Human Reproduction, Vol.24, No.3 pp. 580–589, 2009
Advanced Access publication on December 4, 2008 doi:10.1093/humrep/den404
ORIGINAL ARTICLE Embryology
ROCK inhibitor improves survival
of cryopreserved serum/feeder-free
single human embryonic stem cells
Xiangyun Li1,2, Roman Krawetz1, Shiying Liu 1, Guoliang Meng 1,
and Derrick E. Rancourt 1,3
1
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Canada T2N 4N1 2College of Animal Science and
Technology, Agricultural University of Hebei, Baoding 071001, China
3
Correspondence address. Tel: þ1-403-2202888; Fax: þ1-403-2838727; E-mail: [email protected]
background: Efficient slow freezing protocols within serum-free and feeder-free culture systems are crucial for the clinical application
of human embryonic stem (hES) cells. Frequently, however, hES cells must be cryopreserved as clumps when using conventional slow freezing
protocols, leading to lower survival rates during freeze-thaw and limiting their recovery and growth efficiency after thawing, as well as limiting
downstream applications that require single cell suspensions. We describe a novel method to increase freeze-thaw survival and proliferation
rate of single hES cells in serum-free and feeder-free culture conditions.
methods: hES cells maintained on Matrigel-coated dishes were dissociated into single cells with Accutase and slow freezing. After
thawing at 378C, cells were cultured in mTeSR medium supplemented with 10 mM of Rho-associated kinase inhibitor Y-27632 for 1 day.
results: The use of Y-27632 and Accutase significantly increases the survival of single hES cells after thawing compared with a control
group (P , 0.01). Furthermore, by treatment of hES cell aggregates with EGTA to disrupt cell–cell interaction, we show that Y-27632 treatment does not directly affect hES cell apoptosis. Even in the presence of Y-27632, hES cells deficient in cell– cell interaction undergo apoptosis. Y-27632-treated freeze-thawed hES cells retain typical morphology, stable karyotype, expression of pluripotency markers and the
potential to differentiate into derivatives of all three germ layers after long-term culture.
conclusions: The method described here allows for cryopreservation of single hES cells in serum-free and feeder-free conditions and
therefore we believe this method will be ideal for current and future hES cell applications that are targeted towards a therapeutic end-point.
Key words: human embryonic stem cells / Rho-associated kinase inhibitor Y-27632 / cryopreservation / apoptosis
Human embryonic stem (hES) cells are derived from the inner cell
mass (ICM) of human blastocysts. They have the potential for
unlimited expansion with retention of normal karyotype and the
ability to differentiate into multi-lineage cell types from all three
germ layers (Thomson et al., 1998; Reubinoff et al., 2000). These
properties make hES cells an attractive cell source for a variety of
tissue regeneration applications, and therefore may offer a potential
treatment alternative for many non-curable degenerative diseases
and injuries.
Successful clinical implementation of hES cells-based technologies
will rely on the development of an efficient cryopreservation
method that utilizes serum-free and feeder-free culture conditions.
Current methods involve slow or fast freezing (vitrification) protocols
in the presence of cryoprotectants [dimethylsulphoxide (DMSO),
polyols, etc]. Vitrification (fast freeze, slow thaw) is extremely laborintensive and unsuited for handling bulk quantities of hES cells,
whereas conventional slow freezing (fast thaw) protocols have not
proven to be efficient for hES cells. Here, the typical survival rate is
c.10% (Reubinoff et al., 2001; Fujioka et al., 2004; Richards et al.,
2004; Zhou et al., 2004; Heng et al., 2006).
All current hES cell protocols rely on cryopreserving small clumps of
cells to improve survival rate as hES cells do not passage well as single
cells. However, there are complications to freezing clumps, one being
limitations on cryoprotectant exposure inside the clump. Indeed, only
a handful of cells in the clump may survive cryopreservation, but
remain attached to dead cells, causing a broader cell death response.
The use of hES cell clumps also prevents a good estimation of freezing/passaging efficiency, as precise cell numbers are never truly
known. Additionally, the passaging of hES cells as clumps prevents
other challenges for downstream manipulation. Examples include,
cell transfection, cell separation by flow cytometry and high throughput screening (Ruchi et al., 2008).
& The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Cryopreservation of single human embryonic stem cells
In contrast to clumps of hES cells, single hES cells are more vulnerable
to apoptosis upon cryopreservation (Watanabe et al., 2007). Using
current published methods single hES cells have not been successfully
cryopreserved, although many groups have reported low survival
rates freezing hES cell clumps (Reubinoff et al., 2001; Fujioka et al.,
2004; Richards et al., 2004; Zhou et al., 2004; Heng et al., 2006).
Recently, some reports showed that the application of a selective
Rho-associated kinase (ROCK) inhibitor, Y-27632 (Narumiya et al.,
2000), and Accutase, a formulated mixture of digestive enzymes, to
hES cell passaging permits survival of dissociated hES cells and markedly
increases cloning efficiency (Watanabe et al., 2007; Koyanagi et al., 2008;
Ruchi et al., 2008). In addition, many existing hES cell lines have been
maintained using mouse embryonic fibroblast (MEF) feeder cells and
animal serum medium. Although MEF and MEF-conditioned medium
support hES cells self-renewal, the use of animal-derived components
in hES cell culture precludes the use of these cells in clinical applications.
In the current study, we have demonstrated that Y-27632 treatment along with Accutase can be used to enhance the post-thaw survival rate of single hES cells using conventional slow freezing and rapid
thawing protocols in serum-free and feeder-free culture conditions.
densities. On the first day after replating, the medium was supplemented
with 10 mM of Y-27632. From the second day onward, the medium was
changed daily without Y-27632 supplementation. The experimental
design consisted of five freeze-thaw events that took place within 15 passages of the H9 cells in serum-free and feeder-free culture conditions. The
analysis presented in this study, including karyotype, immunofluorescence,
and in vitro and in vivo differentiation potential analysis, was carried out
after 15 passages.
