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Am J Physiol Heart Circ Physiol 287: H471–H479, 2004;
Cellular Plasticity in the Cardiovascular System
Stable benefit of embryonic stem cell therapy in myocardial infarction
Denice M. Hodgson,1 Atta Behfar,1 Leonid V. Zingman,1 Garvan C. Kane,1
Carmen Perez-Terzic,1,2 Alexey E. Alekseev,1 Michel Pucéat,1,3 and Andre Terzic1
Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology, and Experimental Therapeutics,
and 2Physical Medicine and Rehabilitation, Mayo Clinic College of Medicine, Rochester, Minnesota 55905; and
Centre de Recherches de Biochimie Macromoléculaire, CNRS FRE 2593, 34293 Montpellier, France
Submitted 31 December 2003; accepted in final form 22 March 2004
engraftment; xenotransplant; plasticity; cardiac differentiation; remodeling; heart
varying degrees of cardiogenicity (4, 22, 29, 31, 37), embryonic stem cells derived from the inner cell mass of the blastocyst possess the most recognized capacity to yield cardiomyocytes (6, 12, 23, 34, 38). Because of their ability to indefinitely proliferate in vitro, embryonic stem cells can generate
large colonies and supply a reservoir for extensive tissue
regeneration (15, 35, 45). Indeed, in vitro differentiation of
embryonic stem cells produces cells that recapitulate the cardiac phenotype expressing characteristic cardiac markers and
demonstrating functional excitation-contraction coupling (6,
13, 24, 32, 34). Furthermore, when transplanted into injured
hearts, embryonic stem cells generate cardiomyocytes that
repopulate significant regions of dysfunctional myocardium
and result in improved contractile performance with reduced
mortality (3, 25, 26). This transition of embryonic stem cells to
cardiomyocytes occurs under the direction of host paracrine
signaling that supports cardiac-specific differentiation (3). Although understanding of the molecular mechanisms of stem
cell commitment and integration with host myocardium is
increasing, limited information is available on the natural
history of stem cell therapy outcomes.
We serially monitored the effect of embryonic stem cell
therapy in a model of myocardial infarction and demonstrated
improvement in myocardial function sustained over a 12-wk
follow-up period. The presence of embryonic stem cell-derived
cardiomyocytes within the infarct regions was associated with
preserved left ventricular structure and diminished scar. Thus
embryonic stem cells, through myocardial regeneration and an
impact on ventricular remodeling, can provide stable long-term
benefit after myocardial infarction.
leads to cardiomyocyte loss, ventricular
remodeling, and consequent impairment of myocardial function, whereas the mitotic capacity of cardiomyocytes is too
limited to support adequate myocardial regeneration (5, 7, 39).
Moreover, current therapeutic modalities attenuate disease progression without contributing significantly to myocardial repair
(16). In this regard, an alternative approach to conventional
strategies is emerging with advances in the manipulation of
nonterminally differentiated cells that maintain the potential to
form cardiomyocytes and, thus, the propensity to replace diseased myocardium (27, 28, 30, 36, 42).
Although multiple candidate cell types have been isolated
from cardiac or noncardiac sources and shown to display
Embryonic stem cells. The CGR8 murine embryonic stem cell line
was propagated in baby hamster kidney (BHK21) cells or Glasgow
MEM supplemented with pyruvate, nonessential amino acids, mercaptoethanol, 7.5% fetal calf serum, and the leukemia inhibitory factor
(24, 32). A CGR8 cell clone was engineered to express the yellow
fluorescent protein or the enhanced cyan fluorescent protein (ECFP)
under the control of the cardiac-specific ␣-actin promoter subcloned
upstream of ECFP using XhoI and HindIII restriction sites of the
promoterless pECFP vector (Clontech). This ␣-actin promoter construct was linearized using XhoI and electroporated into CGR8 stem
cells as described elsewhere (3, 24). To image stem cells by field-
Address for reprint requests and other correspondence: A. Terzic, Guggenheim 7, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail:
[email protected]).
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.
