Download Cardiac Stem Cells and Mechanisms of Myocardial Regeneration

Physiol Rev 85: 1373–1416, 2005;
doi:10.1152/physrev.00013.2005.
Cardiac Stem Cells and Mechanisms
of Myocardial Regeneration
ANNAROSA LERI, JAN KAJSTURA, AND PIERO ANVERSA
Cardiovascular Research Institute, Department of Medicine, New York Medical College, Valhalla, New York
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I. Introduction
II. Exogenous Progenitor Cells and Cardiac Repair
A. Human embryonic stem cells
B. Hematopoietic stem cells
C. Mesenchymal stem cells
D. Controversy on bone marrow cell transdifferentiation and cardiac repair
III. Cardiac Progenitor Cells
A. Identification
B. Origin of cardiac stem cells
C. Cardiac stem cell niches
IV. Cardiac Progenitor Cells and Myocardial Repair
A. Myocardial regeneration and ischemic and nonischemic cardiomyopathy
B. Cell fusion and myocardial regeneration
V. Future Directions
Leri, Annarosa, Jan Kajstura, and Piero Anversa. Cardiac Stem Cells and Mechanisms of Myocardial Regeneration. Physiol Rev 85: 1373-1416, 2005; doi:10.1152/physrev.00013.2005.—This review discusses current understanding of the role that endogenous and exogenous progenitor cells may have in the treatment of the diseased heart.
In the last several years, a major effort has been made in an attempt to identify immature cells capable of
differentiating into cell lineages different from the organ of origin to be employed for the regeneration of the
damaged heart. Embryonic stem cells (ESCs) and bone marrow-derived cells (BMCs) have been extensively studied
and characterized, and dramatic advances have been made in the clinical application of BMCs in heart failure of
ischemic and nonischemic origin. However, a controversy exists concerning the ability of BMCs to acquire cardiac
cell lineages and reconstitute the myocardium lost after infarction. The recognition that the adult heart possesses
a stem cell compartment that can regenerate myocytes and coronary vessels has raised the unique possibility to
rebuild dead myocardium after infarction, to repopulate the hypertrophic decompensated heart with new better
functioning myocytes and vascular structures, and, perhaps, to reverse ventricular dilation and wall thinning.
Cardiac stem cells may become the most important cell for cardiac repair.
I. INTRODUCTION
At the boundary of the second and third millennia,
the discovery that hematopoietic stem cells (HSCs) can
acquire cell lineages different from the organ of origin has
started a new intriguing scientific revolution (23, 178, 206,
212, 253, 266, 384, 392, 401, 473). The unpredicted behavior of HSCs has surprised and distressed many of us; they
disobey the dogma of embryonic specification and with
plastic changes undergo unexpected metamorphoses
(150, 384, 438, 487, 502). Because of their potential ability
to transdifferentiate (Fig. 1), HSCs have been proposed as
a novel form of cell therapy for damaged organs (27, 527).
However, this possibility was perceived with excitement
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and mistrust, which equally divided the scientific and
clinical community. The prevailing reaction on the part of
the skeptics was disbelief for this alternative view of stem
cell biology. Studies reporting negative findings were published, and questions were raised about the validity of the
shift in paradigm advanced in the early work (508). This
controversy is feeding a fire that may disrupt the stream
of scientific consciousness and delay the clinical application of a prospectively revolutionary therapeutic tool.
In addition to the plasticity of HSCs and their ability
to regenerate damaged organs, the function of resident
stem cells is to maintain tissue homeostasis in response to
perturbations. The documentation of the existence of organ-specific adult stem cells has created great expecta-
0031-9333/05 $18.00 Copyright © 2005 the American Physiological Society
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LERI, KAJSTURA, AND ANVERSA
tions concerning their utilization as a strategy for the
management of the human disease (36, 51, 59, 66, 139, 343,
374, 387, 457, 500). If the goal of medicine has been to
treat symptoms of diseases and possibly remove their
causes, scientists and physicians point now to a more
ambitious target. The primary objective of regenerative
medicine is the complete structural and functional recovery of the damaged organ (245, 315, 490). Regeneration
coincides with tissue homeostasis and involves the replacement of cells lost by normal wear and tear and,
ideally, following injury (143, 499). The regenerative capacity of organs is a property of particular significance in
organisms with long lifespan; in fact, the preservation of
the components of each tissue and their functional integration is essential for survival. Damage creates a barrier
to restitutio ad integrum and promotes the initiation of a
repair process that leads to the formation of a scar. Scar
formation is crucial for rapid handling of the damage, to
seclude the lesion from healthy tissue and to prevent a
cascade of uncontrolled deleterious events (41, 325).
However, the scar does not possess the biochemical,
physical, and functional properties of the uninjured tissue
and, therefore, negatively affects the overall performance
of the organ (52, 263, 345, 377, 435, 534).
In spite of the presence of adult stem cells in selfrenewing organs, tissue repair involves scar formation
(280, 410, 512). Understanding the factors that dictate the
evolution of a lesion into scarring instead of regeneration
is at its beginning. Some insights come from embryonic
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development. Tissue repair in embryos is rapid, efficient,
and scar free (143). Skin wounds in the early mammalian
embryo heal with restitutio ad integrum, whereas wounds
in late gestational fetuses and adult mammals result in
scarring. This difference may be related to the intrinsic
characteristics of embryonic fibroblasts or to external
stimuli. Fibroblasts in embryos are less prone to synthesize and release collagen, attenuating the amount of fibrosis following injury (287). The presence of hyaluronic acid
and fibronectin in the amniotic fluid may also interfere
with scar formation during wound healing (269). Moreover, the inflammatory response is attenuated; a low number of poorly differentiated inflammatory cells accumulate in the region of damage, and the growth factors
present at the site of healing in embryos are different from
those in the adult organism (143).
The expression of transforming growth factor-␤
(TGF-␤) isoforms, which have powerful profibrotic effects, and their receptors (TBRI, TBRII) vary in the skin of
embryos at different gestational ages (92, 143). TGF-␤3 is
more abundant in early embryo, while the amount of
TGF-␤1 and TGF-␤2 increases progressively with development. Similarly, TBRI and TBRII increase with maturation. The different ratios of TGF-␤ isoforms in the skin
appear to condition the evolution of healing triggering
profibrotic or antifibrotic signals in prenatal and postnatal
life (5). Adult wounds in mice, rats, and pigs, exposed to
the same profile of cytokines present in the embryo,
experience a more effective repair process with reduction
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FIG. 1. Plasticity of adult stem cells. Stem cells acquire cell phenotypes different from their organ of origin. The confocal image illustrates the
transdifferentiation of male c-kit positive bone marrow-derived cells (BMCs) into cardiomyocytes after infarction in a female mouse. Myocytes are
labeled by cardiac myosin heavy chain (red) and nuclei by propidium iodide (PI; green). The Y chromosome in nuclei is shown by white dots.
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II. EXOGENOUS PROGENITOR CELLS
AND CARDIAC REPAIR
In embryonic and early postnatal life, the increase in
cardiac mass reflects the delicate balance between the
addition of new myocytes and the death of unnecessary
cells (102, 393). Shortly after birth, cardiac growth occurs
by an increase in myocyte number and volume, which
together contribute to the development of the adult heart
phenotype (29, 31). In adulthood, the heart is characterized by a surprisingly high and rapid turnover of its parenchymal cells that is regulated by a stem cell compartment (51, 201, 293, 296, 308, 338). Under physiological
conditions, the intrinsic growth reserve of the heart is
sufficient to maintain cell homeostasis and adequate
pump performance. However, when an increase in pressure and/or volume load is imposed on the heart, myocyte
hypertrophy and proliferation in combination with myocyte apoptosis and necrosis constitute the elements of
myocardial remodeling (18, 20, 25, 27, 327).
Novel methodological approaches applied to the
analysis of the myocardium have challenged the paradigm
introduced more than 60 years ago that the heart is a
postmitotic organ and myocytes are terminally differentiated cells that participate in cardiac function throughout
life (7, 97, 288, 329, 339, 340, 354). A subset of parenchymal cells expresses the molecular components mediating
the entry and progression of these cells through the cell
cycle, and karyokinesis and cytokinesis. The recognition
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that myocyte replication, hypertrophy, and death occur in
the pathological heart has significantly enhanced our understanding of the dynamic nature of the myocardium and
the critical role that these variables have in the preservation of cardiac performance or in the onset of ventricular
dysfunction (22, 25, 27, 34, 326, 327). Contrary to the
general belief, the restricted regenerative capacity of the
myocardium does not represent the initial causal event of
impaired cardiac function. The nonischemic failing heart,
in its early phases of decompensation, has a number of
myocytes that often exceeds the number of cells in a
normal heart. Alternatively, the modest reduction in myocyte number chronically is not consistent with the severe
deterioration in ventricular performance. A typical example is found in hypertensive cardiomyopathy or chronic
aortic stenosis in animals and humans (29, 30, 497). The
initiation and evolution of cardiac failure appears to depend mostly on two other crucial factors that are strictly
interrelated in the determination of pump function: the
accumulation of old, poorly contracting cells and the
formation of multiple foci of myocardial scarring (52, 53,
67, 98, 484, 497).
A severe reduction in cell number, however, occurs
acutely after ischemic injury that inevitably results in scar
formation not only in the heart but also in other organs,
whether their parenchymal cells are highly proliferating,
slowly cycling, or terminally differentiated (263, 280, 410,
512). The need to overcome this biological obstacle has
favored the development of strategies aiming at the replacement of dead cells with viable cells. Several interventions have been used in an attempt to promote cardiac
regeneration experimentally. These protocols have employed different cell types, including fetal tissue and fetal
and adult cardiomyocytes (264, 395, 444, 544), skeletal
myoblasts (306, 323, 466), embryonic-derived myocytes
and endothelial cells (109, 142), bone marrow-derived
immature myocytes (194), fibroblasts (165), smooth muscle cells (272), and bone marrow c-kit positive and negative progenitor cells (144, 217, 226, 228, 356, 357, 539, 545).
Unexpectedly, these multiple approaches had a rather
uniform outcome that consisted of variable degrees of
improvement in cardiac contractile function. Most likely,
the implanted cells formed a passive graft, which reduced
negative remodeling by decreasing the stiffness of the
scarred portion of the ventricular wall. The elastic properties of the implanted cells could be the major cause for
enhanced heart function. An active graft, which dynamically contributed to myocardial performance, was observed only in a few cases (144, 228, 356, 357, 539). The
possibility has also been advanced that the implanted
cells exert a paracrine effect on resident cardiac stem
cells (CSCs) activating myocardial regeneration (50, 539).
Despite these efforts, however, the most appropriate form
of cell therapy for actual restitutio ad integrum of the
damaged myocardium has not yet been identified.
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in scar formation (143). Although scar-free regeneration
of damaged organs is well known in amphibians and
zebrafish (103, 385, 451), there is only one example in
mammals in which repair occurs in the absence of fibrosis. Cartilage, skin, hair follicles, and cryoinjured myocardium regrow in the adult MRL mouse (197, 262). However,
ischemia-reperfusion injury leads to comparable infarct
size in wild-type and MRL mice (3), suggesting that the
native capacity to reconstitute dead myocardium after
infarction may be limited in this model.
In summary, modulation of the inflammatory response may improve tissue repair in the adult organism.
However, the removal of these extrinsic signals may not
be sufficient for long-term regeneration of the injured
organ. Resident stem cells in proximity to the lesion can
change the properties of the microenvironment by secreting cytokines that contribute to cell homing, proliferation,
and differentiation (50, 239, 240, 291, 482). Enhancement
of the intrinsic growth reserve of the organ constitutes the
foundation of regenerative medicine and regenerative cardiology. This review discusses current understanding of
the role that endogenous and exogenous progenitor cells
may have in the treatment of ischemic and nonischemic
heart failure.
