Download Factors released from embryonic stem cells inhibit apoptosis of

Am J Physiol Heart Circ Physiol 293: H1590–H1595, 2007.
First published June 1, 2007; doi:10.1152/ajpheart.00431.2007.
Factors released from embryonic stem cells inhibit apoptosis of H9c2 cells
Dinender K. Singla and Debbie E. McDonald
Department of Medicine, Division of Cardiology, Cardiovascular Research Institute, University of Vermont, College
of Medicine, Colchester, Vermont
Submitted 7 April 2007; accepted in final form 26 May 2007
Pluripotent ES cells hold significant appeal for cell therapy
because they have the ability to differentiate into any cell type
in the body, including the various cell types found in the heart
(26). Prior studies have suggested that mouse ES cells transplanted after MI in the rat heart can differentiate into cardiac
myocytes (5), and, as our group (25) recently showed, they can
differentiate into vascular smooth muscle and endothelial cells
as well. We and others have also shown that transplantation of
mouse ES cells improves cardiac function despite low amounts
of regeneration that are likely to be insufficient to compensate
for the cell loss due to apoptosis and/or necrosis (5, 20, 25).
Notably, ⬃80 –90% of the transplanted cells die (7, 24). This
might alter the cardiac microenvironment by accumulation of
factors released from both dying and surviving cells.
Accordingly, we hypothesized that substances released from
ES cells contain cytoprotective factors that inhibit stressinduced apoptosis. To test this hypothesis, we prepared conditioned medium (CM) from cultured ES cells and from ES cells
in which apoptosis was induced by H2O2, which simulates the
oxidative stress typical of the post-MI heart. We delineated the
factors released from ES cells and their effects on H2O2induced apoptosis in the cardiomyocyte H9c2 cell line.
MATERIALS AND METHODS
MYOCARDIAL INFARCTION (MI) is a major cause of heart failure
and death. Death of myocytes within the infarct and periinfarct regions occurs as a result of both necrosis and apoptosis
(1– 4, 13). This cell loss may be partially offset by natural
regeneration of cardiac myocytes in adult hearts (6). Cell
transplantation to treat heart disease has been studied in various
animal models (9, 14, 16, 19, 25). Cell types used include
ventricular cardiomyocytes, skeletal myoblasts, smooth muscle
cells, fetal and embryonic cardiomyocytes, bone marrow stromal, hematopoietic stem cells, and mouse embryonic stem (ES)
cells (9, 14, 16, 19, 25).
Cell lines and CM. Mouse ES cells (line CGR8) were passaged and
maintained in DMEM (Invitrogen) supplemented with leukemia inhibitory factor (LIF), sodium pyruvate, glutamine, penicillin-streptomycin, ␤-mercaptoethanol, nonessential amino acids, and 15% FBS
(Invitrogen) as reported previously (27). The rat cardiomyocytederived cell line H9c2 was obtained from the American Type Culture
Collection (Manassas, VA). Cells were cultured in DMEM containing
10% FBS, 2 mM L-glutamine, and 25 ␮g/ml gentamycin at 37°C in a
humidified atmosphere of 5% CO2.
Mouse ES cells at 9,000 ES cells/cm2 were grown in Petri dishes
containing cell culture medium with LIF for 24 h. Cells were treated
with and without H2O2 (400 ␮M) for 2 h, which was then replaced
with fresh cell culture medium without LIF for 48 h. After 48 h,
supernatants (CM) from both groups were collected and labeled as ES
cell-CM or ES cell-H2O2-CM. To prepare H9c2 cell-CM, 500 cells/
cm2 were grown, and supernatant (CM) was collected after 48 h.
Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1)-CM was
prepared as reported (23) and was kindly provided to us by Dr. David
Denhardt (Piscataway, NJ). In brief, human TIMP-1 was subcloned
into the pIZ/V5-His plasmid in a frame with the COOH-terminal
histidine tag. BTI-TN-5B1-4 insect cells were then transfected with
the pIZ/hTIMP-1V5-His plasmid. The CM from cell culture-maintained transfected cells was collected for up to 2 mo.
