting.
Actinomycin D and cycloheximide showed little protective effects on the dissociation-induced apoptosis of hESC,
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indicating that de novo protein synthesis is not essential for this cell death.
Plasmids and transfection.
The cDNAs for rhoA, rac1, fadd, bcl-xL, ezrin, mrlc2, rock1 and abr (valiant 1) were amplified from a KhES-1
cDNA library. Point or deletion mutants of RhoA (RhoAV14), Rac1 (Rac1V12, Rac1N17), ROCK-I
(ROCK-I-Δ3), Ezrin (EzrinT567D), FADD (dnFADD), MLC2 (MLC2-AA) and Abr (Abr*, Abr-RA/NA,
Abr-ΔDH) were obtained by PCR mutagenesis using wild-type cDNA as a template. cDNAs for H2B-venus,
Lyn-mCherry, actin-venus and eGFP-MLC2 were obtained by fusing the fluorescence proteins to H2B,
N-terminus fragment of Lyn, β-actin and MLC2 by PCR. The active Akt expression plasmid (mΔPH-Akt) was a
gift from Y. Gotoh (Tsuruta et al., 2002; University of Tokyo). All cDNAs was subsequently sequenced and
subcloned into the pCAG-IP or pCAG-IG (in which the puromycin-resistance gene in pCAG-IP was replaced with
nuclear eGFP) expression vector (Niwa et al., 2002). 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 to knockdown the expression of specific genes. An
shRNA expression vector was constructed to generate a short interference RNA (siRNA) corresponding to the
following sequence of nucleotide (nt); nt 1532-1552 (A), 2460-2480 (B), and 4419-4439 (C) of human abr mRNA
(GenBank accession number; NM_021962), nt 2901-2921 of human rock1 mRNA (NM_005406), nt 1458-1478
of human rock2 mRNA (NM_004850), nt 2363-2383 of human cdh1 mRNA (NM_004360), nt 1100-1120 of
human myh9 mRNA (NM_002474), nt 5725-5745 of myh10 mRNA (NM_005964), nt 1890-1910 of myh14
mRNA (NM_001077186) and nt 654-674 of E coli. lacZ mRNA (V00296, negative control). Abr*, a
shAbr(A)-resistant mutant of Abr, is designed to be translated into the normal protein whereas it bears the 4 base
substitution in the wild type mRNA sequence.
The transfection of hESC with cDNA- or shRNA-expression plasmids was performed with the
FuGENE HD transfection reagent (Roche). hES clumps were detached from the feeder layer and contaminating
MEF cells were removed as described above. The MEF-free hESC clumps or dissociated hESC were mixed with
the complex of plasmid/FuGENE HD reagent and seeded onto the MEF feeder layer or Matrigel (when cells were
dissociated, 10 µM Y-27632 was added to culture medium). The following day, the transfected cells were washed
thoroughly with PBS for the complete removal of the transfection reagent and Y-27632, and maintained in fresh
medium until these cells were used for experiments. For the selective elimination of non-transfected cells, the cells
were first seeded onto the puromycin-resistant MEF feeder layer (Cell Biolabs). The medium was switched to 1-2
μg/ml puromycin-containing hESC medium at 48 hours after transfection and the cells were cultured until they were
subjected to experiment (at least, for 24h). In the shRNA-mediated knockdown experiments, the efficiency for
knockdown was confirmed at 72 hour after transfection by quantitative PCR and western blot. In addition to strong
apoptosis suppression by abr shRNA, a moderate suppression was observed with dbl (another Rho-GEF) shRNA.
Annexin V-staining, immunostaining and colony formation assay.
For the evaluation of apoptosis, cells were stained with fluorescence-conjugated Annexin-V/Propidium Iodide
(Biovision) and flow cytometric analysis was performed using FACSAria (BD Biosciences). In the analyses of the
exogenous protein-expressing cells, cell were cotransfected with a H2B-venus expression plasmid for identify the
transfected cells. Venus-negative non-transfected cells were omitted during FACS analysis for Annexin V. In some
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cases, non-transfected cells were selectively eliminated by puromaysin treatement as described above.