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A. Human Embryonic Stem Cells
Embryonic stem cells (ESCs), which are derived
from the inner mass of the blastocyst (337, 369, 397, 477),
possess unique properties. These cells can be grown in
vitro and propagated indefinitely in their undifferentiated
state while retaining a normal karyotype. Additionally,
they maintain over time the property of multilineage commitment and, therefore, can differentiate in vitro into all
cell types present in an organism (49, 88, 337, 525). Because of their enormous potential, human ESC lines have
been obtained from the human blastocyst (369, 477) with
the expectation of a future successful application of their
broad therapeutic potential to patients (124, 233, 370).
However, a controversy exists between the therapeutic
relevance of ESCs and autologous adult progenitor cells
(PCs). The debate concerning the efficacy of ESCs and
adult PCs is unfortunate because the utilization of ESCs in
clinical practice is at least a decade away while adult PCs
present minimal risks and are the only applicable form of
cell therapy for myocardial regeneration today. This does
not exclude that ESCs may become in the future a powerful alternative to adult PCs.
Human ESCs cultured in suspension form spontaneously cellular aggregates called embryoid bodies (EBs).
EBs are composed of cells derived from the three germ
layers (118). These teratoma-like structures consist of
semiorganized tissues composed of cardiomyocytes, striated skeletal muscle cells, neuronal rosettes, and hemoglobin-containing blood islands. However, they lack critical features of embryonic patterning (216, 231). The morphology of solid EBs can be affected by the process of
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cavitation that results in the formation of cystic EBs.
During their formation, markers of the mesoderm (␨globin), ectoderm (neurofilament 68), and endoderm (␣fetoprotein) are expressed (187, 216). Synchronously
pulsing EBs contain myogenic cells positive for ␣-cardiac
actin, desmin, myogenin, and smooth muscle actin (44,
234). Consistent with these in vitro observations, in vivo
delivery of human ESC lines to skeletal muscle or the
testis gives rise to teratomas (397, 459, 477). Teratomas
include endodermal, mesodermal, and ectodermal structures formed by mature cells (Fig. 2). Conversely, teratocarcinomas contain undifferentiated cells and possess a
stem cell compartment (16, 88). Although the malignant
tumorigenic potential of ESCs is not well defined yet, it is
worrisome that ESCs and the embryonal carcinoma (EC)
cells, i.e., the stem cell of teratocarcinomas, share several
markers including Oct4 and Nanog (88, 89, 193, 342, 363).
Moreover, the self-renewal property of ESCs and EC cells
is modulated by similar pathways (88, 191, 458). Conversely, the ability to induce formation of human EBs
containing cells of neuronal, hematopoietic, and cardiac
origins is important for the recognition of early human
embryonic development. In the last 15 years, this accessible in vitro system has been exploited to elucidate differentiation events in several tissues (118, 126, 397, 477).
The study of ESCs may provide a unique understanding of
the mechanisms of embryonic development that may lead
to therapeutic interventions in utero and the correction of
congenital malformations. This may become the most
exciting outcome of ESC research.
In view of the risks associated with the broad differentiation potential of ESCs in vivo, only sporadic reports
employed these cells in their uncommitted state to repair
the damaged heart (203, 243, 244, 312, 313, 509). In sharp
contrast to the pluripotency of ESCs, cardiomyocytes
were detected, but capillary structures and resistance
arterioles were not found. The reconstituted tissue did
not possess the characteristics of functionally competent
myocardium. However, human ESCs differentiate in endothelial cells in vitro and form microvessels in vivo in the
subcutaneous tissue of SCID mice (268). The differentiation of ESCs into cardiomyocytes has been attributed to
the paracrine effects of host signals, including members
of the TGF-␤ superfamily of proteins (50, 244). Although
some degree of functional improvement was observed,
the therapeutic utilization of ESCs can be associated with
dramatic side effects. Teratomas are benign tumors, but
the cardiac localization confers on them a malignant biological behavior. Most importantly, ESCs injected intravenously can engraft and colonize all organs with the possible development of neoplastic lesions (509).
In an attempt to avoid these complications, ESCs
have been partially differentiated in vitro before their
implantation in the injured heart (235, 284, 313, 535, 542).
Some degree of cardiomyogenic commitment was
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It is intuitively apparent that if the adult heart possesses a pool of primitive multipotent cells (51, 201, 293,
296, 338), these cells must be tested first before more
complex and unknown cells are explored. The attraction
of this approach is its simplicity. Cardiac regeneration
would be accomplished by enhancing the normal turnover of myocardial cells. However, major difficulties exist
in the acquisition of myocardial samples and the isolation
and expansion of CSCs in quantities that can be employed
therapeutically. Until protocols capable of resolving these
limitations are implemented, CSC therapy remains a difficult task. Alternatively, CSCs may be activated locally
within the myocardium, but the growth factor-receptor
systems that regulate CSC proliferation and differentiation are largely unknown (277, 335). Although the importance of resident CSCs in organ homeostasis is obvious
and, in the future, CSCs may become the most appropriate form of cell therapy for the diseased heart, currently,
emphasis has been placed on the identification of exogenous sources of highly proliferating cells that can acquire
the cardiac phenotypes.
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claimed to enhance engraftment of the cells into the
myocardium, attenuate the probability of ESCs to acquire
undesired cell lineages, and thereby reduce the risk of
teratoma formation (397, 477, 492). Although immature
myocytes have been obtained from ESCs and their morphological, phenotypical, and functional properties characterized (196, 234, 235), the residual growth potential of
the committed cells was not established. Additionally, the
purity of the preparation remains a major problem since
pluripotent ESCs may persist among the enriched population of developing myocytes and injection of this cell
pool may engender tumor formation. There are no rigorous methodologies for ESC differentiation into cardiomyocytes in vitro (62, 196, 198, 234, 235, 519, 548). It is
emblematic that eight distinct growth factors have forced
ESCs to acquire only in part a specific cell phenotype
while other undesired cells were consistently present in
the preparations (417). None of the growth factors was
capable of driving the commitment to a single cell type,
making it impossible, at present, to obtain a uniform
population of mature progeny from ESCs in culture.
However, the plasticity of ESCs is greater than that of
adult stem cells. The implantation of beating EBs reverses
complete atrioventricular block in pigs (235). The morphology of the grafted cells resembled those of embryonic
cardiomyocytes being of small size with disorganized
myofibrils located at the periphery of the cells. The mechanical and electrical behavior of beating EBs indicates
that human ESCs give rise to cells that display functional
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properties typical of the embryonic human myocardium.
When the electrophysiological analysis of the newly
formed cells was associated with the analysis of their
gene expression profile, the conclusion was reached that
cardiomyocytes derived from ESCs maintain a phenotype
reminiscent of the embryonic heart tube and hardly myocytes develop a chamberlike myocardial phenotype.
Whether these immature myocytes can reach adult characteristics in vivo remains an unanswered question (147).
The immunorejection of the allogeneic ESCs or their
differentiated progeny constitutes an additional limitation
inherent in the use of ESCs (128). Because of their early
stage of development, ESCs were considered “immune-privileged” and therefore not recognizable by the immune defenses of the recipient (477). Conversely, recent evidence
suggests that even in their undifferentiated state, human
ESCs express discrete levels of HLA class I antigens that
increase as the cells mature (127, 294). The immunosuppressive regimen is poorly tolerated, and this treatment severely
impairs a patient’s quality of life. The immunodeficient state
of mice that develop teratomas after ESC implantation (397,
477, 509) closely reflects the immunosuppressed condition
that may have to be reached clinically to avoid rejection of
ESCs. However, these limitations should not discourage the
promoters of ESCs. Intense research in this area is expected
to resolve these current biological problems and change the
skepticism concerning the future use of ESCs for human
disease.
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FIG. 2. Embryonic stem cells. During development, embryonic stem cells
(ESCs) give rise to the ectoderm, mesoderm, and endoderm. ESCs are obtained
from the inner cell mass of the blastocyst
and in vitro form embryoid bodies (EBs)
that contain undifferentiated (yellow),
ectodermal (green), mesodermal (blue),
and endodermal (red) cells. Implantation
of EBs in vivo can repair damaged organs
and generate tumors.
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B. Hematopoietic Stem Cells
Before discussing bone marrow cell (BMCs) transdifferentiation, a few comments concerning the developmental
origin of the hematopoietic system and the heart may be
relevant. Blood cells derive from the mesoderm (328, 516),
and the hematopoietic progenitors accumulate first in blood
islands in the early somites, migrate then to the fetal liver
and, ultimately, reach the bone marrow, which represents
their definitive localization in late embryogenesis and adulthood (328, 431, 516). Concurrently, the primitive heart is
generated within the anterior lateral mesoderm (100, 133,
138). Eisenberg et al. (134) have shown that the early mesoderm has the capability of creating both blood and cardiac
cells. The specification of organs that arise from adjacent
regions in the developing embryo may differ by a single
developmental decision, affecting the expression of a relatively small number of master switch genes (438, 485– 487).
This modification in cell fate has been defined as direct
transdifferentiation (56, 132, 426) to contrast the transdifferPhysiol Rev • VOL
entiation that requires a dedifferentiation step (256, 257, 316)
or the transdifferentiation that occurs through the involvement of a stem cell (10, 27, 178, 327, 392, 438, 487). If this
were also the case for the bone marrow and the heart,
differentiated BMCs could undergo a direct form of transdifferentiation and generate new myocardium. Although this
remains a possibility, committed CD45 positive myeloid
cells appear to be able to give rise to skeletal muscle cells or
hepatocytes (79, 80). However, so far, only c-kit-positive
immature myelomonocytic progenitors can contribute to
muscle fibers while more mature CD11b positive myelomonocytic cells do not transdifferentiate into skeletal
muscle (125).
Embryonic hematopoietic progenitors acquire the cardiomyocyte phenotype in the presence of retinoic acid,
dexamethasone, PGE2, interleukin (IL)-2, fibroblast growth
factor (FGF)4, and bone morphometric protein (BMP)4
(136). Although the efficiency of transdifferentiation is low,
the committed embryonic progenitors express transcription
factors and contractile proteins of cardiac muscle cells. The
creation of cardiomyocytes is markedly increased when lineage negative Sca-1-positive adult BMCs are cocultured with
embryonic mesoderm. The new myocytes integrate functionally with the embryonic tissue (100). Together, these
lines of evidence support the notion that BMCs can form
cardiac and skeletal muscle cells in vitro and in vivo. This
possibility has been strengthened by recent observations in
which c-kit-positive fetal liver hematopoietic progenitor
cells have been employed successfully to regenerate myocytes and coronary vessels in the infarcted heart (258).
These hematopoietic progenitors were obtained from
cloned embryos derived from somatic cell fusion between
nuclei of LacZ-positive fibroblasts and enucleated oocytes of
a different mouse strain (256, 257, 259).
The newly formed myocardium replaced 38% of the
infarct at 1 mo (258). The rebuilt tissue expressed LacZ and
was composed of myocytes and vessels connected with the
primary coronary circulation. Myocytes were functionally
competent and expressed contractile proteins, desmin, connexin43, and N-cadherin. These structural characteristics
indicated that the developing new myocytes were coupled
electrically and mechanically (258). Similarly, the formed
coronary arterioles and capillary structures contained blood
cells and contributed, therefore, to tissue oxygenation. Cardiac replacement resulted in an improvement of ventricular
hemodynamics, attenuation of cardiac remodeling, and reduction of diastolic wall stress. These beneficial effects were
obtained by stem cell transdifferentiation and commitment
to the cardiac cell lineages. In fact, myocardial growth was
independent from fusion of the injected progenitor cells
with preexisting partner cells. Although problems currently
plague nuclear transplantation, including the potential for
epigenetic and imprinting abnormalities, fetal hematopoietic
progenitor cells derived from cloned embryos are sufficiently normal to repair dead myocardium in vivo.