Cell adhesion assays
Cell adhesion assays were performed using the Vybrant Cell Adhesion
Assay Kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions with minor modifications. Briefly, single hES cells
were added to a Matrigel-coated microplate at a density of 1 105 per
well. After 12 h the media, with or without 10 mM of Y-27632, was
replaced with fresh media containing 5 mM of calcein acetoxymethyl
ester and incubated at 378C for 30 min. Non-adherent cells were
removed by gently washing twice with Dulbecco’s Modified Eagle’s
Medium (DMEM)/F12 medium. Adherent cells were quantified using a fluorescence microplate reader (Fluoroskan Ascent, Thermo Labsystems) at
494 nm excitation and 517 nm emission. All experiments were repeated at
least five times. Statistical analysis was performed using the Student’s
t-test. Values of P , 0.05 were considered significant.
Materials and Methods
Cells and animals
The present study adheres to the guidelines for hES cell research established by the Government of Canada and the Canadian Institutes of
Health Research. All experimental protocols and animal handling procedures were reviewed and approved by the Laboratory Animal Care
and Use Committee of the University of Calgary.
Karyotype analysis
Karyotype analysis of hES cells was carried out using the G-banding
method. Briefly, cells were incubated with 0.05 mg/ml of colcemid at
378C for 35 min, trypsinized, resuspended and incubated in 0.56% KCl
for 25 min at 378C, then fixed with 3:1 methanol:glacial acetic acid
three times and then dropped onto slides to spread the chromosomes.
The slides were baked overnight at 558C, then treated with 0.05%
trypsin for 90 s and stained with Giemsa and Leishman’s solution.
Maintenance and passaging of hES cells
H9 hES cells (WiCell Research Institute) were maintained on MEF feeder
cells and transferred onto Matrigel (BD Biosciences) -coated 35 mm dishes
(Nunc) in mTeSR1 media (StemCell Technologies Inc.) following the manufacturer’s instructions. For passaging, hES cells were first washed twice
with Dulbecco’s phosphate-buffered saline (DPBS) without calcium and
without magnesium (Invitrogen) and then dissociated with Accutase (Chemicon) cell detachment solution in a 378C incubator for 8 min. After
gentle pipetting, the cells were collected by centrifugation and replated
on Matrigel-coated dishes at different densities. On the first day after
replating, the culture medium was supplemented with 10 mM of
Y-27632. From the second day onward, no Y-27632 was added and the
medium was changed daily. Cells were passaged as soon as they
reached 80% confluence.
Freezing and thawing of hES cells
hES cell colonies were washed twice with DPBS followed by Accutase
treatment in a 378C incubator for 8 min and gentle pipetting to dissociate
the colonies into single cells. The cells were collected by centrifugation and
resuspended in pre-cooled freezing medium consisting of 90% knockout
serum replacement (Invitrogen) and 10% DMSO. The hES cells and freezing medium were transferred into cryovials (Nunc), and immediately
placed into a freezing container (Nalgene) containing pre-cooled isopropyl
alcohol as cooling agent and then transferred to a 2808C industrial
freezer. After overnight at 2808C, the cryovials were plunged into and
stored in liquid nitrogen. One week later, the cryovials were rapidly
thawed in a 378C water bath. After removing the freezing medium by centrifugation, cells were replated on Matrigel-coated dishes at different
Apoptosis analysis
The apoptosis of hES cells was detected using an Annexin V-fluorescein
isothiocyanate (FITC) Apoptosis Detection Kit (BD Pharmingen) following
the manufacturer’s instructions. Briefly, single cells were harvested and
resuspended in binding buffer at a concentration of 1 106 cells/ml.
One hundred microliters of the solution was transferred to a 5 ml
culture tube and add 5 ml of Annexin V-FITC and 5 ml propidium iodide
(PI). The cell suspension was incubated for 15 min at room temperature
in the dark then analyzed by flow cytometry (FACS Calibur, BD
Biosciences). Differences between treatment groups were assessed by
Student’s t-test and values of P , 0.05 were considered significant.
Immunofluorescence
The characterization of hES cells was carried out by measuring the
expression of specific pluripotent markers including, alkaline phosphatase
(AKP), stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor rejection
antigens (TRA)-1-60, TRA-1-81 and octamer 4 (OCT-4). The immunostaining was done according to published procedures. Briefly, colonies
were fixed in 4% paraformaldehyde for 30 min at room temperature, permeablized with 0.1% Triton-X 100 in DPBS for 15 min at room temperature, washed three times with DPBS and incubated with DPBS containing
10% normal goat serum for 30 min at room temperature. Primary antibody (1:100) was applied at 48C overnight. The negative control consisted
of mouse immunoglobulin (Ig)G2b (Caltag, Burlingame, CA, USA) at the
same concentration as the primary antibody. After washing three times
with DPBS, cells were incubated with either FITC-conjugated goat antimouse IgG (HþL) (1:100, Jackson Immunoresearch Labs) or Alexa
582
Fluorw 546 goat anti-mouse IgG (HþL) (1:300, Invitrogen). Histochemical
staining for AKP was carried out using a commercially available kit following the manufacturer’s instructions (Chemicon).