CM from groups was filtered (0.22-␮m cellulose syringe filter) and
tested for the presence of cytokine and growth factors and used to
assess effects on H2O2-induced apoptosis in H9c2 cells.
Address for reprint requests and other correspondence: D. K. Singla,
Biomolecular Science Center, University of Central Florida, 4000 Central
Florida Blvd., BMS224, Orlando, FL, 32817.
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
hydrogen peroxide; tissue inhibitor of metalloproteinase-1
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0363-6135/07 $8.00 Copyright © 2007 the American Physiological Society
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Singla DK, McDonald DE. Factors released from embryonic
stem cells inhibit apoptosis of H9c2 cells. Am J Physiol Heart Circ
Physiol 293: H1590–H1595, 2007. First published June 1, 2007;
doi:10.1152/ajpheart.00431.2007.—Our recent study (Singla DK,
Hacker TA, Ma L, Douglas PS, Sullivan R, Lyons GE, Kamp TJ, J
Mol Cell Cardiol 40: 195–200, 2006) suggests that transplanted
embryonic stem (ES) cells subsequent to myocardial infarction differentiate into the major cell types in the heart and improve cardiac
function. However, the extent of regeneration is relatively meager
compared with the observed functional improvement. The mechanisms underlying their improved function are completely unknown. In
this report, we provide evidence using a cell culture model system for
novel mechanisms that involve the release of cytoprotective, antiapoptotic factor(s) from ES cells and inhibit H2O2-induced apoptosis
in the rat cardiomyocyte-derived cell line H9c2. Conditioned medium
(CM) from growing mouse ES cells treated with and without H2O2
was generated. Apoptosis was induced after exposure to H2O2 in
H9c2 cells for 2 h followed by replacement with fresh cell culture or
ES cell-CM. After 24 h, H9c2 cells treated with both ES cell-CMs
demonstrated significantly decreased apoptosis, as determined by
terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining, apoptotic ELISA, caspase-3 activity, and DNA ladder. Next,
using Luminex technology, we examined the presence of antiapoptotic
proteins cystatin c, osteopontin, and clusterin and anti-fibrotic, tissue
inhibitor of metalloproteinase-1 (TIMP-1) in both ES cell-CMs. The
levels of released factors were 2- to 170-fold higher than those in
H9c2 cell-CM. Antiapoptotic effects of ES cell-CM were significantly
inhibited with TIMP-1 antibody, suggesting that TIMP-1 is an important factor to inhibit apoptosis. Furthermore, we used CM from an
TIMP-1-overexpressing cell line and demonstrated that H2O2-induced
apoptosis in the H9c2 cells was significantly inhibited. These observations demonstrate that factors released from ES cells contain antiapoptotic factors and that the effects are mediated by TIMP-1. Moreover,
these findings suggest that released factors might be useful for
therapeutic applications in ischemic heart disease as well as for many
other diseases.
EMBRYONIC STEM CELLS RELEASED FACTORS INHIBIT APOPTOSIS
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Cytokine and growth factor analysis. ES cell-CM, H9c2 cell-CM,
or control cell culture medium was prepared and mailed to Rule Based
Medicine (Austin, TX) where cytokine or growth factor analysis using
Luminex technology was performed. In brief, using automated pipetting, an aliquot of each sample is introduced into one of the capture
microsphere multiplexes of the rodent antigen mitogen-activated protein. These mixtures of sample and capture microspheres are thoroughly mixed and incubated at room temperature for 1 h. Multiplexed
cocktails of biotinylated, and reporter antibodies for each multiplex
are then added robotically and, after thorough mixing, incubated for
an additional 1 h at room temperature. Multiplexes are developed
using an excess of streptavidin-phycoerythrin solution, which is thoroughly mixed into each multiplex and incubated for 1 h at room
temperature. The volume of each multiplexed reaction is reduced by
vacuum filtration, and the volume is increased by dilution into matrix
buffer for analysis. Analysis was performed in a Luminex 100
instrument, and the resulting data stream was interpreted by use of
proprietary data analysis software developed at Rule Based Medicine.