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Finally, the potential application of human ESCs poses
profound ethical concerns. A fundamental issue involves the
notion of the moral status of human embryos. However,
when the ethical principles are taken to the extreme, they
threaten to paralyze public funding for ESCs (523), leaving
experimentation to the private sector and, thereby, precluding the possibility to effectively monitor practices or the
search for alternatives (149, 167, 285). The privatization of
ESCs has reached a point that protocols of cryopreservation
have been developed in spite of the fact that we are far from
the actual documentation of the efficacy and safety of ESCs.
Active research on human ESCs is highly desirable, but the
duty to heal sick individuals cannot overcome the moral
imperative to treat human beings as subjects and not as
objects. An interesting viewpoint has recently been proposed (254). In parallel with the definition of organismic
death of the adult, which occurs when the criteria for brain
death are met, an embryo can be considered organismically
dead when it has lost a fundamental function. This consists
of continued, integrated cellular division, growth, and differentiation. This premise implies that irreversible arrest of cell
division rather than the death of each and every cell corresponds to the organismic death of the embryo (189, 254).
Nearly 60% of the in vitro fertilized embryos fail to cleave at
24 h (261). Often, this growth inhibition reflects severe genetic abnormalities but not necessarily all the cells of the
embryo are abnormal. In these mosaic embryos, at least one
normal diploid blastomere is found (254). Thus the ethical
framework for organ donation can be applied to the harvesting of human ESCs from dead embryos. This would provide a common basis in which the need to protect human
dignity and progress in biomedical research are no longer
in conflict.
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Consistently, the injection of BMCs improves the performance of the pathological heart (144, 217, 228, 356, 539),
but the mechanism by which the administration of BMCs
results in enhanced cardiac function remains controversial (135, 137, 200, 247, 295). In spite of these uncertainties, clinical trials have been completed and some are
ongoing (39, 69, 158, 163, 186, 273, 372, 373, 415, 421, 448,
449, 452, 528). The safety of this approach has been
documented, and double-blind clinical studies are in
progress. Limitations in the analysis of myocardial regeneration in humans due to the difficulty in obtaining cardiac biopsies, together with contrasting findings in animals, have prompted different interpretations of the positive outcome of BMC administration. Three possibilities
have been advanced. They include the development of
coronary vessels that rescue hibernating myocardium, de
novo formation of myocytes and vascular structures, or
the activation and growth of resident progenitor cells via
a paracrine effect mediated by the implanted BMCs (17,
50, 135, 144, 217, 226, 228, 239, 356, 357, 460, 539, 545).
These mechanisms of action of BMCs are not mutually
exclusive and may be operative in the rescue of the
injured heart. However, it is relevant to discuss available
information to establish what has been shown so far and
what has to be done to document unequivocally the actual
role of BMCs in the management of cardiac diseases. This
analysis can only be performed by comparing protocols
and data accumulated in animal studies.
The efficacy of adult BMCs for myocardial regeneration
after infarction was documented four years ago (356). BMCs
of male mice heterozygous for EGFP were collected, immunodepleted for lineage markers, and then sorted for the stem
cell antigen c-kit. Enriched lineage negative c-kit-positive
cells were injected in the border zone of infarcted mice
where they colonized to the dead tissue and gave rise to
contracting myocardium occupying 68% of the original infarct. The newly formed EGFP-Y-chromosome positive cells
corresponded to functionally competent myocytes and vascular cells organized in coronary arterioles and capillary
structures. Overall, the aspect of the newly formed myocardium was that of a rather immature tissue with myocytes
small in size, ⬃500 ␮m3, and vessels with a small lumen and
a thick multilayered wall. Myocardial regeneration with
amelioration of cardiac performance was obtained only in
40% of the treated mice. Coronary ligation in mice is a
complex procedure with an inherent variability in infarct
size and a 50% probability of correct injection (228, 356). The
mouse heart beats ⬃600 times/min and has a left ventricular
(LV) wall that is ⬍1 mm thick. These factors make the
injection of cells within the LV wall highly problematic.
When these technical difficulties were overcome by the
mobilization of BMCs with the systemic administration of
stem cell factor (SCF) and granulocyte-colony stimulating
factor (G-CSF), myocardial regeneration was obtained in all
infarcted treated animals. The new myocardium was com-
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The adult bone marrow contains several cell populations. In addition to differentiated cells, such as stroma
cells, vascular cells, adipocytes, osteoblasts, and osteoclasts, primitive cells reside in the bone marrow. This
class of undifferentiated cells with stem cell properties is
heterogeneous (383, 431, 457, 516), being composed of
HSCs and mesenchymal stem cells (MSCs). These two
subsets of cells have been employed in the repair of
damaged organs giving rise to tissues distinct from the
organ of origin (178, 206, 245, 253, 295, 315, 390, 392, 473,
490). However, a mixed population of bone marrow cells
rather than a highly purified stem cell pool has commonly
been used for the restoration of injured organs (64, 68,
131, 211, 275, 357, 360, 388, 475). In the context of this
review, we have defined this heterogeneous cell population as BMCs, i.e., a cell preparation that contains an
enriched pool of HSCs together with variable degrees of
MSCs and endothelial progenitor cells.
In 1961, Till and McCullock discovered clonogenic
bone marrow cells that gave rise to multilineage hematopoietic colonies in the spleen (48, 436, 479). Till and
McCullock (480) proposed that these cells were multipotent HSCs that possess the properties of self-renewal and
multilineage differentiation. The definition of stem cells
has not changed, and bone marrow HSCs are functionally
defined by their capacity to self-renew, form clones, and
differentiate into mature blood cell types (431, 516, 517).
Although HSCs were discovered more than four decades
ago, their isolation has become possible only more recently when surface markers were identified (445, 493,
517). Different epitopes characterize HSCs in different
species. Mouse HSCs correspond to a subpopulation of
BMCs that are c-kit⫹Sca-1⫹Thy-1-low (358, 493). Operationally, this population is multipotent and repopulates
the bone marrow of irradiated mice (493). However, the
c-kit⫹Sca-1⫹Thy-1-low cells are functionally heterogeneous. Based on the identification of additional surface
markers and clonal analysis, the Mac-1-negative-CD4-negative cells within this category are enriched for long-term
reconstituting cells (320). Conversely, the Mac-1-lowCD4-negative pool contains short-term repopulating cells.
Finally, the Mac-1-low-CD4-low cells correspond to transient multipotent progenitors together with B lymphocyte
progenitors (319). The CD34 antigen has been used to sort
human HSCs (331) which, however, are also present in
the CD34 negative fraction (123, 181). AC133 is another
marker of human HSCs (57) but, so far, there is no specific epitope for the true stem cell.
The discovery that adult HSCs or BMCs retain a
remarkable degree of developmental plasticity and may
have the potential to differentiate across boundaries of
lineage and tissue (9, 10, 150, 438, 487, 502) has divided
the scientific and clinical community (324, 507, 508, 518).
Therefore, the therapeutic efficacy of BMCs for the damaged heart has been questioned (112, 137, 295, 309, 508).
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vascularization and cardiomyogenesis (539). Thus these
findings support the notion that BMCs adopt the cardiac
phenotype and potentiate the growth reserve of the adult
heart. Collectively, these observations point to the therapeutic import of BMCs for cardiac diseases in humans
(17, 39, 69, 158, 163, 239, 273, 372, 373, 415, 421, 448, 449,
452, 528).
C. Mesenchymal Stem Cells
There is no accepted definition of MSCs. Originally,
MSCs were thought to correspond to the putative marrow
cells that self-renew and give rise to one or more mesenchymal tissues (314). However, the marrow stromal cell
population that contains the MSC pool can also differentiate into tissues other than those that originate in the
embryonic mesoderm, raising questions about the appropriateness of the term mesenchymal for this type of stem
cell (413, 505, 529). In the majority of studies, the adherent fibroblastic cells obtained from the unfractionated
mononuclear cell class of the bone marrow are termed
mesenchymal stem or progenitor cells (8). But this heterogeneous cell population is recognized to be too “crude”
to represent the purified pool of MSCs (383). A common
and widely used characterization of MSCs is that they are
the nonhematopoietic multipotent stem cells of the bone
marrow (314, 383).
The notion of MSCs was introduced in 1961 by
Friedenstein (154) who documented that marrow stroma
cells contain osteogenic progenitors. Subsequently, the
culture conditions necessary for the expansion and differentiation of this cell category were developed. Marrow
stromal cells were found to be highly proliferative, clonogenic, and capable of forming colonies of different size
and density, composed of several mesenchymal lineages
(86, 87, 155, 453, 465). In the late 1980s and in the 1990s,
MSCs acquired a significant biological role with the work
performed by Caplan (84), who suggested that the actual
MSC is an adherent fibroblastic cell isolated by Percoll
density centrifugation. This postulated MSC expressed
antigens reactive with the monoclonal antibodies SH2 and
SH3 that recognize CD105 and CD73, respectively (195).
The disadvantage of this traditional method of isolation is
the inevitable contamination from HSCs and the cellular
heterogeneity of the preparation. Over the past decade,
Caplan’s definition has been questioned as being not restrictive enough for the ideal MSC. Although CD105 is a
relevant epitope of MSCs and angiogenesis (314, 382),
CD29, CD44, and CD90 have been proposed to be important determinants of the mesenchymal phenotype (382,
383) while STRO-1 may identify an immature state (175).
The immunophenotype of MSCs has only been partially
determined, although the SH2, SH3, CD29, CD44, CD71,
CD90, CD106, CD120a, and CD124 epitopes have consis-
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posed of contracting myocytes and patent coronary vessels
(357).
These studies prompted other investigators to document the ability of BMCs to repair the damaged heart
(144, 217, 226, 228, 334, 411, 539, 545). Initially, the hypothesis tested was whether spontaneous mobilization of
BMCs occurs after infarction and whether these circulating cells home to the region of injury rescuing the dead
tissue (217). Although the degree of engraftment and
transdifferentiation in coronary vessels was low and
markedly exceeded that of cardiomyocytes, this report
confirmed the plasticity of BMCs. The difference in the
degree of myocardial reconstitution between this and the
previous studies can be attributed to several factors. They
include the pathologic model, ischemia-reperfusion injury
in irradiated mice (217) versus permanent coronary occlusion in nonablated mice (228, 356, 357), together with
the modality of intervention, spontaneous mobilization of
BMCs (217) versus intramyocardial injection of BMCs
(228, 258, 356) or cytokine-mediated BMC mobilization
(357), and the type of BMCs, c-kit enriched BMCs versus
peripheral blood cells. The bone marrow of irradiated
mice was repopulated with a subset of cells capable of
excluding the Hoechst 33342 dye (217). These cells are
positive for both c-kit and Sca-1 and are probably more
enriched for true HSCs than the lineage negative c-kit
positive cells (112) implemented in other studies (228,
356, 357). In spite of several unresolved issues, these
observations are consistent with the notion that BMCs
can adopt the cardiogenic fate by forming myocytes and
coronary vessels.
More recently, the possibility that cell therapy of the
infarcted heart exerts its beneficial effects not only by
reconstitution of dead myocardium but also by the activation of resident progenitor cells in the spared portion of
the ventricular wall has been carefully examined (228,
291, 539). To properly evaluate the formation of cardiomyocytes and coronary vasculature in the region bordering the infarct and distant from the infarct, the accumulation of newly formed myocytes and vascular structures
was measured utilizing markers of the cell cycle and
morphometric methods. Although similar protocols were
employed in the analysis of myocyte and vessel growth,
positive and negative results were obtained concerning
the paracrine effects of the administered BMCs (228, 539).