Immunostaining was performed as described previously (Watanabe et al., 2007; Eiraku et al., 2008). The cells were
fixed with 4% PFA at 4°C for 20 minutes. The staining was visualized using secondary antibodies conjugated with
AlexaFluor-488, -546 or -647 (Invitrogen). Experiments were performed at least three times.
Colony formation assay was performed as described before (Watanabe et al., 2007). hESC were
dissociated into single cells, seeded on MEF-coated 96 well plate (500 cells /well). Inhibitors were supplemented
during first 2 days, thereafter removed. Cells were cultured for additional 5 days with dairy medium change until
colonies were formed. Colonies were stained by the Alkaline Phosphatase for Leukocytes kit (Sigma) and the
obvious AP-positive colonies were counted. For Abr-depleted hES cells, the colony formation assay was started
using 2500 cells per well.
Commercial antibodies were purchased from Santa Cruz (RhoA, sc-418, sc-179; Hsc70, sc-7298), Cell
Signaling Technology (Akt, #9272; phospho-Akt, #9271; COX-4, #4844; MLC2, #3672; phospho-MLC2, #3674,
#3671; phospho-FAK, #3283), BD Pharmingen (cytochrome c, 556433; Rac1, 610650; ROCK-I, 611146;
ROCK-II, 610623; Paxillin, 610051; Abr, 611122; Oct-3, 611203), Takara (E-cadherin, M108, M126), Invitrogen
(V5, R960-25), Covance (NMMHC-IIC, PRB-444P), Upstate (Myc, 05-724; Phospho-tyrosine, 05-321X),
Nakalai (GFP, 04404-26), MBL (GFP, 598), Chemicon (TRA-1-60, MAB4360), Repro Cell (Nanog,
RCAB0003P) and Sigma (α-catenin, C2081; β-catenin, C2206; NMMHC-IIA, M8064; NMMHC-IIB, M7939;
Flag, F1804). Antibodies against ROCK-I (from M. Takeichi, RIKEN CDB, Nishimura and Takeichi, 2008) and
ROCK-II (Thumkeo et al., 2005) were also used. For F-actin staining, AlexaFluor-conjugated phalloidin
(Invitrogen) was used.
Rho/Rac pull-down assay, ROCK kinase assay, western blot and immunoprecipitation.
The Rho/Rac activity was evaluated with a GST pull-down assay. Cell lysate prepared in the MLB solution
(Upstate) was incubated at 4°C for 1 hour with Rhotekin-RBD (Upstate, for Rho activation) or Pak1-RBD (Upstate,
for Rac activation). ROCK kinase assay was performed using the ROCK activity immunoblot kit (Cell Biolabs).
For the detection of protein expression, cell lysates were made in RIPA lysis buffer (50 mM Tris-HCl pH 7.6, 1.0 %
TritonX-100, 0.1 % SDS, 150 mM NaCl, 1 mM EDTA, 0.5 % Sodium Deoxycholate and protease inhibitor
cocktail). For dissection of protein-protein interaction, cell lysates were made in NT lysis buffer (20 mM Tris-HCl
pH 7.6, 1.0 % TritonX-100, 0.5 % NP-40, 150 mM NaCl, 1 mM EDTA and protease inhibitor cocktail) and
immunoprecipitation was performed as described before. The cell lysates and immunoprecipitates were analyzed by
SDS-PAGE and sequential western blot. For detection of endogenous protein expression, MEF-feeder cells were
excluded as described above. Specifically, when the efficiency of shRNA-mediated knockdown of endogenous
proteins was evaluated by western blotting, hESC were once transferred onto the Matrigel in MEF-conditioned
medium to exclude the possible contamination of MEF-expressing proteins into hESC lysates.