This technology detected a total of 69 factors present in the CMs.
TIMP-1 ELISA assay. The detection of TIMP-1 in CMs was
performed with the mouse TIMP-1 ELISA sandwich kit (RayBiotech)
in accordance with the kit instructions. In brief, 100 ␮l of CMs (1:200
dilution) and standards (recombinant mouse TIMP-1 provided with
the kit) were added to a 96-well ELISA plate. After washings, plates
were incubated with 100 ␮l of biotinylated anti-mouse TIMP-1
antibody for 1 h at room temperature followed by horseradish peroxidase-conjugated streptavidin. The reaction was detected with tetramethylbenzidine one-step substrate reagent followed by stop solution.
The 96-well ELISA plate was read at 450 nm in a microtiter plate
reader. TIMP-1 concentration in the samples was analyzed from the
standard curve performed according to the kit directions.
Data analysis. Significance of differences between values was
assessed with the Excel program t-test. All values are means ⫾ SE.
Statistical significance was assigned at P ⬍ 0.05.
RESULTS
We first determined the optimal concentration of H2O2 for
induction of apoptosis. H9c2 cells treated with 50 – 400 ␮M of
H2O2 demonstrated morphological changes as shown in Fig. 1,
A and B, and had decreased cell survival at tested concentrations (Fig. 1C). The maximum decrease in cell survival (58%)
occurred at 400 ␮M H2O2 (P ⬍ 0.05).
Using TUNEL staining, we determined the effect of ES
cell-CM and ES cell-H2O2-CM on H2O2-induced apoptosis in
H9c2 cells. Figure 2, A–C, demonstrates the presence of total
nuclei stained with DAPI, and Fig. 2, D–F, shows TUNELstained nuclei. Merged nuclei are shown in Fig. 2, G–H. The
percentage of TUNEL-stained nuclei was 28% in H2O2-treated
H9c2 cells compared with ⬍1% in the control group (P ⬍
0.05, Fig. 3A). The percentage of apoptotic nuclei induced by
H2O2 was significantly reduced (P ⬍ 0.05) after treatment with
both ES cell-CMs (Fig. 3A). Quantitative apoptosis measured
by the ELISA assay was significantly reduced (Fig. 3B; P ⬍
0.05) with both ES cell-CMs. However, apoptosis was not
inhibited with H9c2 cell-CM used as control (data not shown).
With the use of gel electrophoresis, DNA fragmentation was
observed in the H2O2-treated H9c2 cells, whereas no DNA
fragmentation was seen in the untreated control group (Fig.
3C). Importantly, DNA fragmentation was inhibited after treatment with both ES cell-CMs (Fig. 3C).
It has been reported that caspase-3 activity plays a major role
in stress-induced apoptosis in H9c2 cells. We examined
whether ES cell-CMs had an effect on caspase-3 activity in
H9c2 cells induced to undergo apoptosis. Our data demonstrate
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For blocking experiments, ES cell-CM was incubated with antiTIMP-1 antibody or control non-TIMP-1 IgG antibody (Santa Cruz
Biotechnology) for 1 h at 4°C. Immunoprecipitation was performed
by further incubation with 20 ␮l of protein A-agarose on a rotating
platform at 4°C for overnight. Supernatant was collected by centrifugation at 3,000 rpm for 5–10 min and used to examine its effect on
apoptosis.
Preparation of apoptotic cell culture model. H9c2 cells were
cultured (500 cells/cm2) for 24 h. We assessed the optimal dose of
H2O2 (Sigma) by exposing the cells to concentrations ranging from 50
to 400 ␮M for 2 h followed by replacement with fresh cell culture
medium. Cells were cultured for an additional 24 h and then counted
in each of 24 wells based on morphology. Rod-shaped cells were
considered to be alive, whereas round cells were considered to be
dead. The 400 ␮M H2O2 concentration was found to produce the
greatest amount of apoptosis and was used in subsequent experiments
in which H9c2 cells were exposed to H2O2 (400 ␮M) for 2 h and then
replaced with fresh cell culture medium, ES cell-CM, ES cell-H2O2CM, H9c2 cell-CM, or TIMP-1-CM. Apoptosis was examined by
terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) staining, apoptotic ELISA kit, caspase-3 activity, and
DNA laddering.