0363-6135/04 $5.00 Copyright © 2004 the American Physiological Society
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Hodgson, Denice M., Atta Behfar, Leonid V. Zingman, Garvan
C. Kane, Carmen Perez-Terzic, Alexey E. Alekseev, Michel
Pucéat, and Andre Terzic. Stable benefit of embryonic stem cell
therapy in myocardial infarction. Am J Physiol Heart Circ Physiol
287: H471–H479, 2004; 10.1152/ajpheart.01247.2003.—Conventional therapies for myocardial infarction attenuate disease progression without contributing significantly to repair. Because of the
capacity for de novo cardiogenesis, embryonic stem cells are considered a potential source for myocardial regeneration, yet limited
information is available on their ultimate therapeutic value. We
treated infarcted rat hearts with CGR8 embryonic stem cells preexamined for cardiogenicity, serially probed left ventricular function,
and determined final pathological outcome. Stem cell delivery generated new cardiomyocytes of embryonic stem cell origin that integrated
with host myocardium within infarct regions. This resulted in a
functional benefit within 3 wk that remained sustained over 12 wk of
continuous follow-up and included a vigorous inotropic response to
␤-adrenergic challenge. Integration of stem cell-derived cardiomyocytes was associated with normalized ventricular architecture, little
scar, and a decrease in signs of myocardial necrosis. In contrast,
sham-treated infarcted hearts exhibited ventricular cavity dilation and
aneurysm formation, poor ventricular function, and a lack of response
to ␤-adrenergic stimulation. No evidence of graft rejection, ectopy,
sudden cardiac death, or tumor formation was observed after therapy.
These findings indicate that embryonic stem cells, through differentiation within the host myocardium, can contribute to a stable beneficial outcome on contractile function and ventricular remodeling in
the infarcted heart.
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calculate EF as follows: EF ⫽ 100 䡠 (D2 ⫺ S2)/D2, where D is the
end-diastolic cavity diameter and S is the end-systolic cavity diameter
(3, 14).
Histopathology. On autopsy, gross pathological examination was
performed on 4% formalin-fixed transverse-cut hearts, and then 0.5␮m-thick paraffin sections stained with hematoxylin and eosin were
examined by light microscopy (46). The fraction of chamber circumference with residual postinfarction left ventricular scar was determined in standardized sections through the left ventricular base at the
midlevel of the infarct. Fluorescent microscopy (Zeiss) was performed
on unstained paraffin sections. For transmitted electron microscopy,
myocardial specimens were postfixed in phosphate-buffered 1%
OsO4, stained en bloc with 2% uranyl acetate, dehydrated in ethanol
and propylene oxide, and embedded in low-viscosity epoxy resin.
Thin (90-nm) sections were cut on an ultramicrotome (Reichert
Ultracut E), placed on 200-␮m mesh copper grids, and stained with
lead citrate. Micrographs were taken on an electron microscope
(JEOL 1200 EXII) operating at 60 kV (14).
Statistics. Values are means ⫾ SE. Embryonic stem cell- vs.
sham-treated groups were compared using Student’s t-test, with P ⬍
0.05 considered significant. Wilcoxon’s log-rank test was used for
nonparametric evaluation of randomization.
All protocols were approved by the Mayo Clinic Institutional
Animal Care and Use Committee.
Cardiogenic potential of embryonic stem cells and delivery
into infarcted heart. The CGR8 embryonic stem cell colony
demonstrated typical features of undifferentiated cells, including a high nucleus-to-cytosol ratio, prominent nucleoli, and
mitochondria with few cristae (Fig. 1, A and B). The cardiogenic capacity of this embryonic stem cell line was probed by
in vitro differentiation, with cells readily derived that express
the cardiac transcription factor MEF2C (19), the cardiac contractile protein ␣-actinin, and sarcomeric striations (Fig. 1C).
Consistent with proper differentiation toward cardiac lineage,
stem cell-derived cardiomyocytes demonstrated action potential activity associated with prominent inward Na⫹ and Ca2⫹
currents (Fig. 1D), critical for excitation-contraction coupling
manifested as rhythmic intracellular Ca2⫹ transients (Fig. 1E).
Injection of CGR8 cells into the myocardium resulted in local
retention of these embryonic stem cells (Fig. 1F) without
detectable dispersal into noncardiac tissues (Fig. 1G). To
determine the outcome of stem cell therapy for cardiac repair
in myocardial infarction, rats were randomly assigned to stem
cell- or sham-treatment groups. At 8 wk after left anterior
descending coronary artery ligation, infarction was confirmed
by electrocardiographic evidence of myocardial necrosis (Fig.