Live imaging.
For the live single-cell imaging, dissociated cells were seeded onto a Matrigel-coated 35-mm glass bottom dish. The
recording was started at 15 min after the first contact with the dissociation reagent, since it took a few minutes to
dissociate, seeding, focusing on the cells as well as to select a proper recording field. For the EGTA experiments,
hESC, mESC, or mEpiSC 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
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medium. In the case of E-cadherin-knockdown experiment, the recording was started at 48 hour after the plasmid
transfection.
The cells were imaged on an inverted microscope (IX81-ZDC, Olympus) that was equipped with a
stepper filter wheel (Ludl), a cooled EM-CCD camera (ImagEM, Hamamatsu Photonics), 75W Xenon lamp and
the appropriate excitation and emission filters (Chroma). For confocal observation, the images were collected using
a CSU-X1 unit (Yokogawa) configured with an IX81-ZDC microscope. The obtained Images were analyzed with
MetaMorph 7.5 software (Molecular Devices). The blebbing movement and sequential cell death were similarly
observed on MEF or on collagen-coated dishes as well as on non-adhesive dishes (Lipidure-coated; NOF
CORPORATION).
Epiblast culture.
E6.0-6.25 embryos from pregnant female ICR mice were dissected in HEPES-buffered DMEM medium
(dissection medium). The proximal portion of the embryo was cut off at the boundary between the extra-embryonic
ectoderm and the epiblast and then the Reichert membrane was removed by manual dissection using tungsten
needles. The embryonic fragment containing the epiblast was incubated in 2 U/ml Dispase solution for 5 min at
37°C and subsequently returned to the dissection medium containing 20% KSR. The visceral endoderm was peeled
away to isolate the epiblast tissues. Then, the isolated epiblast was incubated in the TrypLE select solution for 10 min
at 37°C and then dissociated into single cells by gentle pipetting. The dissociated epiblast cells were suspended in the
hESC medium supplemented with recombinant human bFGF (10 ng/ml) and activin A (20 ng/ml) and seeded onto
a Matrigel-coated dish or a glass-bottomed dish (for imaging).
Culture on E-Cadherin-coated dishes.
35-mm petri dishes were coated with recombinant human E-Cadhein-Fc chimera protein (R&D systems) as
described previously (Nagaoka et al, 2006). Dissociated hESC were plated onto the coated dish and PlasDIC images
were obtained by an inverted microscope (Carl Zeiss).
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 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-hour point were
analyzed unless otherwise mentioned) with a post-hoc Tukey’s (among all groups) or Dunnett’s test (vs. control)
using the Prism 4 program (GraphPad).
Supplemental References
Nagaoka, M., Koshimizu, U., Yuasa, S., Hattori, F., Chen, H., Tanaka, T., Okabe, M., Fukuda, K., and Akaike, T.
(2006). E-cadherin-coated plates maintain pluripotent ES cells without colony formation. PLoS One 1, e15.
Nishimura, T., and Takeichi, M. (2008). Shroom3-mediated recruitment of Rho kinases to the apical junctions
regulates epithelial and neuroepithelial planar remodeling. Development 135, 1493-1502.
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Niwa, H., Masui, S., Chambers, I., Smith, A.G., and Miyazaki, J. (2002). Phenotypic complementation establishes
requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells. Mol.
Cell. Biol. 22, 1526-1536.
Thumkeo, D., Shimizu, Y., Sakamoto, S., Yamada, S., and Narumiya, S. (2005). ROCK-I and ROCK-II
cooperatively regulate closure of eyelid and ventral body wall in mouse embryo. Genes Cells. 10, 825-834.
Tsuruta, F., Masuyama, N., and Gotoh, Y. (2002). The phosphatidylinositol 3-kinase (PI3K)-Akt pathway
suppresses Bax translocation to mitochondria. J. Biol. Chem. 277, 14040-14047.
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