Detection of apoptosis by TUNEL staining. Cells were fixed in 4%
paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100
in 0.1% sodium citrate followed by proteinase K (25 ␮g/ml in 100
mM Tris 䡠 HCl). An in situ apoptotic cell death detection kit (TMR red,
Roche Applied Bio Sciences) based on TUNEL assay was used as per
manufacturer’s instruction to detect apoptotic cells. Negative controls
were included in each case by omitting TUNEL enzyme terminal
deoxynucleotidyl transferase (TdT) reaction mixture and incubating
the cells with label solution. Sections were mounted with Antifade
Vectashield mounting medium containing 4⬘-6-diamidino-2-phenylindole (DAPI; Vector Laboratories) to stain nuclei and examined with a
Zeiss Axiovert 200 microscope. The percentage of apoptotic nuclei
was calculated by counting the total number of TUNEL-stained nuclei
divided by total DAPI-positive nuclei.
Cell death detection ELISA. The cell death detection ELISA plus
kit (Roche Applied Sciences) was used to measure histone-bound
DNA fragments in an ELISA format. Medium was used as reported in
a previous study (18) to examine apoptosis collected at 24 h from
different groups.
Caspase-3 activity assay. H9c2 cells (1 ⫻ 106) were cultured in
150-mm2 petri dishes for 2–3 days. Cells were treated with and
without H2O2 and then replaced with cell culture medium or CM from
the different groups. Caspase-3 activity was measured by a caspase-3
colorimetric activity assay kit from BioVision. Cells were washed
with PBS and enzymatically dissociated with trypsin-EDTA (Invitrogen) and then collected in the cell lysis buffer provided in the kit. The
cell lysate was centrifuged for 1–2 min at 10,000 g. Protein concentrations were measured in the supernatant by a Bio-Rad assay.
Caspase-3 activity was performed as per the instructions provided in
the kit and measured at 405 nm in a microtiter plate reader.
Agarose gel electrophoresis for DNA fragmentation. To characterize the extent of DNA fragmentation by agarose gel electrophoresis
after treatment with H2O2 and CM, we used the apoptotic DNA ladder
kit (Roche Applied Diagnostics). H9c2 cells (1 ⫻ 106), treated or
untreated, were suspended in lysis buffer, and DNA was extracted
according to the manufacturer’s instructions. DNA concentrations
were measured with a spectrophotometer. In brief, 5 ␮l of DNA were
diluted in 995 ␮l of water, and optical density was read at 260 nm with
the use of water as a blank. DNA concentration (␮g/␮l) was calculated as optical density at 260 nm ⫻ 9.88 (9.88 is a multiplication
factor that accounts for the 1:200 dilution and the optical density of
DNA in water). DNA sample (3 ␮g) was used to analyze DNA
fragmentation on 1% agarose gels stained with ethidium bromide. The
gels were visualized under UV light, and photographs were taken.
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EMBRYONIC STEM CELLS RELEASED FACTORS INHIBIT APOPTOSIS
that caspase-3 activity was significantly higher (P ⬍ 0.05) in
H2O2-induced apoptosis in H9c2 cells. This increase was
inhibited by both ES cell-CMs (P ⬍ 0.05) (Fig. 3D).