However, an enriched population of mouse c-kit-positive
BMCs was used in one case (228) and a novel human bone
marrow stem cell in the other (539). Both cell populations
differentiated into cardiac cell lineages and repaired the
infarcted heart, but the latter also promoted a robust
regenerative response in the surviving myocardium. Thus
a specific human BMC has the ability to transdifferentiate
in cardiac muscle cells, smooth muscle cells, and endothelial cells in vitro and in vivo (144, 539). Additionally,
this unique cell population can induce endogenous neo-
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telomerase incompetent and have short telomeres. The
senescent phenotype can be reversed by forced expression of human telomerase (2).
During the phase of amplification, MSCs do not differentiate spontaneously but do so in the presence of
growth factors and cytokines (72, 85, 456). They can
acquire multiple phenotypes including osteoblasts, chondrocytes, adipocytes, endothelial cells, and neuronal-like
cells (99, 221, 286, 382, 383, 405, 412, 422, 538). Bone
marrow MSCs differentiate into cardiomyocytes in the
presence of 5-aza-cytidine (159, 185, 290); the morphology
of the cells changes from a spindle-shaped to a ball-like
form and, subsequently, a rod-shaped configuration. The
cells then fuse in a syncitium-like structure that resembles
a myotube (160). Although these characteristics mimic
the organization of skeletal muscle cells, the committed
progeny expresses markers commonly found in cardiomyocytes at different stages of fetal development (185).
Transcription factors of the cardiac and myocyte lineage,
GATA4, Nkx2.5, and HAND1/2, have been detected (159).
Interestingly, a switch in MEF2 isoform occurs. From
early to late passages, MEF2C is replaced by MEF2A and
MEF2D. Additionally, the ␤-isoform of cardiac myosin
heavy chain is more abundant than the ␣-isoform. Similarly, ␣-skeletal actin predominates, and ␣-cardiac actin is
moderately present. Myosin light-chain 2v is also expressed. Functionally competent ␣- and ␤-adrenergic and
muscarinic receptors have been found (185). Cells beat
spontaneously and synchronously, and the rate of contraction increases with stimulation by isoproterenol. Conversely, contractile activity is inhibited by ␤1-blocking
agents (290). Thus the differentiation process of these
cardiomyogenic cells recapitulates in part the developmental program of gene expression of prenatal life.
An elusive stem cell that may or may not belong to
the MSC category is the primitive cell identified by Verfaillie and co-workers (225). This human or mouse multipotent adult progenitor cell (MAPC) can be obtained by
growing CD45 and glycoprotein A-depleted BMCs in selective culture conditions (Fig. 3). When this cell population is plated at low density in laminin-coated dishes with
a low serum medium containing EGF and PDGF, MAPCs
appear several months later after ⬃20 population doublings. In this in vitro system, MAPCs differentiate into
cells of the three germ layers including hepatocytes, endothelial cells, and neurons (398, 419). This multilineage
differentiation occurs also in vivo when MAPCs are injected in the early blastocysts of mice. However, in adult
sublethally irradiated immunodeficient mice, the distribution of systemically delivered MAPCs is much more restricted. MAPCs engraft only in hematopoietic tissues,
lung, liver, and intestine but not in the heart (Fig. 3).
Whether MAPCs injected locally in the myocardium acquire the myocyte lineage has not been tested. In sum-
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tently been found in MSCs (85, 383, 456). Moreover, MSCs
are negative for markers of the hematopoietic lineage,
including CD34 and the common leukocyte antigen CD45
(43, 383). However, Prockop and others (105, 423, 439)
support the notion that MSCs cannot be distinguished
solely by antigen expression but necessitate functional
assays demonstrating their multipotent growth and differentiation behavior. Thus a definitive consensus on the
properties of MSCs has not been reached yet.
Putative MSCs have been identified in embryonic,
fetal, and postnatal organs (84, 389). During prenatal life,
mesenchymal progenitors accumulate in sites harboring
HSCs (307). MSCs appear in the aorta-gonad-mesonephros region and colocalize with HSCs (117). Additionally, MSCs are found in the embryonic circulation including the cord blood and amniotic fluid (213, 214, 400, 489).
In adulthood, the bone marrow and the systemic circulation constitute the main sources of MSCs (389, 402),
although they have also been detected in tissues distant
from the bone marrow. The oval cells of the liver, prostatic stem cells, metanephric mesenchymal cells, precursors of the Leydig cells in the testis, primitive osteoprogenitors, preadipocytes, and satellite cells of the skeletal
muscle have been classified as MSCs (13, 114, 399, 488,
543, 552). Because not all studies agree that MSCs reside
in these mesenchymal tissues, the possibility has been
raised that a long-distance traffic of MSCs occurs between
the bone marrow and distant sites through the bloodstream. Alternatively, embryonic primordia of MSCs may
be stored in organs of mesodermal origin and, in response
to injury, may participate in tissue repair (383).
Characteristically, MSCs adhere quickly to the culture dish and grow, forming colonies that become visible
1 wk after plating (314, 383, 389). Only 0.001– 0.01% of the
initial unfractionated bone marrow cell population consists of MSCs (383, 389). Some preparations of MSCs can
be expanded over 15 cell doublings, while others cease
replicating after ⬃4 cell doublings (389). This difference
may depend on several determinants, including the procedure used to harvest the marrow (314), the low frequency of MSCs in marrow harvests (84), and the age or
condition of the donor from which MSCs were prepared
(450). This limited growth potential is associated with
preservation of the karyotype, telomerase activity, and
telomere length (381, 382). The constant level of telomerase activity and the absence of telomeric shortening have
been interpreted as unique properties of MSCs, which
were considered capable of escaping replicative senescence. However, MSCs from young donors experience
⬃20 population doublings while MSCs from old donors
are compromised in their proliferative capacity (381, 450).
Extensive subcultivation impairs cell function, and evident signs of senescence (119) and/or apoptosis (314)
become apparent. Both young and old MSCs undergo
senescence-associated growth arrest (314); they become
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LERI, KAJSTURA, AND ANVERSA
mary, MAPCs could represent a rare population of MSCs
or a by-product of long-term culture.
Although MSCs are capable of self-renewal and multilineage differentiation, whether they are involved in the
physiological turnover of cells in organs in which they
reside remains unclear. However, MSCs are considered a
powerful cell for reconstructive and gene-targeting therapy (245). They are easily transfected with engineered
DNA (15, 291) and possess an extended survival after
transplantation (461) together with the inherent ability to
differentiate into several cell classes. The clinical application of MSCs for the regeneration of cartilage and bone
is a good example of MSC safety and efficacy (40, 46).
Clinical trials based on the therapeutic potential of MSCs
have been conducted or are in progress throughout the
world; MSCs have been injected intravenously in children
with a genetic disorder of the bone, osteogenesis imperfecta (207–209). This was the first study demonstrating
that bone marrow MSCs engraft outside of the organ of
origin and produce a beneficial effect (208). Recently,
autologous MSCs have been given to patients affected by
cancer in an effort to enhance hematopoietic recovery
during chemotherapy (242). Reports in animals have
shown that bone marrow MSCs home to the heart and
promote regeneration of the infarcted myocardium in
different species (434, 483), possibly involving a paracrine
effect (169, 291). Whether MSCs will be implemented in
the management of ischemic heart failure in humans is
difficult to predict, but it is a likely possibility.
Autologous MSC-derived myogenic cells (321) and
autologous undifferentiated MSCs (424) have been emPhysiol Rev • VOL
ployed for cardiac repair after infarction or ischemiareperfusion injury in pigs. Cells were injected directly in
proximity or within the injured myocardium, acutely or
chronically after the ischemic event, and the effects of
these interventions were determined several weeks later.
In all cases, myocardial regeneration was documented
and the reconstitution of dead myocardium correlated
with the improvement in ventricular function and reappearance of wall motion activity (321, 424). Moreover,
cotransplantation of human MSCs and human fetal cardiomyocytes resulted in an amelioration of cardiac performance greater than with MSCs alone (311). Formation
of myocytes and coronary vessels has also been observed
in rats after cryoinjury and MSC implantation (355). However, the intracoronary delivery of MSCs after infarction
led to the differentiation of MSCs into fibroblasts in the
scarred region and to the regeneration of myocytes in the
surviving, unaffected portion of the ventricular wall (506).
Importantly, in the absence of injury, MSCs engraft into
the myocardium but remain in a viable quiescent state and
do not participate in the physiological turnover of myocytes as well as vascular smooth muscle and endothelial
cells. Thus the microenvironment significantly conditions
the developmental pathway of MSCs in vivo.
D. Controversy on Bone Marrow Cell
Transdifferentiation and Cardiac Repair
Stem cell transdifferentiation in the adult organism is
a highly questioned mechanism of growth (9, 10, 150, 178,
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FIG. 3. Multipotent adult progenitor
cells (MAPCs). MAPCs are isolated from
the bone marrow and have the potential
to differentiate in many different cell
types in vitro and in vivo. However,
MAPCS give rise to a more limited number of somatic cells when injected in vivo
in the adult mouse than in the early
blastocyst.
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Physiol Rev • VOL
cardium to a modest recovery of ventricular performance
in the absence of tissue regeneration. The lack of beneficial consequences on the heart has also been reported.
However, predominant observations have been positive
supporting the feasibility of this form of cell therapy for
the failing heart.
Although these experimental results and the therapeutic efficacy of BMCs in patients with ischemic and
nonischemic heart failure were indicative of BMC transdifferentiation (Fig. 4), the plasticity of BMCs has been
questioned. Recent publications in mice have emphasized
negative results, criticizing the documentation that BMCs
can convert into cardiac cell lineages. The ability of BMCs
to regenerate dead myocardium in the mouse heart was
questioned, and the claim was made that the fate of the
injected cells was to acquire only the hematopoietic lineages (45, 324). An additional report suggested that the
engraftment of BMCs in the damaged heart is transient
and hematopoietic in nature. BMC-derived cardiomyocytes were observed at low frequency and only outside
the infarcted myocardium (336).
Six possibilities can account for these contrasting
results: 1) differences in the experimental protocol, 2)
differences in the methodology of detection of newly
formed structures, 3) differences in the viability of the
injected cells, 4) artifacts due to autofluorescence and
erroneous interpretation of immunolabeled structures, 5)
relevance of animal models of parabiosis and repopulation of ablated bone marrow with an ideal HSC, and 6) a
combination of these factors. We will discuss each of
these variables.
The intramyocardial delivery of BMCs in the mouse
heart is complex and faced with difficulties dictated by
the extremely high heart rate and very thin LV wall.
Laboratories with years of experience in inducing coronary artery occlusion, coronary artery narrowing, or ischemia-reperfusion injury in mice are well aware that the
successful imposition of myocardial infarction is ⬃80%,
with ⬃20% unsuccessful surgery (115, 228, 356, 357, 539).
Additionally, in positive cases, infarct size can vary from
⬃20 to ⬃90% of the entire LV inclusive of the interventricular septum. Infarcts involving ⬃50 – 60% of the LV
carry a nearly 50% mortality within 24 – 48 h after surgery
(33). Even more problematic is the recognition of a successful administration of BMCs within the LV myocardium, ⬃40 –50%, and the identification of the site of injection in proximity of the infarcted tissue (228, 356).
Because of these unpredictable factors, methodologies have been developed in which rhodamine-labeled
polystyrene spheres are mixed with BMCs to have a reliable reference point for the recognition of the actual
positioning of BMCs in the LV wall and with respect to the
infarct. When this simple protocol was applied, myocardial regeneration was demonstrated in 100% of mice,
which had rhodamine particles and BMCs implanted in
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206, 253, 279, 384, 392, 438, 473, 487, 502, 507, 508, 518).