In vitro and in vivo differentiation
Multilayer hES cell clumps were aggregated using 35 mm agar-coated
dishes (Nunc) to produce embryoid bodies (EBs) in suspension culture
which were then cultured in differentiation medium. The differentiation
medium consisted of 80% DMEM (Invitrogen), 20% fetal bovine serum
(Invitrogen), 1 mM L-glutamine, 0.1 mM b-mercaptoethanol and 0.1 mM
non-essential amino acids. After 7 – 14 days, the cells aggregated and generated cystic EBs. RT – PCR analysis on undifferentiated cells, Day 7 and
Day 14 EBs were performed by using primers specific for the three
germ layers as previously described (Yoo et al., 2005), namely, ectoderm
(neurofilament-68 and keratin), mesoderm (enolase and kallikrein) and
endoderm (a-fetoprotein and a1-antitrypsin). Also, the EBs of Day 14
were transferred onto gelatin-coated 4-well plates (Nunc) and cultured
for an additional 4 days. The resulting cell types were analyzed by immunofluorescence using monoclonal antibodies against ectoderm (b-tubulin
III, Sigma, 1:1000), mesoderm (smooth muscle actin, Sigma, 1:1000) and
endoderm (a-fetoprotein, Sigma, 1:1000) as previously described (Li
et al., 2005). In addition, cells were injected into severe combined
immunodeficient-beige mice (5 106 cells per injection site) in order to
examine the in vivo differentiation potential of the freeze-thawed hES
cells. Teratomas were removed after 6 weeks, fixed overnight in 4% paraformaldehyde, embedded in paraffin, sectioned and examined histologically after being stained with eosin and haematoxylin.
Influence of EGTA on hES cells treated with
the rock inhibitor Y-27632
EGTA was added into mTeSR medium to chelate extracellular calcium
ions. The hES colonies were dissociated into single cells by Accutase
and replated on Matrigel-coated and agar-coated dishes at a density of
1 106 cells per 35 mm dish. Agar-coated dishes were prepared as
follows: a 1% agar solution was autoclaved and allowed to slightly cool
at room temperature, then applied to culture dishes to completely coat
the bottom of the dish with the excess being aspirated immediately.
The thin coat of agar was allowed to solidify and was then ready for
use. The media were supplemented with different concentrations of
EGTA (1 mM; 2 mM; 5 mM) and 10 mM of Y-27632. Each treatment
group was cultured for 24 h at 378C and the cell attachment in Matrigelcoated dishes, and cell aggregation in agar-coated dishes, was observed.
Results
ROCK inhibitor permits survival of single hES
cells after cryopreservation
In this study, hES cells were freeze-thawed for five rounds during 15
passages as single cell suspensions. When frozen cells were thawed
and cultured, most cells in the Y-27632 treatment group attached
well to the culture dishes (Fig. 1A –C), while fewer colonies formed
without Y-27632 treatment (Fig. 1D). When 5 105 hES cells were
plated per dish with Y-27632 treatment, the cells reached 80% confluence by Day 3 after thawing (Fig. 1K), while fewer colonies are
observed in the non-treated control group (Fig. 1L). To control for
effects of freezing not regulated by Y-27632 we compared all
freeze-thawed cells to a non-cryopreserved, passaged hES group on
Day 1 and Day 3 after replating (Fig. 1E– H and M –P, respectively).
Li et al.
The passaged cells demonstrated a similar response to Y-27632 treatment with increased growth rates. Although it was clear that the duration of each passage depended on the initial plating density, when
initial cell density was between 1 105 and 5 105 cells per dish,
the passage duration was 3–5 days, with plates not allowed to
exceed 80% confluence. When compared with control groups
(Fig. 1D and H), Y-27632 treatment increased the attachment of
single hES cells to the dish surface under the same seeding density
(Fig. 1C and G). These attached cells merged and formed monolayer
colonies (Fig. 1K and O). Using the Vybrant Cell Adhesion Assay Kit,
we quantified the increased hES cell attachment observed with 10 mM
of Y-27632 supplementation. In the presence of Y-27632, approximately 10 times as many hES cells remained attached to the culture
vessel after washing with fresh medium. We obtained mean (+SD)
values of 2.61 + 0.68 without Y-27632 treatment, compared with
20.63 + 3.04 with 10 mM of Y-27632. Since treatment with the
ROCK inhibitor increased the number of attached cells present after
freeze-thawing, we next decided to tease out the mechanism behind
this observation.
ROCK inhibitor Y-27632 increases adherent
properties of hES cells
When hES cells are cultured for 1 h in the presence of 10 mM of
Y-27632 on Matrigel-coated dishes, we found that hES cells become
more adherent, as it required more time in the presence of Accutase
to detach cells from the culture dishes. When the cells reached 80%
confluence and required passaging, they normally require 8 min of
Accutase treatment at 378C to completely dissociate into single
cells. However, cells treated with Y-27632 required 16 min of Accutase treatment at 378C for complete dissociation. To elucidate the
mechanism behind this observation, hES cells were treated with
EGTA to chelate calcium in the presence or absence of Y-27632 to
disrupt cadherin activity. As a note, mTeSR medium contains 1 mM
calcium, and as one molecule of EGTA binds four calcium ions,
1 mM EGTA should be sufficient to sequester most extracellular
calcium. After 24 h on Matrigel-coated dishes, cells attached and
formed monolayer colonies with Y-27632 treatment (Fig. 2A). The
addition of 1 mM EGTA did not affect the cell to dish attachment,
however, the attached cells were far more dispersed than the
Y-27632 treatment group alone (Fig. 2B). As expected, EGTA-treated
cells displayed reduced cell–cell attachment and hES cells had a
reduced colony formation capacity compared with control groups.
When EGTA was removed from medium, the effect was reversed
and cells adhered to each other with the colonies becoming tight
and compact. However, fewer cells attached with 2 mM (Fig. 2C)
and 5 mM (data not shown) of EGTA supplement. At higher concentrations of EGTA we can assume that intercellular calcium levels are
severely affected.
To further study the effect of cell–cell attachment on cell survival,
we cultured post-thawed cells in suspension culture prior to replating.