Next, we determined the presence of cytokine or growth
factors in the CM that provided protection from H2O2-induced
apoptosis. The 69 cytokine or growth factors analyzed in the
present study are listed in Table 1 in the supplemental data
(supplemental data for this article is available online at the
Am J Physiol Heart Circ Physiol website). Proteins released in
the CM by ES cells, in ES cells exposed to H2O2, and from the
H9c2 cells that were present at levels greater than those found
in the control cell culture medium (containing serum back-
Fig. 2. Effects of embryonic stem (ES) cell-conditioned medium (CM) on H9c2 cell apoptosis after exposure to H2O2. Representative photomicrographs show
total nuclei stained with 4⬘-6-diamidino-2-phenylindole (DAPI) in blue (A–C), terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling
(TUNEL)-stained nuclei in red (D–F), and merged images (G–I). ⫹ve, Positive. Magnification ⫽ ⫻10.
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Fig. 1. Effects on H9c2 cell viability after exposure to H2O2. A: representative photomicrograph of untreated H9c2 cells grown in control medium for 24 h.
B: H9c2 cells exposed to 50 – 400 ␮M H2O2 for 2 h and then placed in normal cell culture control medium for 24 h. C: histogram showing number of H9c2 cells
exposed to different concentrations of H2O2.
EMBRYONIC STEM CELLS RELEASED FACTORS INHIBIT APOPTOSIS
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ground levels) are listed in Table 2 in the supplemental data
(supplemental data for this article is available online at the
Am J Physiol Heart Circ Physiol website). Four major antiapoptotic proteins were released in the ES cell-CM or ES cellH2O2-CM at these levels, specifically, cystatin-c (2- to 4-fold),
clusterin (3- to 5-fold), osteopontin (16- to 31-fold), and
TIMP-1 (107- to 171-fold), which were significantly different
from their levels in H9c2 cell-CM (Table 1).
TIMP-1 is a potent inhibitor of matrix metalloproteinases
(MMPs) and also plays a major role in adverse cardiac remodeling in the heart. Accordingly, we determined whether
TIMP-1 plays a role in ES cell-CM inhibition of apoptosis in
H9c2 cells. TIMP-1 ELISA data were in concordance with the
Luminex data, demonstrating significant amounts (P ⬍ 0.05)
of TIMP-1 in both ES cell-CMs and 10% TIMP-1-CM used in
the study (Fig. 4). We then blocked TIMP-1 containing ES
cell-CM with an anti-TIMP-1 antibody, using a non-TIMP-1
IgG antibody as a control. Figure 5 demonstrates that the
TIMP-1 antibody abolished the protective antiapoptotic effects
of ES cell-CM. Non-TIMP-1 IgG antibody did not inhibit the
antiapoptotic effects of ES cell-CM (data not shown). These
Table 1. List of anti-apoptotic factors released in the ES
cell- and H9c2 cell-derived CM
Released Factor
Cystatin-C
Clusterin
Osteopontin
TIMP-1
Control
H9c2 Cell-CM,
pg/ml
ES Cell-CM,
pg/ml
ES Cell-H2O2-CM,
pg/ml
L
L
L
L
21,000
2,200
3,900
28
41,000
7,450
66,000
3,000
77,000
11,000
123,000
4,800
L, lowest detectable dose by the assay (see MATERIALS AND METHODS);
TIMP-1, tissue inhibitor of metalloproteinase-1. Control indicates cell culture
medium used to grow embryonic stem (ES) cells.
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Fig. 4. Mouse tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) ELISA
kit was used to measure the total amount of TIMP-1 protein present in the ES
cell-CM, ES cell-H2O2-CM, and 10% TIMP-1-CM. Note that TIMP-1 was
undetectable in the control cell culture medium. Data are from set of 4 – 6
independent experiments. *P ⬍ 0.05 vs. control (C).
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Fig. 3. Effects of both of the ES cell-CMs on H9c2
cell-induced apoptosis, as determined by TUNEL
staining (A), apoptotic ELISA (B), DNA fragmentation (C), and caspase-3 activity (D) after exposure
to H2O2. A: histogram shows quantitative apoptotic
nuclei (n ⫽ 4 – 6 fields/well in each condition). Data
are from set of 3 or 4 independent experiments.