The most versatile cell is the bone marrow progenitor
cell. The documentation that adult BMCs are capable of
generating mature cells beyond their own tissue boundaries has provided an unexpected and powerful new form
of cell therapy. The wave of enthusiasm created by this
discovery and the lack of effective new drugs for the
treatment of heart failure has prompted cardiologists to
the rapid implementation of BMCs in the management of
the infarcted human heart. Currently, bone marrow cells
or circulating bone marrow cells, including endothelial
progenitor cells (EPCs), are utilized in patients, and several initial clinical trials have been performed (39, 69, 158,
163, 273, 372, 373, 415, 421, 448, 449, 452, 528). Because of
the compelling need to seek new treatments for severely
ill patients, clinicians are leading the field of cell therapy
and myocardial regeneration. Conversely, statements of
caution have been made concerning the necessity to acquire a better understanding of the mechanisms of recovery of cardiac function before these protocols are introduced in the treatment of the diseased human heart. The
fundamental question to be addressed is whether this
more conservative position is justified because strong
criticisms have been made not only against the notion of
stem cell plasticity (45, 95, 279, 324, 474, 507, 508, 518) but
also against observations that have challenged the perennial view of the heart and the brain as postmitotic organs
incapable of regenerating after birth (508). In fact, the
revolutionary work on the brain and the heart that has led
to the identification of neural stem cells (139, 500) and
cardiac stem cells (51, 201, 293, 296, 338) has been attacked as inconclusive, methodologically incorrect (508),
and, more recently, a collection of artifacts (45, 95, 324, 495).
The early studies on BMCs and cardiac repair were
followed by numerous reports in which different types of
BMCs and modalities of interventions were employed for
the treatment of the damaged heart (24, 26, 34). These
cells of bone marrow origin included several subsets making it difficult to dissect the impact that each cell type had
on the function and structure of the diseased heart. For
example, lineage-negative c-kit-positive cells (228, 258,
356), lineage-negative c-kit-positive Sca-1-positive cells,
c-kit-positive-Thy1.1-low-lineage-negative Sca-1-positive,
long-term reconstituting HSCs (45), CD34-positive cells
(545), mesenchymal-like progenitor cells (424, 434), EPCs
(281, 282), mononuclear BMCs, and clonally expanded
human multipotent stem cells (539) have been tested.
Moreover, BMCs with the potential of restoring the dead
myocardium have been mobilized from the bone marrow
into the systemic circulation utilizing several cytokines;
SCF, G-CSF, and SDF-1␣ have been administered alone or
in combination in various animal species (1, 38, 162, 188,
334, 357, 462). The effects of these various modalities of
therapy ranged from improvement in regional cardiac
function together with the restoration of the dead myo-
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the border zone of the infarcted myocardium (228).
Therefore, differences in the protocols employed in the
administration of BMCs could account for the differences
observed experimentally between positive studies of
myocardial regeneration (228, 356, 539) and reports challenging the ability of BMCs to form myocytes and coronary vessels (45, 324). Moreover, the negative studies
claim that cell transplantation was 100% successful, that a
mortality of 8% occurred with infarcts of 60%, and that a
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FIG. 4. Myocardial regeneration by BMCs. A: newly formed myocytes (troponin I; red) created by the injection of mouse male EGFP
tagged c-kit positive BMCs in a female infarcted mouse heart. Nuclei are
stained by PI (blue). Laminin defines the boundaries of the cells (white
lines). The male phenotype of the regenerated myocytes is demonstrated
by the presence of the Y chromosome in nuclei (yellow dots). B: a
significant fraction of new myocytes expresses EGFP in the cytoplasm
(green). C: the merge of A and B shows the colocalization of troponin I
and EGFP (yellow-green) in the male myocytes.
normal LV end-diastolic pressure was found with infarcts
commonly incompatible with life in this model (45).
A critical issue in studies of myocardial regeneration
mediated by transplantation of BMCs involves the morphological analysis of the heart. This is an important point
because one of the reports questioning the ability of
BMCs to form myocytes and coronary vessels utilized
conventional light microscopy and immunoperoxidase
staining to demonstrate the presence or absence of newly
formed structures within the infarcted myocardium (324).
This approach reflects the contention that conventional
light microscopy is superior to fluorescence labeling and
confocal microscopy (250, 467). The resolution in images
obtained by light microscopy is markedly inferior to that
provided by confocal microscopy (28). Additionally, only
the very superficial layer of the section can be examined
by light microscopy (Fig. 5), whereas the entire thick
histological section can be viewed by confocal microscopy (391). At high-magnification light microscopy, the
observer is faced with a blurred image with loss of detail
and undefined cell boundaries. This inherent problem
with light microscopy was recognized immediately when
confocal technology became available (521). Clear examples were published documenting the impossibility of
identifying microtubules, mitotic spindle, cytoplasmic
proteins, and chromosomal structures by light microscopy of cultured cells. Conversely, these morphological
details were apparent when the same cells were evaluated
by confocal microscopy. The difference between epifluorescence microscopy or bright-field microscopy and confocal microscopy is magnified in tissue sections. In fact,
focal depth in a 5- to 6-␮m tissue section examined by
epifluorescence microscopy or bright-field microscopy is
only 8%, while the focal depth in a 0.5-␮m optical section
analyzed by confocal microscopy is 12.5-fold higher, i.e.,
100% (29, 283; Fig. 5).
Problematic is the decision whether frozen tissue
samples or paraffin-embedded specimens have to be employed for the immunohistochemical analysis of the myocardium by confocal microscopy. Both preparations have
their advantages and their limitations. Perfusion fixation
and paraffin processing of the heart facilitates morphological studies but inevitably leads to the extraction of
some proteins and RNAs complicating the interpretation
of negative results. Conversely, frozen section immunolabeling preserves better antigenicity but compromises
structural detail. For the injured heart in particular, frozen
samples introduce artifacts affecting the quality of the
sections and microscopic resolution. The infarct is rarely
preserved in frozen sections, and this problem interferes
with the recognition of the region of the ventricle being
examined.
Paraffin-embedded tissue sections result, at most, in
an underestimation of specific immunostaining not in an
overestimation and, therefore, differences in the technical
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ADULT STEM CELLS
approach could explain why positive and negative results
concerning BMC transdifferentiation have been obtained.
Hearts studied immunocytochemically should be the
same examined functionally and with routine histology.
This was not the case in the negative studies (45, 324).
Whether coronary ligation was unsuccessful or a small or
large infarct was generated was not determined. This is
critical, because of the complexity of the model and the
difficulty of producing an infarct and a correct injection of
BMCs. Moreover, the utilization of frozen sections may
interfere with the recognition of small cells of the size of
newly formed myocytes (115, 228, 258, 356). Double labeling for the identification of the transgene present in the
administered BMCs and the detection of myocyte cytoplasmic proteins was never performed on the same section (324), and negative claims were based on only two
animals (45) that were supposedly properly infarcted and
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FIG. 5. BMC transdifferentiation in the infarcted heart: light microscopy histochemistry and confocal microscopy immunofluorescence. A
comparison is made between a section of 5– 6 ␮m in thickness examined
by light microscopy (A) obtained from Reference 324 and optical sections of 0.5 ␮m analyzed by confocal microscopy (B). The image in A
was used to demonstrate the lack of transdifferentiation of BMCs into
myocytes. Conversely, the image in B documents the ability of BMCs to
adopt the cardiomyogenic fate. The brown cells in A should correspond
to EGFP-positive cells that were not stained by any myocyte marker and
were considered adequate for the unequivocal demonstration of the
absence of BMC transdifferentiation. There is no double staining in this
preparation for EGFP and myocyte cytoplasmic proteins. In contrast, B
illustrates newly formed myocytes, ⬃400 ␮m3 in volume, that are positive for EGFP (yellow-green). These cells express GATA-4 in their nuclei
(yellow), and connexin43 is detected at the cell boundary (white, arrows). Focal depth in A is 8% and in B is 12.5-fold higher, 100%.
injected with cells comparable to those used in the positive studies (356). This limited cohort calls the statistical
validity of this type of experimentation into question, an
issue that has recently been recognized in a large number
of published reports in Nature and Nature Medicine
(496). When these problems are dealt with, myocardial
regeneration mediated by BMC transdifferentiation can
also be documented by immunolabeling of frozen sections of infarcted myocardium (539).
The animal models of parabiosis with complete
blood chimerism or with reconstituted bone marrow
through the injection of a single EGFP positive HSC (45,
507, 508) have been introduced to question the ability of
BMCs to acquire a cardiomyocyte lineage. The utilization
of the irradiation protocol in studies aiming at the documentation of the transdifferentiation potential of adult
stem cells has been criticized (4, 297). Irradiation represents a form of injury that complicates the interpretation
of the results. Most importantly, it cannot be used as
documentation of physiological trafficking of cells from
the bone marrow to distant organs. In fact, bone marrow
transplantation in mice affected by amyotropic lateral
sclerosis typically shows cell engraftment in the central
nervous system (CNS) shortly after the intravenous injection of BMCs. But this process decreases with time (110).
Because the damage is not repaired and the CNS continues to send the same signals for eventual cell recruitment,
the progressive decline in BMCs colonized to the CNS
strongly suggests that homing of these cells occurred only
during the phase of high concentration of repopulating
cells in the bloodstream. The administered BMCs targeted
the depleted bone marrow, and their circulating pool
markedly decreased with time. After the initial engraftment, there was no longer translocation of cells from the
bone marrow to the CNS (110). Thus positive and negative results obtained with this protocol are difficult to
interpret.
The limitation of the therapeutic potential of circulating BMCs is not new. If circulating BMCs had the ability
to spontaneously repair damaged organs, infarcts of the
heart, brain, skin, kidney, and intestine would be easily
reconstituted and the majority of current human diseases
would not exist. These models are of little value to resolve
the controversy at hand and can only add confusion to the
confusion. The issue in need of resolution is whether
BMCs injected directly in the infarct, in the border zone or
systemically, differentiate into cardiac cell lineages and
contribute to myocardial regeneration. The paradigms offered by the models of parabiosis and repopulated bone
marrow with single cell injection are nonphysiological.
More than 90% of the animals die, and the question is how
relevant is an experimental procedure that represents the
exception rather than the rule.
Intrinsic genetic markers have been proposed as today’s gold standard for these types of studies (45, 95, 218,
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to false collection of data and erroneous interpretations
and conclusions. Collectively, the data available so far
demonstrate that myocardial regeneration occurs and reestablish the plasticity of BMCs and their ability to acquire a phenotype different from the organ of origin.
Whether the process of cardiac repair occurs independently from the formation of synkaryons and heterokaryons or requires fusion of the progenitor cell with a preexisting partner cell is discussed in section IV.B of this
review.
III. CARDIAC PROGENITOR CELLS
A. Identification
Traditionally the heart has been viewed as a static
organ incapable of repairing any form of damage. According to this paradigm, the number of myocytes is established at birth, and this population of terminally differentiated myocytes is irreplaceable throughout the life of the
organ and the organism (7, 97, 288, 329, 338, 339, 354).
Myocyte aging is dictated by the age of the organism, and
myocytes can only increase in size or die. In agreement
with this notion of the heart, myocytes are permanently
lost, and the heart has no reserve mechanism that compensates for cell death and the wear and tear resulting
from the physiological demands of daily life. There are
men and women 100 years old and older, and according to
this paradigm, all of their myocytes would have lived 100
years or more. In other words, the age of the individuals
and the age of their myocytes should coincide, and myocytes would survive by continuously replacing intracellular organelles, leaving intact the viability of the cells. The
demonstration that small amplifying myocytes can divide
after infarction (54, 229) or pressure overload (30, 497) in
humans has been vigorously challenged, and criticisms
varied from technical errors in the identification of replicating cells (443) to the lack of relevance of the findings
(329). In spite of the evidence of myocyte regeneration
(27), the concept of the heart as a postmitotic organ is still
widely accepted.