When single hES cells are plated in agar-coated dishes, the cells do not
adhere and will normally undergo apoptosis. However, in the presence of Y-27632, the hES cells aggregate into small clumps which
are viable. When post-thawed single hES cells were cultured in suspension for 24 h on agar-coated dishes, no viable cells remained
(Fig. 2D). However, when the medium was supplemented with
Cryopreservation of single human embryonic stem cells
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Figure 1 The attachment and morphology of freeze-thawed and regularly passaged H9: human embryonic stem (hES) cells at different replating
densities with or without Y-27632 treatment.
The plating densities used were 1 105 cells/dish (A, E, I and M), 3 105 cells/dish (B, F, J and N), 5 105 cells/dish with Y-27632, the
Rho-associated kinase (ROCK) inhibitor, treatment (C, G, K and O) and 5 105 cells/dish without Y-27632 treatment (D, H, L and P).
Freeze-thawed cells, were froze (one week) as single cells, rapidly thawed, and seeded on Matrigel-coated 35 mm dishes. Passaged control cells
were dissociated into single cells and immediately plated on Matrigel-coated 35 mm dishes. Day 1 and Day 3 represent days after replating in cryopreserved and passaged H9 cells. Scale bar indicates 100 mm.
10 mM of Y-27632, small aggregates formed and viability increased
(Fig. 2E). The aggregates were tight, spherical, smooth, and were difficult to dissociate into single cells by Accutase. When EGTA 1 mM
was added into medium with Y-27632, aggregates were present, but
they did not form a compact structure, with individual cells clearly
visible within each aggregate (Fig. 2F, inset). The aggregates formed
in the presence of EGTA required only gentle pipetting to dissociate
and most importantly produced no viable colonies when replated
onto Matrigel-coated dishes. Because of recent reports concerning
the ROCK inhibitor and reduction of apoptosis (Watanabe et al.,
2007; Koyanagi et al., 2008), our observation prompted us to
explore the effect of Y-27632 on apoptosis seen during the
freeze-thaw of hES cells.
ROCK inhibitor protects single hES cells
from apoptosis after cryopreservation
Freeze-thawed single hES cells treated with or without Y-27632 were
cultured for 24 h on Matrigel-coated and agar-coated dishes and subjected to apoptosis analysis using Annexin V-FITC Apoptosis
Detection Kit (Fig. 3). The apoptosis analysis was repeated at least
three times and demonstrated consistent results. In the passaged
control cells that were not cryopreserved, 80.34% cells were viable
(both Annexin V-FITC and PI negative), 5.90% cells were in early
apoptosis (Annexin V-FITC-positive and PI-negative) and 11.07%
cells were in late apoptosis or dead (both Annexin V-FITC and PIpositive; Fig. 3A). This is in contrast to hES cells that had been
freeze-thawed without ROCK inhibitor and not cultured after freezing
prior to analysis, where only 65.53% cells were viable, 15.70% cells
were in early apoptosis and 16.80% cells were in late apoptosis or
dead (Fig. 3B).
We next tested the effect of ROCK inhibitor on apoptosis when
cells where cultured in agar-coated dishes for 24 h after freeze-thaw
before analysis. When hES cells were cultured without ROCK inhibitor
in suspension for 24 h, 0.72% cells were viable, 5.81% cells were in
early apoptosis and 92.54% cells were dead (Fig. 3C). However,
when the suspension culture was supplemented with 10 mM of
Y-27632, cells adhered together and formed compact aggregates
(Fig. 2E). The aggregates were dissociated into single cells by Accutase
and immediately subjected to apoptosis analysis. Data showed that
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Li et al.
Pluripotency of cryopreserved and passaged
single hES cells
Figure 2 Effect of EGTA on hES cell attachment.
Cryopreserved H9 cells were replated directly after thawing (A – C)
or cultured in suspension (D – F) for 24 h in the presence (B, C
and F) or absence (A, D and E) of EGTA. Cells were plated at a
density of 1 106 cells/dish and cultured for 1 day on Matrigelcoated (A– C) or agar-coated dishes (D– F). With the exception of
(D) all medium was supplemented with 10 mM of Y-27632. EGTA
was added to (B) (1 mM), (C) (2 mM) and (F) (2 mM). The inset in
(F) is an enlarged representation taken from the center of the field
of view of the same panel. Scale bar indicates 100 mm.
53.03% cells were viable, 14.76% cells were in early apoptosis and
27.29% cells were dead (Fig. 3D). Statistical analysis demonstrated
that Group A versus Group B, and Group C versus Group D, were
significant (P , 0.01). Therefore, more hES cells were viable with
Y-27632 treatment compared with the non-cryopreserved or
freeze-thaw groups without Y-27632 treatment. Y-27632 significantly
decreased apoptosis and enhanced the post-thaw survival rate of
cryopreserved single hES cells.
In order to confirm that the hES cells maintain pluripotent characteristics after slow freezing as single cells in serum-free and feeder-free
culture conditions, we examined six key pluripotent markers, karyotype, and the in vitro and in vivo differentiation potential of hES cells
after five rounds of freeze-thaw. hES cells maintained their characteristic undifferentiated morphology, and immunofluorescence staining
for the key markers of pluripotent hES cells (AKP, OCT-4, SSEA-3,
SSEA-4, TRA-1-60, TRA-1-81) confirmed that cells within hES cell
colonies were positive for these markers (Fig. 4). The undifferentiated
cells proliferated and formed characteristic undifferentiated hES cell
colonies when they were replated on Mitomycin C-treated MEF
feeder layer (Fig. 5A).