2A) as well as by direct visual inspection of the myocardium
after thoracotomy (Fig. 2B). Embryonic CGR8 stem cells from
the pretested colony or acellular preparations (sham controls)
were then injected into the peri-infarct zone (Fig. 2B) for
assessment of functional and structural impact over time.
Sustained functional benefit of stem cell- vs. sham-treated
infarcted hearts. Cardiac contractile function, assessed by
echocardiography at 3 wk after injection, was superior in
embryonic stem cell- compared with sham-treated infarcted
hearts (Fig. 3A). On average, left ventricular EF was 0.80 ⫾
0.05 vs. 0.52 ⫾ 0.05 in the stem cell- vs. the sham-treated
group (P ⬍ 0.05). Moreover, although sham-treated infarcted
hearts failed to augment function under inotropic challenge,
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emission scanning electron microscopy, cells were fixed in phosphatebuffered saline containing 1% glutaraldehyde and 4% formaldehyde
(pH 7.2), dehydrated with ethanol, and dried in a critical point dryer.
Cells, coated with platinum with use of an indirect argon ion-beam
sputtering system (Ion Tech, VCR Group) operating at accelerating
voltages of 9.5 kV and 4.2 mA, were then examined on a fieldemission scanning microscope (model 4700, Hitachi) (33). For transmitted scanning electron microscopy, stem cells were postfixed in
phosphate-buffered 1% OsO4, stained en bloc with 2% uranyl acetate,
dehydrated in ethanol and propylene oxide, and embedded in lowviscosity epoxy resin. Thin (90-nm) sections were stained with lead
citrate, and micrographs were taken on an electron microscope (JEOL
1200 EXII) (14).
Stem cell-derived cardiomyocytes. CGR8 embryonic stem cells
were differentiated in vitro using the previously established hangingdrop method to generate embryoid bodies (3, 20). After enzyme
dissociation of embryoid bodies, Percoll gradient was used to isolate
a highly enriched population of stem cell-derived cardiomyocytes as
described elsewhere (32). The presence of cardiac markers in purified
cells was probed by laser confocal microscopy (LSM 510 Axiovert,
Zeiss) using anti-MEF2C (Cell Signaling Technology) and anti-␣actinin (Sigma) antibodies. Membrane electrical activity was determined by patch-clamp recording in the whole cell configuration using
the current- or voltage-clamp mode (Axopatch 1C, Axon Instruments). Action potential profiles and voltage-current relation were
acquired and analyzed with Bioquest software from cells superfused
with Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 2 CaCl2,
1 MgCl2, 10 HEPES, and 10 glucose (with pH adjusted to 7.4 with
NaOH) using patch pipettes (5–10 M⍀) containing (in mM) 140 KCl,
1 MgCl2, 10 HEPES, and 5 EGTA and supplemented with 5 mM ATP
(with pH adjusted to 7.2 with KOH). Electrophysiological measurements were performed at 31 ⫾ 1°C with a temperature controller
(model HCC-100A, Dagan) equipped with a Peltier thermocouple
(47). To assess intracellular Ca2⫹ dynamics, cells were loaded with
the Ca2⫹-fluorescent probe fluo 3-AM (Molecular Probes), line
scanned with a Zeiss laser confocal microscope, and analyzed using
imaging software (LSM Image Browser, Zeiss) as described elsewhere (32, 46).
Myocardial infarction model. Myocardial infarction was induced at
6 wk of age by ligation of the left anterior descending coronary artery
in male Sprague-Dawley rats, resulting in an established model with
an ⬃30% infarcted left ventricle (Charles River). Consequently, left
ventricular ejection fraction (EF) was depressed from 75 ⫾ 2% at
baseline to 47 ⫾ 3% after infarct. Infarcted animals were randomized
into sham- and embryonic stem cell-treatment groups. At 8 wk after
infarct, animals were anesthetized with isoflurane (3% induction,
1.5% maintenance), 12-lead electrocardiography was performed, and
the heart was exposed by thoracotomy. Medium (20 ␮l of Glasgow
MEM) without cells (sham) or CGR8 embryonic stem cells (3 ⫻ 105
in 20 ␮l of medium), engineered to express ECFP under control of the
cardiac-specific actin promoter, were injected through a 28-gauge
needle at three sites (at the left ventricular base just below the left
atrium, in the midanterior region, and at the apex) along the border of
the left ventricular infarcted areas.