The static view of the myocardium implies that myocyte death and regeneration have little role in cardiac
homeostasis. Although stem cells have been identified in
several organs including the blood, skin, central nervous
system, liver, gastrointestinal tract, and skeletal muscle
(59), the search for a CSC was perceived as a futile effort
given the accepted lack of regenerative potential of the
myocardium. However, data challenging this belief began
to accumulate (63, 322, 391). In the past several years, the
existence of cycling ventricular myocytes in the normal
and pathological heart has been published (21, 29, 54, 229,
391, 497). Although these data provided an alternative
understanding of cardiac homeostasis, they also raised
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324, 495). Although the detection of the Y chromosome
and EGFP protein in the regenerated myocytes (228, 356)
falls within this category, recent experiments were performed with a genetic marker consisting of a cardiomyocyte-restricted transgene. Donor c-kit-positive BMCs
were obtained from male transgenic mice carrying a cmyc tagged nuclear Akt under the control of the cardiac
specific ␣-myosin heavy chain promoter (430). BMCs
from these animals were injected acutely after infarction
in wild-type female mice, and myocardial regeneration
within the infarct was identified a few days later. The
band of formed myocardium contained small myocytes
that expressed ␣-sarcomeric actin, troponin I, ␣-actinin,
connexin43, and N-cadherin. In all cases, the nuclei of
these developing myocytes were positive for the c-myc
tag (Fig. 6A). Vascular structures and CD45 labeled cells
in the reconstituted tissue were negative for the c-myc
tag. The Y chromosome was detected in endothelial and
smooth muscle cell nuclei but not in CD45 positive cells.
Therefore, BMCs injected in the dead myocardium do not
adopt the hematopoietic fate (45) but assume the cardiac
phenotype and repair the infarcted heart.
Recently, a new technology was implemented. By
this novel approach, antibodies are labeled by small semiconductor particles termed quantum dots. These particles
are 10 nm in diameter and have a unique property, which
is dictated by quantum mechanics of electrons confined to
a small space (11, 71). The excitation wavelength of quantum dots is widely separated from the emission wavelength. The common site of excitation by confocal microscopy using blue diode laser is 405 nm, while the emission
wavelength of quantum dots varies and the most frequently used have emissions between from 525 to 705 nm.
The spectrum of autofluorescence under this setting totally escapes the emission wavelength; autofluorescence
ranges between 440 and 520 nm. The advantage of this
approach is remarkable; it is impossible to detect
autofluorescence of tissue sections using this combination of excitation and emission wavelengths (Fig. 6B).
Conversely, a minimal level of autofluorescence cannot
be avoided when standard fluorochromes such as rhodamine or fluorescein are used. However, the degree of
autofluorescence is always much lower than the emission
peak. By employing primary and secondary antibodies
conjugated directly with distinct quantum dots, the ability
of BMCs to regenerate infarcted myocardium was confirmed in the absence of autofluorescence. Importantly,
this protocol was combined with a gene reporter assay
(Fig. 6C).
The emphasis placed on genetic markers for the “objective” recognition of newly formed structures is only in
part correct. Any genetic marker requires its subsequent
identification by histochemical (218, 324) or immunocytochemical (45, 218) procedures. If limitations exist in
these protocols, then the powerful genetic markers lead
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FIG. 6. BMC transdifferentiation in the infarcted heart by quantum dot (Qdot) analysis. A: confocal microscopy with organic fluorescent dyes:
BMCs from transgenic mice overexpressing c-myc tagged nuclear Akt, driven by the ␣-myosin heavy chain promoter, formed myocytes (␣sarcomeric actin; red) in the infarcted mouse heart. Nuclei are stained by DAPI (blue). Laminin defines the boundaries of the cells (green lines). The
donor origin of the regenerated myocytes is demonstrated by c-myc in nuclei (yellow speckles). B: the excitation and emission wavelengths of the
semiconductor particles Qdot 655 are distinct from the autofluorescence wavelength of tissue sections. This cannot be accomplished by organic
fluorochromes (TRITC). The dotted lines indicate the excitation wavelength for blue diode laser and Qdots, 405 nm, or for the argon laser and TRITC,
568 nm. C: a field of myocardial regeneration comparable to that shown in A is illustrated in C by Qdot labeling of antibodies. The new myocytes
are recognized by ␣-sarcomeric actin conjugated with Qdot 655 (red), c-myc in nuclei by Qdot 605 (yellow speckles), and laminin by Qdot 525 (green
lines). Nuclei are stained by DAPI (blue).
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organ, the replenishment of its parenchymal and nonparenchymal cells is regulated by a stem cell compartment and by the ability of these primitive cells to selfrenew and differentiate. Regeneration conforms to a hierarchical archetype: slowly dividing stem cells give rise
to highly proliferating, lineage-restricted progenitor cells,
which then become committed precursors that, eventually, reach growth arrest and terminal differentiation.
Although stem cells have been known for quite some
time, there is little understanding of the significance of
stem cell surface antigens in the growth and differentiation potential of these cells (76, 113, 246). These limitations are not restricted to the heart and the CSC but apply
to stem cells in all self-renewing organs including the
bone marrow. Moreover, the surface antigens c-kit,
MDR1, or Sca-1 are present in cells undergoing lineage
differentiation, complicating recognition of the actual
primitive cells in the population. FACS analysis is extremely helpful in separating stem cells in various categories according to the expression of one or more surface
epitopes (101, 146, 481), but the distinction between undifferentiated and early committed cells necessitates caution. Sophisticated analyses of nuclear and cytoplasmic
proteins can be performed by FACS, but they require
fixation of the cells to make them permeable and amenable to the detection of intracellular components (174, 192,
255). However, the viability of the cells is lost precluding
any subsequent in vivo or in vitro study. Similar problems
are encountered by immunocytochemistry that also demands cell fixation before the antibody or a cocktail of
antibodies is utilized to determine the stemness or commitment of the cells (305, 318, 447).
The first attempt to describe an endogenous myocardial stem cell was made by Rudnicki and co-workers
(201), who isolated a Hoechst effluxing cell population
from the heart of 2-mo-old mice. Similar cells, which
express MDR1 or a comparable ABC transporter, have
been identified in other organs, and they correspond to
the so-called side population (37, 73, 172, 299, 328, 546).
These putative CSCs are negative for markers of hematopoietic cells including CD34, c-kit, Sca-1, Flk-2, and
Thy1.1. Additionally, they give rise to colonies in methylcellulose medium and, in coculture with mature myocytes, differentiate into cardiac muscle cells expressing
connexin43. Their pool size is markedly reduced in hearts
of mice overexpressing a dominant negative form of
MEF2C, suggesting that under this stressful condition
there is a massive recruitment and commitment of these
primitive cells to the myocyte lineage. These Hoechst
effluxing cells also display a robust capacity to form
hematopoietic colonies and to fuse with skeletal myoblasts in the absence of cell transdifferentiation (201).
Although the characterization of these primitive cells as
true resident CSCs remained incomplete, this work introduced the concept of a myocardial stem cell pool that
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questions concerning two apparent contradictory lines of
evidence: the well-documented irreversible withdrawal of
cardiac myocytes from the cell cycle soon after their
formation and the presence of cycling myocytes undergoing mitosis and cytokinesis. These results raised the question of the origin of dividing myocytes and their increase
in response to an acute or chronic overload, pointing to
the presence of a CSC.
The pool size of adult stem cells is most likely set
during prenatal life or early after birth, and stem cell
migration, proliferation, maturation, and death regulate
the homeostasis of the adult heart. Stem cells divide
rarely while committed transient amplifying cells are the
actual group of replicating cells. The less primitive amplifying cells possess a unique property; they undergo
rounds of doublings and simultaneously differentiate
(431, 516). The most relevant observation that dramatically challenged the old paradigm of the heart as a postmitotic organ was the identification of male cells in female hearts transplanted in male recipients (391). In these
cases of sex-mismatched cardiac transplants in humans,
the female heart in a male host had a significant number
of Y-chromosome positive myocytes and coronary vessels. Although discrepancies existed among groups in
terms of the degree of cardiac chimerism (116, 168, 322,
391, 471, 472), these results raised the possibility that
these male cells colonized the female heart and subsequently differentiated into myocytes and functional vascular structures. The presence of male cells in the female
heart was consistent with the contention that stemlike
cells can migrate to the cardiac allograft and give rise to
the three main cardiac cell progenies: myocytes, smooth
muscle cells, and endothelial cells.
Importantly, primitive cells of donor and recipient
origin that expressed the stem cell surface antigens c-kit,
Sca-1-like, and MDR1 were identified. Of relevance, identical cells were found in human control hearts (391).
Since in fetal life, c-kit-positive cells colonize the yolk
sack, liver, and probably other organs, and the colonized
organs express SCF, the ligand of the c-kit receptor (249,
470), it was reasonable to assume that stemlike cells are
present in the heart from fetal life. Additionally, the rapid
induction of SCF during myocardial ischemia (152)
strengthened the notion that SCF was involved in the
activation of resident primitive c-kit-positive cells and,
thereby, in the increased formation of myocytes in the
acutely infarcted heart (54). The presence of these undifferentiated cells together with early committed progenies
was suggestive of a true CSC as the critical modulator of
the homeostasis of the normal and stressed myocardium.
These observations were the foundation for the work that
ultimately led to the identification and characterization of
a resident CSC pool in the adult heart (51). A new, more
biologically interesting model of cardiac growth, aging,
and death was emerging. If the heart is a self-renewing
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adapts in response to growth modifications. These observations have been confirmed (293).
The c-kit-positive CSC was the first stem cell identified in the rat heart and, up to date, the c-kit-positive CSC
is the most extensively characterized. These CSCs do not
express transcription factors or membrane and cytoplasmic proteins of bone marrow cells, neural cells, skeletal
muscle cells, or cardiac cells and are, therefore, lineage
negative cells (51). Cardiac lineage negative c-kit-positive
cells possess the three properties of stemness: clonogenicity, self-renewal, and multipotentiality (Fig. 7). In vitro,
these cells grow as a monolayer when seeded in substrate-coated dishes or form spheroids when cultured in
suspension, mimicking the biological behavior of neural
stem cells (82). Transplantation of clonogenic c-kit-positive cells in the infarcted ventricle confirmed the in vitro
characteristics of this cell population. This intervention
led to engraftment, migration, proliferation, and multilineage differentiation of CSCs resulting in the replacement
of dead tissue with new functional myocardium (51).
When this class of CSCs is delivered intracoronarily after
ischemia reperfusion injury, it promotes myocardial repair, limits infarct size, attenuates ventricular remodeling,
Physiol Rev • VOL
and ameliorates cardiac function (115). Thus c-kit-positive CSCs are effective when administered in a clinically
relevant manner, pointing to an approach in which the
heart’s own stem cells could be collected, expanded, and
stored for subsequent therapeutic repair.
Recently, CSCs have been defined as undifferentiated
cells that express on the membrane the stem cell-related
antigens, c-kit, MDR1, and Sca-1, in variable combinations
(277). CSCs sorted separately for each of the stem cell
antigens appear to have comparable growth and differentiation behavior in vitro. Whether the three classes of
CSCs have the same potential in vivo is currently unclear.
MDR1 belongs to the class of ABC transporters that mediate the Hoechst dye efflux in the side population of the
bone marrow (73, 74) and skeletal muscle (172, 289).