For in vitro differentiation examination, the hES cells were
re-cultured on MEF feeder layers to form multilayer colonies. These
colonies were cut into small clumps and cultured in agar-coated
dishes. After 7–14 days in suspension, the cells aggregated and generated cystic EBs (Fig. 5B). RT–PCR analysis was performed on RNA
extracted from undifferentiated cells, Day 7 EBs and Day 14 EBs
(Fig. 5C). The mRNA levels for specific genes for three germ layers
(neurofilament-68 for ectoderm, enolase and kallikrein for mesoderm,
a-fetoprotein and a1-antitrypsin for endoderm) increased during
differentiation (Fig. 5C). The EBs from Day 14 were transferred
onto gelatin-coated 4-well plates and cultured for an additional 4
days. At this point, the EBs were positively stained for b-tubulin III
(ectoderm), smooth muscle actin (mesoderm) and a-fetoprotein
(endoderm) (Fig. 5D– F).
In order to examine the in vivo differentiation potential of the
freeze-thawed hES cells, they were injected into severe combined
immunodeficient-beige mice and teratomas were removed after 6
weeks and examined histologically. Rosettes of neural epithelium
Figure 3 Flow cytometric analysis of apoptotic H9 cells using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI).
After cryopreservation regularly passaged control cells (A), freeze-thawed hES cells without further culture (B), freeze-thawed cells with a further 24 h
culture on agar-coated dishes (C), freeze-thawed cells with a further 24 h culture on agar-coated dishes with 10 mM of Y-27632 supplement (D), were
subjected to apoptosis detection using flow cytometry. For (A – D), quadrant III indicates that the cells are viable and not undergoing apoptosis (both
Annexin V-FITC and PI negative). Quadrant IV indicates that the cells are in an early stage of apoptosis and are still viable (Annexin V-FITC positive and
PI negative). Quadrant I indicates that the cells are in a later stage of apoptosis (no longer viable) or already dead (both Annexin V-FITC and PI positive). The flow cytometry data is also presented in chart form (E). Group A versus Group B, Group C versus Group D, were significant (P , 0.01).
Values shown on graphs represent the mean + SD.
Cryopreservation of single human embryonic stem cells
585
Figure 4 Pluripotent marker expression in freeze-thawed H9 cells cultured on Matrigel-coated dishes.
H9 cells after freeze-thaw with Y-27632 were stained for alkaline phosphatase (A), octamer 4 (B), stage-specific embryonic antigen (SSEA)-3 (C),
SSEA-4 (D), tumor rejection antigen (TRA)-1-60 (E) and TRA-1-81 (F). Scale bar indicates 100 mm.
(ectoderm, Fig. 6A), adipose cells (mesoderm, Fig. 6B) and gut-like
epithelium (endoderm, Fig. 6C) were all observed indicating pluripotency of the injected cells. Karyotye analysis of hES cells was performed using G-banding method. These cells exhibited normal
karyotypes and a representative karyotype from H9 cells is shown
in Fig. 6D. Our data strongly suggest that hES cells maintain the characteristic morphology, normal karyotype and pluripotent potential after
cryopreservation as single cells and long-term culture in serum-free
and feeder-free culture conditions.
Discussion
Apoptosis is a regulated physiologic process which occurs during
embryonic development and is seen within maintenance of tissue
homeostasis and self renewal, where it plays very important roles in
cell proliferation and differentiation. Embryonic stem cells removed
from the developing ICM can be cultured in vitro under specific
culture conditions, however, hES cells are very sensitive to their
environment including medium composition, temperature, CO2
levels, pH and attachment (Ludwig et al., 2006). When these environmental factors fall outside of a narrow window we observe dramatic
cell death. Even during regular passaging of hES cells we can always
observe significant cells in suspension and dead single cells. If regular
maintenance of hES cells results in significant apoptosis and cell loss
then it serves that cryopreservation, dissociation and mechanical
manipulation induce significant apoptosis, and can render a culture
non-viable (Katkov et al., 2006).
Recent reports have demonstrated that Y-27632 is a potent inhibitor of apoptosis and permits the survival of dissociated hES cells
(Watanabe et al., 2007; Koyanagi et al., 2008). In these studies both
groups determined that apoptosis of dissociated hES cells was markedly decreased with Y-27632 treatment in vitro and in vivo. Moreover,
recent papers by Peerani et al. (2007) and Harb et al. (2008) have
brought to light the role of ROCK within the maintenance of hES
cell pluripotency. Peerani et al. (2007) demonstrated that Y-27632
treatment increased levels of Oct-4 expression, and Harb et al.
(2008) demonstrated that hES cells can be grown without the need
for niche-forming feeder layers or animal-derived matrices with the
addition of Y-27632. Although, we do not directly demonstrate
increased pluripotency with Y-27632 treatment, we do observe
Oct-4 staining consistent with un-frozen controls in our study and
decreased apoptosis with Y-27632 treatment.
In this study, after freeze-thawed cells were cultured in suspension
for 24 h, we found dramatic differences in the level of apoptosis in
cryopreserved cells with and without Y-27632 treatment (Fig. 3C
and D). Our results demonstrate that suspension culture can induce
cell apoptosis of hES cells and, consistent with previous reports
(Watanabe et al., 2007; Koyanagi et al., 2008), we found that
Y-27632 treatment decreased the level of apoptosis. When regularly
passaged hES cells were dissociated into single cells and immediately
subjected to apoptosis analysis, a small percentage of cells were
dead and/or apoptotic (Fig. 3A), with this cell death most likely
induced by Accutase dissociation. When these dissociated single
cells were cryopreserved in liquid nitrogen (for 1 week), thawed
and then immediately subjected to apoptosis analysis, we observed
an increase in cell mortality (Fig. 3B), however, these results signify
only a small percentage of cells died as a result of cryopreservation.