Electrocardiography. Twelve-lead electrocardiography was performed under isoflurane anesthesia with subcutaneous needle electrodes (Grass Instruments) and a differential electrocardiographic
amplifier (model RPS312, Grass Instruments). Standard and augmented limb leads (I, II, III, aVR, aVL, and aVF) as well as precordial
leads (V1–V6) were recorded before sham treatment or stem cell
injection and serially thereafter.
Echocardiography. Under isoflurane anesthesia, two-dimensional
M-mode echocardiographic images were obtained from the parasternal short-axis view with a 5-MHz probe at the ventricular base
(Vingmed System FiVe, GE Medical Systems). The leading-edge
convention of the American Society of Echocardiography was used to
stem cell-treated infarcted hearts demonstrated a significant
positive inotropic response. Pharmacological stress testing by
injection of the ␤-adrenergic agonist isoproterenol (3 ␮g/kg)
produced a 12% increase in the EF of stem cell-treated infarcted hearts vs. no significant response in the sham-treated
group (Fig. 3A). M-mode imaging under stress further demonstrated that, in contrast to the hypokinetic or akinetic anterior
left ventricular walls in sham-treated infarcted hearts, stem
cell-injected infarcted hearts exhibit dynamic anterior wall
motion with vigorous ventricular function (Fig. 3B). Long-term
follow-up found no decay in the contractile advantage of stem
cell therapy (Fig. 4). Indeed, the contractile performance benefit of stem cell- vs. sham-treated infarcted hearts was maintained at 3, 6, 9, and 12 wk after injection, such that at 3 mo
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after cell delivery left EF was 83 ⫾ 4% and 62 ⫾ 4%,
respectively (P ⬍ 0.05; Fig. 4, A and B). On M-mode images
3 mo after therapy, the left ventricular dilation and the anterior
regional wall motion abnormalities persisted in the shamtreated group but were not seen in the stem cell-treated group
(Fig. 4A). Furthermore, electrocardiography performed at 3 mo
after therapy revealed in the stem cell-treated group a 33%
decrease in the total number of anterior and lateral leads, with
Q waves reflecting net reduction in myocardial necrosis (P ⬍
0.05) not seen in the sham-treated group (Fig. 4, C–E).
Throughout the follow-up period, serial electrocardiograms did
not document ventricular ectopy, and no animal experienced
sudden cardiac death. Thus delivery of embryonic stem cells
into infarcted hearts was associated with a functional benefit at
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Fig. 1. Validation of cardiogenic potential
and myocardial delivery of embryonic stem
cells. A and B: field emission scanning (A)
and transmission (B) electron microscopy of
undifferentiated CGR8 embryonic stem cells
in culture. In B, N denotes nucleus; n, nucleolus; and m, mitochondria. C: cardiomyocytes, derived in vitro from the CGR8 stem
cell colony, express cardiac-specific transcription factor MEF2C and contractile protein ␣-actinin demonstrated by immunofluorescence. 4⬘,6-Diamidino-2-phenylindole (DAPI)
staining is used as a nuclear marker. D: stem
cell-derived cardiomyocytes exhibit action potential activity under current-clamp mode (top);
current-voltage relation was obtained under
voltage-clamp mode in response to a ramp
stimulus (rate of 1.2 V/s) and displays characteristic peaks corresponding to Na⫹ and Ca2⫹
inward conductances (bottom). Em, membrane
potential. E: Ca2⫹ transients were probed by
fluo 3-assisted laser confocal microscopy from
a stem cell-derived cardiomyocyte (inset) and
recorded at 35 ⫾ 2°C. AU, arbitrary units. F:
local retention of CGR8 stem cells after intramyocardial delivery into mouse heart (inset).
Cryosection (at ⫻40 magnification) of myocardium with an overlay of fluorescing stem cells.
G: lack of stem cell dispersion into noncardiac
tissues with absence of fluorescence or cell
hyperproliferation in mouse brain (left), kidney
(center), and liver (right) shown at low (top)
and ⫻40 magnification (bottom).