ABCG2 represents a more specific marker of this cell
population, since mice defective for ABCG2 do not possess SP cells in the bone marrow (547). MDR1-ABCG2positive cells in the bone marrow give rise to the myeloid,
lymphoid, and erythroid cell lineages (73) while in skeletal muscle they regenerate muscle fibers (184). Additionally, MDR1-ABCG2-positive cells from skeletal muscle are
of bone marrow origin and can repopulate a depleted
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FIG. 7. Cardiac stem cells (CSCs). A:
large clone of c-kit positive cells (green)
generated by a single rat c-kit positive
CSC. B–D: clonogenic cells in differentiating medium acquire the myocyte (B;
␣-sarcomeric actin, red), smooth muscle
cell (C; ␣-smooth muscle actin, magenta), and endothelial cell (D; von Willebrand factor, yellow) phenotype.
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atria, and outflow tract have been linked to the homozygous deletion of Isl1 (77). The expression of Isl1 corresponds to the onset of myocyte commitment since Isl1,
together with GATA-4, is a transcriptional activator of the
myocyte transcription factor MEF2C (121). Recently, the
claim has been made that Isl1 positive cells are the “true”
CSCs and are highly important for myocardial regeneration in the diseased adult human heart (260). Conversely,
the expression of Isl1 in progenitor cells clustered in the
niches or scattered throughout the atrial and ventricular
myocardium of the adult mouse heart is, at best, extremely rare. In our hands, not a single convincing example has been found. Similarly, no Isl1 cells have been
detected in the failing human heart. Although these initial
results do not exclude the possibility that some Isl1 cells
may be present in the adult myocardium, the functional
impact of these cells on cardiac pathology, if it exists, is
debatable. Even during development, Isl1 cells are not
implicated in the formation of the left ventricle.
Myocardial repair requires the formation of myocytes
and coronary vessels, and it cannot be accomplished by a
cell already committed to the myocyte lineage. In the
presence of an infarct, the generation of myocytes alone
cannot restore contractile performance in the akinetic
region; myocytes would not grow or survive in the absence of vessel formation. Resistance arterioles are critical for blood supply, and oxygen delivery is controlled by
the capillary network. Similarly, the creation of vessels
alone would not restore the dead myocardium or reinstitute contractile activity in the infarcted portion of the
ventricular wall; vessels do not produce force. Myocardial
regeneration necessitates the utilization of a more primitive multipotent cell such as the c-kit-positive CSC (51,
277). In vitro data suggest that the growth potential of
c-kit-positive CSCs is greater than that of Sca-1-like or
MDR1-positive cells, although these cell categories give
rise to all cardiac cell lineages (277).
B. Origin of Cardiac Stem Cells
Following the recognition of a tissue-specific stem cell
in the adult heart, the question concerned whether this
primitive cell population originates, lives, and dies within the
myocardium or whether other organs continuously replenish the heart with an undifferentiated stem cell pool that
subsequently acquires cardiac characteristics. The bone
marrow constitutes the main reservoir of primitive cells in
the organism, and these cells can egress from the marrow
niches, enter the systemic circulation, and chronically repopulate the heart and other organs (178, 253, 392, 473).
However, this possibility has never been definitely proven.
Under the condition of parabiosis and in the absence of
myocardial injury, circulating HSCs do not home to the heart
(45, 507, 508) or engraft efficiently in the bone marrow (300),
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bone marrow (289). In humans (391, 497, 498) and animal
models (277), MDR1-positive cells differentiate in progenitors and precursors of myocytes, endothelial and vascular smooth muscle cells, and fibroblasts. Sca-1 has been
considered a marker of stem cells restricted to the mouse
bone marrow (328, 431, 516). Contrary to expectation,
Sca-1-like protein has been found by confocal microscopy
and Western blot in primitive cells of the mouse, rat, dog,
and human heart (277, 497, 498). Most likely, this epitope
belongs to the large family of Ly6 proteins and corresponds to a known or unidentified member of these antigens. The recognition of this antigen by a Sca-1 antibody
is not surprising given the high degree of homology
among Ly6 proteins.
Recent studies have indicated that Sca-1-positive progenitor cells are the predominant stem cell population in
the mouse heart (296, 338). According to these observations, Sca-1-positive cells are 100- to 700-fold more frequent than c-kit-positive cells (51). Similarly, MDR1-positive cells have been found to represent a small fraction of
all CSCs in the mouse heart (293). Comparable results
about HSCs have been reported in the mouse bone marrow (219). Quantitative data in the mouse, rat, dog, and
human heart from our laboratory have demonstrated that
there is one CSC per ⬃30,000 – 40,000 myocardial cells:
⬃65% of all CSCs possess the three stem cell antigens
(c-kit, MDR1, Sca-1-like); ⬃20% two stem cell antigens
and ⬃15% only one. Approximately 5% each of all CSCs
express exclusively c-kit, MDR1, or Sca-1 (277, 498). However, this work did not resolve the issue of whether the
variability in the distribution of these surface markers
actually reflects cells with distinct functional import.
Some information can be obtained from the available
transgenic mice: the Sca-1⫺/⫺ mouse and the Mdr1⫺/⫺
mouse (215, 416) have essentially a normal phenotype
with modest defects of the hematopoietic system and
other organs. In contrast to the deletion of Sca-1 or MDR1,
mutation of the c-kit receptor in the W/WV mouse has
profound effects on the phenotype of the animal. The
W/WV mouse has a spontaneous point mutation in one
allele coupled with an amino acid deletion in the other
allele of the c-kit receptor (166, 333, 396). The homozygous W/W mouse dies shortly after birth, while the heterozygous W/WV has defects of the hematopoietic system
with the development of anemia, melanocytes with loss of
skin pigmentation, and mastocytes with altered immunoresponse (75, 476). The W/WV mouse reaches adulthood,
and the heart shows alterations in cardiac anatomy and
function at 8 –9 mo that precede the overt manifestations
of anemia. Older W/WV mice develop anemia with painful
skin lesions and behavioral abnormalities (94, 476).
Nearly 5 years ago, the Isl1 transcription factor was
shown to be associated with cardiac progenitor cells that
condition heart morphogenesis in the mouse embryo
(541). Defects in the development of the right ventricle,
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ADULT STEM CELLS
Physiol Rev • VOL
mented (302, 304). According to this theory, the primitive
left ventricle and the outflow tract are derived from a
single lineage, while the other regions including the primitive atria and the right ventricle are colonized by both
lineages. The differential expression of transcription factors, Nkx2.5, Tbx5, Isl1, eHAND, and dHAND in distinct
regions of the heart supports this possibility (364, 440).
The common origin of cardiomyocytes was recognized
by retrospective clonal analysis of the cells composing the
mouse embryonic heart. Although clones of the founder
cells appear in a temporal and spatial sequence, i.e., in
different days of development and in distinct regions, the
uniform genetic background of developing myocytes documented their derivation from a shared pool of founder cells.
These precursors, which are ⬃140 at E8.5, participate in the
formation of the entire length of the heart tube (302–304).
Therefore, the initiation of separate transcriptional programs in the cells of the cardiac chambers (440) is an
independent process and not the consequence of a diverse
clonal origin from unipotent progenitors. To confirm this
possibility, the EGFP gene was introduced at the stage of the
morula-blastocyst transition (Fig. 8). EGFP was placed under the control of the desmin or ␣-cardiac actin promoter
leading to the accumulation of EGFP in cells that express
these myocyte-specific proteins (129).
By this sophisticated technology, the patterns of
myocardial histogenesis have been defined and the existence of an individual common progenitor of cardiomyocytes in prenatal and postnatal life has been demonstrated
(129). Most importantly, this study has led to the unequivocal documentation of the intracardiac origin of adult
myocytes and has excluded the possibility that a secondary immigration of stem cells from distant organs occurs
in adulthood under physiological conditions (Fig. 8).
Homing and engraftment of stem cells through the systemic circulation and the blood vessels would lead to a
scattered localization of extra-cardiac-derived myocytes
with small accumulation in the proximity of capillaries
and arterioles (129, 391). However, single cardiomyocytes
or miniclones distributed independently from the original
embryonic EGFP-positive patches were never observed.
Thus “late comers” do not contribute to the postnatal
growth of the heart and, most likely, progenitor cells from
other organs do not have a direct spontaneous role in
myocardial homeostasis.
C. Cardiac Stem Cell Niches
Although stem cell antigens have been unequivocally
detected in CSCs, there is no single marker capable of
providing an absolute identification of stem cells in vivo.
Stem cells are stored in niches that are located deep in the
tissue for protection from damaging stimuli (157, 361, 362,
446, 491, 513). The niche constitutes the microenviron-
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supporting the notion that bone marrow homeostasis is
primarily maintained by division of HSCs in situ (431, 516).
In this regard, the model of heterochronic parabiosis (298,
464) has confirmed that rejuvenation of aged muscle mass
depends on circulating molecules contained in the peripheral blood of young animals and not on young HSCs homed
to old skeletal muscle (106 –108, 524). This argues against
the bone marrow as the source of primitive cells in the heart.
The contribution of bone marrow cells to cardiac
chimerism has been proposed (247). Interestingly, a comparison has been made between the degree of chimerism
in cardiac allografts and in hearts of patients who received allogeneic bone marrow transplantation (472). In
the latter case, only 2–5% chimeric myocytes were detected, while 14 –16% of chimeric myocytes and endothelial cells were found in transplanted hearts. These observations suggest the intracardiac origin of the recipient
cells in the donor heart and the extracardiac origin of
chimeric cells in the resident heart following bone marrow transplantation. In the first case, host cells may have
migrated from the residual atrial stumps to the donor
heart (391) and, in the second, donor cells may have
reached the myocardium because of the high level of
blood chimerism (472). Thus blood-borne cardiac cells
may be detected exclusively when the peripheral blood
contains a large number of HSCs. Experimental results
support this contention (217, 357).
Whether HSCs represents the precursors of CSCs during the regenerative response of the damaged heart remains
an unresolved issue. The striking discrepancy between the
incidence of heart failure and bone marrow failure and the
lack of comorbidity of these disease states in the same
patient indicates that HSCs do not typically migrate from the
bone marrow and repopulate the decompensated heart. If
the bone marrow continuously replenishes the heart with
new functionally competent HSCs, the decline in myocyte
number with cardiac diseases would not occur, and the
poorly contracting myocytes would be constantly replaced
by a bone marrow-derived progeny.
Current knowledge supports the notion that primitive cells are present in the heart during embryonic life
and regulate heart morphogenesis and postnatal development. Organogenesis begins with the specification of a
progenitor cell population (364). The pool size of embryonic cardiac progenitors conditions the final dimension of
the organ. For example, retinoic acid reduces the number
of prenatal cardiac progenitors leading to a hypoplastic
heart (232). Heart morphogenesis is a complex process
that results from the assembly of subsets of myocardial
cells that exert contractile function but express different
genes (432, 440, 520). Initially, the hypothesis was advanced that distinct unipotent progenitors give rise to the
myocytes that populate the different parts of the heart
(310). More recently, the early segregation of two lineages
of myocytes from a common precursor has been docu-
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ment in which primitive cells divide, differentiate, and die.
The recognition of stem cells within their natural milieu is
of crucial importance; stem cells do not exist in the
absence of supporting cells within the niche (446). The
structure of a niche appears to be specifically tailored to
suit the particular needs of its resident stem cells. In turn,
stem cells may play an important role in the organization
and specification of a niche (157). Niches share similarities in the activation of common signal transduction pathways that regulate the slow-cycling, self-renewing, undifferentiated state of their resident cells (12, 55, 83, 140,
202, 220, 223, 361, 446, 491, 513). However, niches in
various self-renewing organs may differ in architectural
organization and cellular composition that establish a
tissue-specific microenvironment.