Other groups have demonstrated in mouse ES cells that Accutase
dissociation and single cell suspension induces more cell death than
that of cryopreservation alone in the recovery of freeze-thawed hES
cells (Koyanagi et al., 2000). Moreover, this result by itself does not
explain why we observed fewer viable cells after freeze-thaw, although
most of the freeze-thawed cells were viable (Fig. 3B), and fewer cells
can attach and form colonies when they were replated and cultured
for 24 h (Fig. 1D). Whereas apoptosis is the major cause of the low
survivability of hES cells after freezing-thawing (Heng et al., 2006),
we believe that hES cells are sensitive to anokis (detachment
induced apoptosis), and that the increased levels of apoptosis we
and others have observed in suspension culture without Y-27632
treatment results from the loss of adherence to the substrate.
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Li et al.
Figure 5 In vitro differentiation of freeze-thawed H9 cells.
hES cells were replated on a mouse embryonic fibroblast feeder layer and formed multilayer undifferentiated colonies (A). Embryoid bodies (EBs) after
14 days suspension culture (B). RT– PCR analysis of undifferentiated H9 cells, and Day 7 and Day 14 EBs during in vitro differentiation using primers
specific for markers in all three germ layers, namely, endoderm (a-fetoprotein and a1-antitrypsin), mesoderm (enolase and kallikrein) and ectoderm
(neurofilament-68 and keratin). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. Immunofluorescence of
a-fetoprotein (D), smooth muscle actin (E) and b-tubulin III (F) in EBs after 18 days suspension culture. Scale bar indicates 100 mm.
However, with Y-27632 treatment we do not see apoptosis in suspension culture after cryopreservation. In addition, Y-27632 treatment
allows for stronger cell –cell interaction between hES cells, which is
evident during Accutase dissociation, and aggregate formation in suspension culture (Fig. 2E).
Our data indicates that increased cellular adhesion induced by
Y-27632 enhances the survival of single hES cells in feeder-free and
serum-free culture conditions as well as single cell cryopreservation.
The potential of Y-27632 to enhance cellular adhesion has also
been observed in other cell types, for example, adhesion of human
trabecular meshwork cells to fibronectin or collagen type I was
increased by the addition of Y-27632 (Koga et al., 2006). Y-27632
is a selective inhibitor of ROCK (Rho kinase). Rho accepts signals
from G-protein-coupled receptors as well as other signaling pathways
that originate in the extracellular matrix (ECM) as well as intracellularly
(Leung et al., 1996; Amano et al., 1997; Ishizaki et al., 1997; Kawaguchi
et al., 2000; Honjo et al., 2001). Rho activation of ROCK leads to the
phosphorylation of a number of downstream targets which are
involved in diverse signaling pathways.
To generalize one role of ROCK is that this protein transduces
signals from the cortical actin cytoskeleton and ECM to the nucleus,
leading to changes in cell morphology as well as transcriptional regulation. We hypothesize that by interrupting signals from the cellular
environment by inhibiting ROCK, hES cells are no longer aware of
their current environment. Therefore, hES cells treated with ROCK
inhibitor are no longer able to detect that they have been dissociated
Cryopreservation of single human embryonic stem cells
587
Figure 6 In vivo differentiation and karyotype of cryopreserved H9 cells.
In vivo teratoma analysis of cryopreserved H9 cells demonstrates that the hES cells are capable of differentiating into tissue types derived from all three
germ layers (A– C). Rosettes of neural epithelium (ectododerm, A), adipose (mesoderm, B); gut like epithelium (endoderm, C). Karyotype analysis of
hES cells after cryopreservation shows that they have a normal chromosome complement of 46XX (D). Scale bar indicates 100 mm.
into single cells or the current state of the plasma membrane or actin
cytoskeleton during/after cryopreservation. In effect we do not
believe that Y-27632 blocks apoptotic pathways but more accurately
causes hES cells to become ‘blind’, ‘deaf’ and ‘dumb’ in respect to
their current environment, and this naivety allows hES cells to aggregate into small colonies and this cell –cell interaction is responsible
for the reduction in apoptosis observed in Y-27632 treatment
groups. This hypothesis is supported when Y-27632 suspension aggregates that are normally observed after cryopreservation can be dissociated with the addition of EGTA and gentle pipetting.
EGTA is a chelating agent that has been employed for many
decades for sequestering free calcium within a micro-environment.
Calcium, a co-factor for many proteins is essential for the proper function of classical cadherin molecules. Cadherins play an important role
in cell and developmental biology, where they bind only to identical
cadherin molecules bound within the plasma membrane of adjoining
cells. These homophillic interactions are responsible for cell –cell interactions between cells/tissues expressing similar cadherins. In development, cadherin molecules are responsible for the correct patterning of
many tissues and organs. With the addition of EGTA to Y-2732 treatment groups to chelate calcium, thereby inactivating cadherins, we
observed no viable cells present after replating. This result signifies
that Y-27632 does not block apoptosis in dissociated hES cells, but
allows cells to aggregate and escape anokis. We recognize that
more testing is required to support this hypothesis, however, our
current and previous studies point in this direction. Aside from
Y-27632 treatment, initial cell plating density affects proliferation of
hES cells.
There are approximately 5 106 hES cells on a 90% confluent
35 mm dish. When the cells are dissociated, frozen, thawed and
replated at a density of 1 105 cells per 35 mm dish, the split rate
will be 1:50. These cells can be re-passaged after 5 days culture.
This high proliferation efficiency demonstrates that hES cells have
been successfully cryopreserved as compared with other cell lines
using the standard slow freezing and rapid thawing protocols. The
time of passaging depended on the initial plating density. At the
same densities, the passaging time of freezing-thawing single cells
was shortened by 0.5 days compared with regularly passaged hES
588
cells. Increasing cell plating density showed that more cells will attach,
merge and then accelerate monolayer colony formation (Fig. 1). The
formed monolayer colonies on Matrigel express pluripotent markers
and maintain differentiation potential similar to multilayer colonies
maintained on a MEF feeder layer (Li et al., 2008). No adverse
effect of monolayer cultures on hES cell viability or pluripotency was
detected, and is consistent with previous reports (Ruchi et al., 2008).