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Fig. 2. Myocardial infarction and stem cell therapy. A and B: infarction of rat
hearts confirmed by the presence of anterior and lateral Q waves on 12-lead
electrocardiogram (A) and by visual inspection after thoracotomy (B). In A,
arrowheads indicate location of Q waves on electrocardiography. In B, arrowheads indicate sites of stem cell injection at the base, midventricle, and apex in
the peri-infarct zone. Inset in B shows injection of stem cells into infarcted
baseline and with stress and was sustained on follow-up without evidence of proarrhythmia in this model.
Stem cell engraftment associated with de novo cardiogenesis
and normalized myocardial architecture. On pathological examination, the entire group of infarcted hearts treated with
stem cells (n ⫽ 4) demonstrated a population of cyan fluorescent myocytes dispersed within the nonfluorescent host myocardium (Fig. 5, 1st and 4th rows). This fluorescent population,
absent from the sham-injected group (n ⫽ 3), indicates the
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Fig. 3. Stem cell therapy of infarcted hearts is associated with improved left
ventricular function and response to stress. A: at 3 wk after injection, left
ventricular ejection fraction, measured by M-mode echocardiography, was
significantly greater in stem cell-treated (n ⫽ 4) than in sham-treated (n ⫽ 3)
infarcted hearts (⫺stress; P ⬍ 0.05). After stress induced by isoproterenol (3
␮g/kg ip), left ventricular ejection fraction increased in the stem cell- but not
the sham-treated group (⫹stress; P ⬍ 0.05). *Significant difference between
stem cell and sham groups; **significant difference between stem cell and
sham groups as well as significant difference in the stem cell group with and
without stress. B: representative M-mode image under stress reveals a hypokinetic-to-akinetic anterior wall and left ventricular dilation in the sham-treated
heart in contrast to normal anterior wall motion and vigorous contractile
function in the stem cell-treated heart. Dashed and solid lines indicate diastolic
and systolic left ventricular dimensions, respectively. MI, myocardial infarction.
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embryonic stem cell origin through expression of the cyan
fluorescent protein under control of the cardiac actin promoter
(3). In contrast to sham-treated infarcted hearts that demonstrated markedly altered ventricular architecture with thinned
free walls and fibrotic scar or aneurysmal areas comprising
34 ⫾ 11% of the ventricle, the presence of stem cell-derived
cardiomyocytes was associated with residual adverse remodeling in only 6 ⫾ 4% of the ventricle (P ⬍ 0.05) and a
myocardial appearance more comparable to that of control
noninfarcted heart (Fig. 5, 2nd and 3rd and 5th and 6th rows).
Stem cell-injected hearts did not demonstrate inflammatory
infiltrates that would otherwise suggest an immune response
toward the engrafted cells (Fig. 6A). On high magnification, the
fluorescent pattern of stem cell-derived cardiomyocytes revealed distinct sarcomeric striations indicating development of
the contractile apparatus (Fig. 6, B and C). Sarcomeres in the
infarct area of stem cell-treated hearts showed normal cardiac
ultrastructure on electron microscopy, in contrast to acellular
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infarct areas of sham-treated hearts (Fig. 6, D and E). Thus
embryonic stem cells were able to incorporate within host
infarct territory, demonstrate cardiogenic differentiation, and
contribute to myocardial repair.
The present study of myocardial infarction shows a stable
favorable impact of embryonic stem cell therapy. This manifested as a sustained benefit on cardiac contractile performance
and ventricular remodeling associated with documented cardiogenesis in the infarct zone from injected stem cells. These
findings indicate that the advantage of embryonic stem cell
delivery occurs early, as first evidenced in the present design at
3 wk after therapy, and is not compromised by spontaneous
failure of stem cell-derived cardiomyocytes and/or by rejection
of this allogenic transplant by the host. The lack of diminishing
effect over time suggests the potential for therapeutic use of
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Fig. 4. Stable benefit of stem cell therapy in infarcted hearts
over 12-wk follow-up. A: representative M-mode images at
12 wk demonstrate left ventricular dilation and lower ejection fraction in sham- than in stem cell-treated infarcted
hearts. Dashed and solid lines indicate diastolic and systolic
left ventricular dimensions, respectively. B: serial echocardiographic assessments at 3, 6, 9, and 12 wk after therapy
indicate that the benefit of stem cell therapy over sham
therapy occurs as early as 3 wk and is stable over the entire
period of follow-up. C: at 12-wk, electrocardiographic evidence of myocardial necrosis as manifested by Q waves
was decreased compared with initial electrocardiograms in
the stem cell- but not the sham-treated group (P ⬍ 0.05).