B: histogram shows quantitative apoptotic ELISA
(see MATERIALS AND METHODS). Data are from set of
8 –10 independent experiments. C: representative
photomicrographs of DNA fragmentation isolated
from different preparations of H9c2 cells performed
on 1% agarose gel electrophoresis (A, 1-kb molecular marker; B, H9c2 cells grown in control cell
culture medium shows no DNA fragmentation; C,
H9c2 cells exposed to H2O2 demonstrate clear
DNA ladder and DNA ladder is inhibited when cells
are grown in the presence of CMs; D, ES cell-CM;
E, ES cell-H2O2-CM). Data are from set of 2– 4
independent experiments. D: histogram shows
quantitative caspase-3 activity examined by ELISA
kit. Data are from set of 8 –10 independent experiments. In A, B, and D, C represents control. *P ⬍
0.05 vs. H2O2 group.
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EMBRYONIC STEM CELLS RELEASED FACTORS INHIBIT APOPTOSIS
data suggest that TIMP-1 is the major factor present in the ES
cell-CM that inhibits apoptosis.
Next, we determined the direct role of TIMP-1 in H2O2induced apoptosis in H9c2 cells. TIMP-1-CM obtained from
TIMP-1-overexpressing cells demonstrated significant (P ⬍
0.05) inhibition of H2O2-induced apoptosis in H9c2 cells. This
was confirmed by TUNEL staining (Fig. 6A), ELISA (Fig. 6B),
and caspase-3 activity (Fig. 6C). Importantly, the amount of
apoptosis inhibited with ES cell-CM determined with apoptotic
ELISA kit was significantly lower (P ⬍ 0.05) than with
TIMP-1-CM (Fig. 6B). Apoptotic ELISA data suggest that
other factors present in the ES cell-CM may also contribute to
inhibition of H2O2-induced apoptosis in H9c2 cells.
DISCUSSION
The major new findings of the present study are as follows:
1) H2O2-induced cell death in H9c2 cells is apoptotic in nature;
Fig. 6. Effects of TIMP-1-CM on H9c2 cell apoptosis after exposure to H2O2. A: histogram shows quantitative apoptotic nuclei per well (n ⫽ 4 – 6 fields/well
in each condition and data are from set of 3 or 4 independent experiments). B: histogram shows quantitative apoptotic ELISA (see MATERIALS AND METHODS).
Data are from set of 8 –10 independent experiments. *P ⬍ 0.05 vs. H2O2 group; #P ⬍ 0.05 vs. H2O2 group and ES cell-CM. C: histogram shows quantitative
caspase-3 activity examined by ELISA kit. Data are from set of 4 – 6 independent experiments. *P ⬍ 0.05 vs. H2O2 group.
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Fig. 5. Histogram shows that TIMP-1 antibody blocks the antiapoptotic effects
of ES cell-CM on H9c2 cells exposed to H2O2. ES cell-CM was blocked by
incubation with anti-TIMP-1 antibody at different concentrations, as explained
in MATERIALS AND METHODS. ES cell-CM containing TIMP-1 shows no decrease in apoptosis compared with H2O2 group. ES cell-CM without TIMP-1
antibody inhibits apoptosis, as assessed by the apoptotic ELISA kit. Data are
from set of 4 – 8 independent experiments. *P ⬍ 0.05 vs. H2O2 group, #P ⫽
not significant vs. H2O2 group.
2) CM from ES cells or ES cells treated with H2O2 inhibits
H2O2-induced apoptosis in H9c2 cells; 3) both of the ES
cell-CMs contained antiapoptotic (osteopontin, clusterin, cystatin) and antifibrotic (TIMP-1) factors; 4) TIMP-1 present in
the ES cell-CM inhibited H2O2-induced apoptosis in H9c2
cells; and 5) TIMP-1-CM obtained from TIMP-1-overexpressing cells also inhibited H2O2-induced apoptosis.