In addition to Y-27632 treatment effects, we also believe that Accutase increased viability rates due to quicker dissociation times. Accutase is a solution of proteolytic, collagenolytic enzymes without
mammalian or bacterial-derived products and increased cell survival
is obtained when Accutase, instead of trypsin, was used for enzymatic
dissociation of neural stem cell cultures (Wachs et al., 2003). Using
Accutase for passaging cells reduced cell death and improved culturing
conditions (Ruchi et al., 2008). The cooperation of ROCK inhibitor
Y-27632 and Accutase dissociation significantly increase the survival
rate of single hES cells in cryopreserved or regularly passaged cells.
We noted that undifferentiated and differentiated hES cells
expressed similar mRNA amounts of the germ layer-specific genes
neurofilament-68 (ectoderm) and enolase (mesoderm), however,
this has also been reported in a previous study (Yoo et al., 2005).
Moreover, we can find a small quantity of differentiated cells in
Fig. 4B, as evident by their lighter immunostaining and irregular cell
nuclei. Quantitative analysis of the hES cell surface markers (SSEA-3,
SSEA-4, TRA-1-60, TRA-1-81, OCT-4) by fluorescence-activated
cell sorting also demonstrated that apparent undifferentiated hES
cell colonies always contain small amounts of differentiated cells
(Katkov et al., 2006; Liu et al., 2006; Ludwig et al., 2006; Sidhu and
Tuch, 2006). The cryopreservation, passaging and maintenance
culture of hES cells is always accompanied by a degree of spontaneous
differentiation (Reubinoff et al., 2001; Fujioka et al., 2004; Richards
et al., 2004; Zhou et al., 2004; Heng et al., 2006; Katkov et al.,
2006; Li et al., 2008), which results from numerous accumulated
subtle negative impacts induced by all manipulations in vitro.
However, Y-27632 treatment does not induce the spontaneous differentiation of hES cells based on our present and previous data (Li et al.,
2008), as well as other groups (Watanabe et al., 2007; Martin-Ibañez
et al., 2008). We have not observed obvious adverse effects of
Y-27632 treatment on pluripotent marker expression in maintenance
culture even after a substantial number of passages. Although the H9
cell line was described in this study, Y-27632 treatment also had a
similar effect on the cryopreservation and passaging of H1, CA1 and
CA2 hES cell lines (data not shown). Further study is required to
reveal whether subtle genetic alterations can be induced by the
single cell cryopreservation, Accutase dissociation and Y-27632 treatment. In the present study, single hES cells can be effectively cryopreserved and propagated by incorporating the ROCK inhibitor Y-27632
and Accutase into the standard slow freezing and rapid thawing protocol in serum-free and feeder-free culture system. A recent study
has also demonstrated that Y-27632 increased the survival rate of
cryopreserved hES cells (Martin-Ibañez et al., 2008); however, we
have demonstrated that Y-27632 treatment is also effective in
serum/feeder-free conditions, and we have also described a partial
mechanism for the increased survival observed in Y-27632-treated
cultures. Furthermore, we have demonstrated that following
Y-27632 treatment, the hES cells can be cryopreserved and passaged
using similar methods to mouse ES cells and transformed cell lines.
Li et al.
Throughout repeated freeze-thaw events and long-term culture, hES
cells retain the key properties such as, typical morphological characteristics, marker expression (AKP, SSEA-3, SSEA-4, TRA-1-60,
TRA-1-81, OCT-4), normal karyotype and the potential to differentiate into derivatives of all three germ layers. The convenient, reliable
and effective cryopreservation and propagation of single hES cells
provide a standard in vitro manipulation protocol and bring a bright
future for the clinical application of hES cells.
Acknowledgements
The authors would like to thanks Dr Peter Andrews (The University of
Sheffield) for providing antibodies for immunofluorescence.
Funding
This work was supported by the Canadian Stem Cell Network and a
Canadian Institutes of Health Research Regenerative Medicine Team
Grant. D.E.R. is a Senior Scholar of the Alberta Heritage Foundation
for Medical Research.
References
Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, Matsuura Y,
Kaibuchi K. Formation of actin stress fibers and focal adhesions
enhanced by Rho-kinase. Science 1997;275:1308– 1311.
Fujioka T, Yasuchika K, Nakamura Y, Nakatsuji N, Suemori H. A simple
and efficient cryopreservation method for primate embryonic stem
cells. Int J Dev Biol 2004;48:1149 – 1154.
Harb N, Archer TK, Sato N. The Rho-Rock-Myosin signaling axis
determines cell – cell integrity of self-renewing pluripotent stem cells.
PLoS ONE 2008;3:e3001.
Heng BC, Ye CP, Liu H, Toh WS, Rufaihah AJ, Yang Z, Bay BH, Ge Z,
Ouyang HW, Lee EH et al. Loss of viability during freeze-thaw of
intact and adherent human embryonic stem cells with conventional
slow-cooling protocols is predominantly due to apoptosis rather than
cellular necrosis. J Biomed Sci 2006;13:433 – 445.
Honjo M, Tanihara H, Inatani M, Kido N, Sawamura T, Yue BY,
Narumiya S, Honda Y. Effects of rho-associated protein kinase
inhibitor Y-27632 on intraocular pressure and outflow facility. Invest
Ophthalmol Vis Sci 2001;42:137 – 144.