*Significant difference between stem cell-treated and shamtreated groups. D and E: representative electrocardiograms
from sham-treated (D) and stem cell-treated (E) rats at 12
wk indicate fewer Q waves in stem cell-treated hearts,
suggesting less extensive area of infarct. Arrowheads indicate location of Q waves.
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embryonic stem cells in the chronic management of myocardial
Several potential mechanisms may account for the demonstrated benefit of embryonic stem cell therapy. Specifically,
embryonic stem cell-derived cardiomyocytes, through electrical and mechanical coupling with native myocardium, could
contribute to a net increase in contractile tissue. Here, stem
cell-derived cardiomyocytes aligned with and integrated within
host myocardial fibers. In fact, the host myocardium has been
shown to secrete cardiogenic growth factors that interact in a
paracrine fashion with receptors on stem cells, supporting
cardiac differentiation with expression of cardiac contractile
and gap junction proteins (3, 23). The present failure to observe
ectopy is further consistent with electrical integration of stem
cell-derived cardiomyocytes and host tissue. The stem cellderived cardiomyocyte effect on active myocardial properties
is moreover evidenced here by improved inotropic response to
␤-adrenergic challenge. A synergistic potential mechanism for
functional improvement by stem cell-derived cardiomyocytes
is through alteration of myocardial passive mechanical properties (2), as shown here by the limited appearance of scar and
less dilation of the left ventricle than in sham-treated infarcted
hearts. This may occur through direct repopulation of scar by
stem cell-derived cardiomyocytes, as well as limitation of
adverse remodeling (8, 21). Moreover, cell fusion after grafting
in vivo has been recently documented with adult stem cells in
Fig. 5. Infiltration of stem cell-derived cardiomyocytes into infarct zone diminishes scar and preserves left ventricular architecture.
The presence of cardiomyocytes derived from injected embryonic stem cells can be demonstrated through expression of enhanced
cyan fluorescent protein under control of the ␣-actin promoter. Fluorescence is not seen in untreated noninfarcted or sham-treated
infarcted hearts (1st row) but is seen within the infarct zone of stem cell-treated hearts (4th row; ⫻10 magnification). Compared
with noninfarcted hearts, gross specimens and hematoxylin-and-eosin-stained cross sections at the base of each heart within the
sham-treated group demonstrate dilated left ventricular cavities and prominent anterolateral scar with aneurysms (2nd and 3rd
rows). In contrast, left ventricular cavity size and wall thickness are largely preserved in the stem cell-treated hearts, which also
demonstrate less gross evidence of scar (5th and 6th rows).
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Fig. 6. In vivo stem cell-derived cardiomyocytes integrate with host myocardium. A:
representative hematoxylin-and-eosin-stained
myocardial sections without interstitial infiltration of small round basophilic mononuclear cells consistent with absence of lymphocytes and chronic inflammation in control
(top) or stem cell-injected (bottom) infarcted
hearts. B and C: high-magnification fluorescence confocal microscopy of stem celltreated hearts reveals fluorescent cardiomyocytes indicating expression of enhanced cyan
fluorescent protein under control of the ␣-actin promoter and, thus, injected embryonic
stem cell origin. Stem cell-derived cardiomyocytes organize to form regular fibers (B),
oriented along the same axis as host cardiomyocytes (B and C), display typical sarcomeric striations, and form junctions with
nonfluorescent host cardiomyocytes (C). D
and E: representative transmission electron
micrographic images of biopsies taken from
the infarct zone reveal acellular regions with
pronounced collagen deposition in shamtreated heart (D) compared with cellular areas with normal subcellular architecture in
stem cell-treated heart (E).
This study was supported by National Institutes of Health Grants HL64822, HL-07111, GM-65841, and GM-08685, the American Heart Association, Marriott Foundation, Miami Heart Research Institute, Mayo-Dubai
Healthcare City Research Project, Mayo Clinic CR20 Program, and Association Francaise contre les Myopathies and Fondation de France. M. Pucéat is an
Established Investigator of Institut National de la Santé et de la Recherche
Médicale. A. Terzic is an Established Investigator of the American Heart
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