Apoptosis has been shown to be an important factor in the
development and progression of MI, cardiac remodeling, and
eventually chronic heart failure (1– 4, 13). It is well established
that H2O2 induces apoptosis in isolated adult cardiomyocytes and
in H9c2 cells (10, 22). In the present study, we showed that
treatment of H9c2 cells with H2O2 induces dose-dependent decreases in cell survival. Apoptotic detection methods, including
TUNEL staining, ELISA, DNA ladder, and caspase-3 activity,
indicate that the cell death observed in the H9c2 cells is apoptotic
in nature. We also demonstrated for the first time that cytoprotective factors were released in both of the ES cell-CMs and that
these inhibit H2O2-induced apoptosis in H9c2 cells. Notably, our
data suggest that exposure of ES cells to H2O2 further increases
the amount of antiapoptotic factors (Table 1) present in the CM.
These findings suggest that factors released from surviving and
from dying cells after exposure to H2O2 protect against stressinduced apoptosis. Our data are consistent with recent findings
suggesting that factors released from hypoxia-induced Akt-mesenchymal stem cells subjected to hypoxia inhibit apoptosis in
isolated cardiomyocytes (8). In the present study, we demonstrated effects of released factors from ES cells. The recent reports
suggest that progenitor cell-specific markers such as Flk-1 (endothelial cells) and Nkx2.5 (cardiac myocyte) could be used for the
preselection of endothelial or cardiomyocyte cell-specific lineage
from mouse ES cells (11, 29). Importantly, both of the cellspecific, marker-isolated preselected cells could be significantly
different in characteristics vs. ES cells used in the present study.
However, future studies are warranted to isolate CM from preselected ES cells to determine the released factors and their protective effects on apoptosis.
Next, we used cell culture model system to establish that
released factors from ES cells inhibit H9c2 cell-induced apoptosis. The present study opens new avenues to investigate
whether protective effects of released factors can also be seen
EMBRYONIC STEM CELLS RELEASED FACTORS INHIBIT APOPTOSIS
GRANTS
We acknowledge partial support provided by American Heart Association
Scientist Development Grant 0430227N (to D. K. Singla).
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in other major cell types present in the heart, such as endothelial or vascular smooth muscle cells.
We identified four major antiapoptotic factors, osteopontin,
clusterin, cystatin-c and TIMP-1, in the ES cell-CM with and
without exposure to H2O2. These factors have been shown to be
antiapoptotic in cancer cells (15, 17), neuroblastoma cells (30),
PC-12 cells (21), and endothelial cells (12) but not in isolated
cardiomyocytes or in the rat cardiomyocyte-derived cell line
H9c2. TIMP-1 is antifibrotic in the heart because of inhibition of
MMPs (28). In the present study, the antibody-blocking experiments suggest that TIMP-1 is the key factor present in the ES
cell-CMs that inhibit apoptosis. Furthermore, we also demonstrated for the first time that TIMP-1-CM inhibits apoptosis in
H9c2 cells. However, further mechanisms of inhibited H9c2 cell
apoptosis are required. Moreover, our data are consistent with the
antiapoptotic properties of TIMP-1 observed in cancer and endothelial cells (15, 17). Further studies are required to delineate
whether the antiapoptotic effects of TIMP-1 on cardiac cell types
are independent of its ability to inhibit MMP activity. Importantly,
we have previously shown that transplanted, undifferentiated ES
cells can be successfully engrafted and differentiated and that they
are associated with significant improvement in cardiac function;
however, the extent of regeneration itself does not appear sufficient to account for these observations (25). Thus our data generated in the stress-induced cell culture model system suggest
inhibition of apoptosis typical of the oxidative stress-induced
post-MI heart following ES cell transplantation. We hypothesize
that improved function in our previous study (25) could be caused
by factors released from transplanted ES cells that inhibit cardiomyocyte apoptosis.
In conclusion, we report that ES cells treated with and
without H2O2 release factors that provide protection against
stress-induced apoptosis in a cell culture model system. We
also demonstrate that TIMP-1 is a key factor present in ES
cell-CM or ES cell-H2O2-CM responsible for inhibition of
apoptosis. However, further transplantation studies with ES
cells and ES cell-derived CM are required to identify the
released factors in vivo, levels of TIMP-1, and the potential
mechanisms of action of the released factors, which may have
important implications for treatment of various heart diseases.
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