Ishizaki T, Naito M, Fujisawa K, Maekawa M, Watanabe N, Saito Y,
Narumiya S. p160ROCK, a Rho-associated coiled-coil forming protein
kinase, works downstream of Rho and induces focal adhesions. FEBS
Lett 1997;404:118– 124.
Katkov II, Kim MS, Bajpai R, Altman YS, Mercola M, Loring JF, Terskikh AV,
Snyder EY, Levine F. Cryopreservation by slow cooling with DMSO
diminished production of Oct-4 pluripotency marker in human
embryonic stem cells. Cryobiology 2006;53:194 – 205.
Kawaguchi A, Ohmori M, Harada K, Tsuruoka S, Sugimoto K, Fujimura A.
The effect of a Rho kinase inhibitor Y-27632 on superoxide production,
aggregation and adhesion in human polymorphonuclear leukocytes. Eur J
Pharmacol 2000;403:203 – 208.
Koga T, Koga T, Awai M, Tsutsui J, Yue BY, Tanihara H. Rho-associated
protein kinase inhibitor, Y-27632, induces alterations in adhesion,
contraction and motility in cultured human trabecular meshwork cells.
Exp Eye Res 2006;82:362 – 370.
Koyanagi R, Akimitsu N, Arimitsu N, Hatano T, Sekimizu K. Apoptosis of
mouse embryonic stem cells induced by single cell suspension. Tissue
Cell 2000;32:66 – 70.
Cryopreservation of single human embryonic stem cells
Koyanagi M, Takahashi J, Arakawa Y, Doi D, Fukuda H, Hayashi H,
Narumiya S, Hashimoto N. Inhibition of the Rho/ROCK pathway
reduces apoptosis during transplantation of embryonic stem
cell-derived neural precursors. J Neurosci Res 2008;86:270 – 280.
Leung T, Chen XQ, Manser E, Lim L. The p160 RhoA-binding kinase ROK
alpha is a member of a kinase family and is involved in the reorganization
of the cytoskeleton. Mol Cell Biol 1996;16:5313– 5327.
Li Y, Powell S, Brunette E, Lebkowski J, Mandalam R. Expansion of human
embryonic stem cells in defined serum-free medium devoid of
animal-derived products. Biotechnol Bioeng 2005;91:688 – 698.
Li X, Meng G, Krawetz R, Liu S, Rancourt DE. The ROCK Inhibitor
Y-27632 Enhances the Survival Rate of Human Embryonic Stem Cells
Following Cryopreservation. Stem Cells Dev 2008;17:1 – 8.
Liu Y, Song Z, Zhao Y, Qin H, Cai J, Zhang H, Yu T, Jiang S, Wang G,
Ding M et al. A novel chemical-defined medium with bFGF and
N2B27 supplements supports undifferentiated growth in human
embryonic stem cells. Biochem Biophys Res Commun 2006;
21:346:131– 139.
Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL,
Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS et al. Derivation of
human embryonic stem cells in defined conditions. Nat Biotechnol
2006;24:185 – 187.
Martin-Ibañez R, Unger C, Strömberg A, Baker D, Canals J, Hovatta O.
Novel cryopreservation method for dissociated human embryonic
stem cells in the presence of a ROCK inhibitor. Hum Reprod 2008;
23:2744– 2754.
Narumiya S, Ishizaki T, Uehata M. Use and properties of ROCK-specific
inhibitor Y-27632. Methods Enzymol 2000;325:273 – 284.
Peerani R, Rao BM, Bauwens C, Yin T, Wood GA, Nagy A, Kumacheva E,
Zandstra PW. Niche-mediated control of human embryonic stem cell
self-renewal and differentiation. EMBO J 2007;26:4744– 4755.
Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem
cell lines from human blastocysts: somatic differentiation in vitro. Nat
Biotechnol 2000;18:399 – 404.
589
Reubinoff BE, Pera MF, Vajta G, Trounson AO. Effective cryopreservation
of human embryonic stem cells by the open pulled straw vitrification
method. Hum Reprod 2001;16:2187 – 2194.
Richards M, Fong CY, Tan S, Chan WK, Bongso A. An efficient and safe
xeno-free cryopreservation method for the storage of human
embryonic stem cells. Stem Cells 2004;22:779 – 789.
Ruchi B, Jacqueline L, Min K, Alexey VT. Efficient propagation of single cells
accutase-dissociated human embryonic stem cells. Mol Reprod Dev 2008;
75:818– 827.
Sidhu KS, Tuch BE. Derivation of three clones from human embryonic
stem cell lines by FACS sorting and their characterization. Stem Cells
Dev 2006;15:61 –69.
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ,
Marshall VS, Jones JM. Embryonic stem cell lines derived from human
blastocysts. Science 1998;282:1145 – 1147.
Wachs FP, Couillard-Despres S, Engelhardt M, Wilhelm D, Ploetz S,
Vroemen M, Kaesbauer J, Uyanik G, Klucken J, Karl C et al. High
efficacy of clonal growth and expansion of adult neural stem cells. Lab
Invest 2003;83:949 – 962.
Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T,
Takahashi JB, Nishikawa S, Nishikawa S, Muguruma K et al. A ROCK
inhibitor permits survival of dissociated human embryonic stem cells.
Nat Biotechnol 2007;25:681 – 686.
Yoo SJ, Yoon BS, Kim JM, Song JM, Roh S, You S, Yoon HS. Efficient
culture system for human embryonic stem cells using autologous
human embryonic stem cell-derived feeder cells. Exp Mol Med 2005;
37:399– 407.
Zhou CQ, Mai QY, Li T, Zhuang GL. Cryopreservation of human
embryonic stem cells by vitrification. Chin Med J 2004;117:
1050 – 1055.
Submitted on August 18, 2008; resubmitted on October 9, 2008; accepted on
October 15, 2008