Stem cells divide rarely, and cell replication is mostly
confined to the niches (446). Stem cells undergo symmetPhysiol Rev • VOL
ric or asymmetric division (141, 220, 223, 513). When stem
cells divide symmetrically, two self-renewing daughter
cells are formed, and the purpose of this mechanism of
growth is the expansion of the stem cell compartment
(513). When stem cells engage in asymmetric division, one
daughter stem and one daughter amplifying cell are obtained. The objective of this division is cell differentiation,
i.e., the production of a committed progeny. Stem cells
can also divide symmetrically into two committed amplifying cells, decreasing the number of primitive cells (513).
The developmental choice made by stem cells at any
given time has a direct impact on the stem cell pool size,
the number of progenitors and precursors and, ultimately,
the number of mature cells (157, 251, 446, 513).
The systematic identification of niches within an organ requires that stem cells be recognized together with
the cells responsible for their anchorage to the niche
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FIG. 8. Heart histogenesis and mouse chimeras. The
combination of two 8-cell morulas of different genetic
origin (blue ⫽ LacZ; green ⫽ EGFP) gives rise to a chimeric morula that evolves into a chimeric blastocyst. The
implantation of the early chimeric blastocyst in pseudopregnant mice results in balanced (equal parental contribution) or unbalanced (unequal parental contribution)
mouse chimeras. By this approach, that is termed aggregation method, the number of progenitor cells that contribute to the development of the heart or other organs can
be determined. The heart of these chimeric mice in which
the two morulas carry either EGFP or LacZ originates
from a single progenitor cell through a mechanism of
coherent expansion of clones. In this case, patchy areas of
the heart will be ␤-gal positive (blue) and others EGFP
positive (green). However, intermingling of clonogenic
cells occurs and foci of ␤-gal-positive cells (blue) are
found in EGFP clonal patches (green) or vice versa. Limited intermingling of clonogenic cells and their progeny
interferes partly with the coherent expansion of clonal
patches. This pattern of embryonic heart growth is shown
within the red box. The possibility that this model of
cardiac growth is mediated by a large number of progenitors would require a massive loss of clones early in development, a phenomenon that cannot be excluded. If
thousands of progenitors were implicated in embryonic
cardiac growth, a mechanism of coherent expansion together with extensive intermingling of clonogenic cells
would lead to a dispersed pattern of cardiomyocyte formation illustrated in the scheme. Importantly, the persistence of the embryonic clonal patches with small, intermingled foci in the adult myocardium excludes that new
clonal regions (red foci) contribute to the growth of the
heart postnatally as graphically represented in the red box.
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advantage with respect to the remaining endogenous
CSCs. Stem cells in solid organs are dispersed in the
tissue and, after their identification, it is critical to analyze
the microenvironment where they reside and whether,
upon activation, they can repair the damaged organ.
Recently, CSC clusters have been found in the adult
heart (22, 51, 98, 326, 498). Although CSC clusters are
scattered throughout the myocardium, their distribution
appears to be conditioned by the distinct levels of wall
stress. In fact, the frequency of CSC clusters is inversely
related to the hemodynamic load sustained by the anatomical regions of the heart: they accumulate in the atria
and apex and are less numerous at the base and midportion of the left ventricle (Fig. 9). Physical forces, mechanical deformation, and high wall stress can be transduced
in intracellular responses that regulate cell behavior and
fate. In a fashion similar to chemical cues, i.e., cytokines
and hormones, local forces modulate cell migration, proliferation, differentiation, and death (93, 140, 265, 407).
CSCs nested in the atrial microenvironment may be implicated in the preservation of the CSC pool through
symmetric division and the formation of self-renewing
daughter cells. Conversely, the very high degree of stress
at the base and midregion of the LV wall may condition
the turnover of mature progeny and CSC commitment.
Ventricular niches could be mostly involved in the formation of transient amplifying cells while apical niches may
exert a dual function.
Lineage negative cells expressing stem cell antigens,
separately or in combination, have been detected, clustered together with cardiac progenitors and precursors. In
close proximity to lineage negative cells, myocytes or
other cells in an early stage of differentiation are commonly visible. The structural organization of these sites of
accumulation of poorly differentiated cells differs from
that described in the bulge region of the skin (65, 491,
513), seminiferous tubes of the testis (429), and gut crypts
(386). In contrast, similarities in the spatial arrangement
of candidate stem cells and committed cells can be found
in the mouse bone marrow (540) and rat brain (122, 361).
However, nurse cells in these organs have been characterized while they remain to be identified in the heart.
Stem cells and supporting cells in the niches interact
structurally and functionally through specialized gap and
adherent junctional proteins (157). Connexins are gap
junction channel proteins that mediate passage of small
molecules and signals involved in cell-to-cell communication (170). Additionally, these gap junctions interfere with
the activation, commitment, and migration out of the
niches of stem cells (157, 317, 365). Survival factors and
mitogens traverse gap junctions to oppose cell death and
favor cell growth (Fig. 9). The identity of these molecules
is largely unknown. However, Ca2⫹, ATP, adenosine, and
cyclic nucleotides can translocate from one cell to another via gap junctions (170). Ca2⫹ influx or release from
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structure, i.e., the supporting cells. Additionally, the existence of a progenitor-product relationship has to be established (157, 446). The direct physical interactions between stem cells and their non-stem cell neighbors in the
niche are critical in keeping stem cells in this specialized
compartment and in maintaining stem cell characteristics
(35, 78, 276). The identification of a stem cell niche is a
difficult challenge in mammals. This search is more
readily accomplished in simpler organisms such as the
Drosophila (276, 341, 446, 531). The precise architectural
organization of germ cells in the Drosophila gonads simplifies the recognition of the niches (171, 441, 442, 532,
533, 549). In the fruit fly, there is a consistent reference
point for the identification of the supporting cells. Mammalian stem cell niches have been documented in some
organs. The better defined are those in organs that possess an epithelial lining, such as the skin niches in the
bulge of the hair follicles and the intestinal niches in the
crypts. In these cases, the supporting cells or nurse cells
are the dermal papilla cells and the mesenchymal cells
underlying the crypt (238, 491).
The niche characteristics are applicable to all organs
whether bone marrow, brain, or heart. The niches define
the growth potential of an organ, and the recognition of
their microenvironment is more important than the requirements postulated to be fundamental for the definition of stem cells. This is relevant because the criteria
employed in the study of HSCs cannot be transferred to
neural stem cells or CSCs without caveats. For example,
the radiation protocol commonly used for lethal irradiation and bone marrow reconstitution would not be effective in the heart. In fact, the radiation dose required to
reach and kill CSCs is 30 Gy. The need for 30 Gy is
dictated by the structural and physical properties of the
cardiac muscle (511). This very high dose, even though
restricted to the heart, results in profound alterations of
the entire organ, diffuse apoptosis, and death of the animals in congestive heart failure in 3–5 days. Also, these
data demonstrate that the circulating pool of hematopoietic progenitor cells cannot replenish a depleted tissue
such as the myocardium.
The limitations inherent in the therapeutic potential
of the circulating blood should not come as a surprise. If
this were not the case, spontaneous regeneration would
occur throughout the organism, and ischemic foci in all
organs would be rapidly repaired. Models of parabiosis
and bone marrow transplantation with a single ideal HSC
(45, 324, 507) have little to contribute to our understanding of the human disease and the future impact of regenerative medicine (228). A similar comment can be made
concerning the viewpoint that the “true” CSC has to be
identified and a single CSC has to be shown to possess the
ability to repopulate the depleted heart (95, 508). The
heart cannot be ablated of its CSC population, and the
injected single cell would have no competitive growth
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LERI, KAJSTURA, AND ANVERSA
intracellular stores activates proliferation of neural stem
cells. Also, Ca2⫹ is implicated in their differentiation and
survival (406).
The molecular glue that holds stem cells within the
niches is represented by the cadherin family of proteins,
which, together with ␤-catenin, participate in the formation of specialized intercellular junctions, called adherens
junctions (536). When differentiating stem cells lose contact with the neighboring cells, they proliferate more
easily and change location within the niche. Cadherins are
calcium-dependent transmembrane adhesion molecules
(371, 536), which have a dual function; they anchor stem
cells to their microenvironment and promote interaction
between stem cells and between stem cells and supporting cells. Stem cell anchorage depends also on integrin
receptors that bind cells to a basal lamina composed of
extracellular matrix (222). Elevated levels of integrins are
often characteristic of stem cells. Both integrins and adherent junctions play a critical role in the maintenance of
Physiol Rev • VOL
adhesiveness and quiescent status of stem and early committed cells within the organ (371). Thus the recognition
of cardiac niches and the identity of the supporting cells
constitute a major challenge for the definitive proof that
the heart is a self-renewing organ in which cardiac homeostasis is regulated by a stem cell compartment (Fig.
9). The niches are expected to control the physiological
turnover of myocardial cells and the growth, migration,
and commitment of primitive cells leaving the niches to
replace old dying cells in the myocardium.
IV. CARDIAC PROGENITOR CELLS
AND MYOCARDIAL REPAIR
The recognition that the mammalian heart possesses
a stem cell compartment that regenerates myocytes and
coronary vessels raises the unique possibility to reconstitute dead myocardium after infarction, to repopulate the
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FIG. 9. Schematic representation of CSC niches. Cardiac niches are prevalently located in the atria and apex
and consist of differentiated myocytes that surround clusters of CSCs and highly dividing amplifying cells. The
amplifying cells are committed cells and express transcription factors of the cardiac (GATA-4), myocyte (MEF2C),
smooth muscle cell (GATA-6), and endothelial cell (Ets1)
lineages. The interaction among CSCs, early committed
cells (ECCs), and supporting cells occurs via junctional
proteins (cadherins and connexins).
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ADULT STEM CELLS
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A. Myocardial Regeneration and Ischemic
and Nonischemic Cardiomyopathy
Myocardial regeneration occurs in humans following
ischemic (52, 54, 229, 346, 498) and nonischemic injury
(53, 229, 346, 348, 349, 497). However, myocyte proliferation appears to be restricted to the viable myocardium
adjacent to and remote from the infarct or to areas of
intact myocardium in other pathological states (497). The
identification of CSCs is consistent with the notion that
replicating myocytes constitute a subpopulation of rapidly growing amplifying cells originated from progenitors
or more primitive cells. CSCs are distributed in all regions
of the heart, suggesting that the reconstitution of parenchymal cells and coronary vessels can occur throughout
the myocardium offering great potential for cardiac repair. CSCs located within the infarct or in its proximity
could divide and differentiate reconstituting dead myocardium (Fig. 10). If this were the case, strategies can be
implemented to enhance myocyte and vascular growthpromoting partial restoration of the infarct. This response
would increase the number of myocytes and vessels, reduce infarct size, improve function, and decrease mortality. An identical argument can be made for other cardiac
diseases associated with increases in pressure and/or volume loads on the heart (346, 348, 349, 497). Before discussing the role that CSCs may have in the treatment of
the pathological heart, some comments have to be made
concerning the complexity of the problem and the heterogeneity of cardiac pathology in the patient population
that inevitably conditions the feasibility and efficacy of
CSC therapy.
Currently, it is difficult to anticipate how the etiology
of heart failure and the unpredictable path of the disease
influence the CSC compartment and, thereby, cardiac
reserve. Ischemic heart disease and hypertension are the
major causes of congestive heart failure (224, 274, 514,
551). However, myocardial infarction and high blood
pressure lead to heart failure by mechanisms that are
initially different, remain distinct during the progression
of the pathological processes, and may become comparable only in the late evolution to terminal failure experimentally (345, 352, 353, 377) and in humans (54, 156, 179,
229, 344, 346, 348, 351, 497). Myocardial infarction results
in acute ventricular decompensation (351, 359, 378) if the
loss of mass and, thereby, of myocytes, reaches a critical
value (Fig. 11). If cardiac dysfunction does not occur, the
segmental loss of myocardium conditions ventricular remodeling and the clinical course of the disease (104). The
postinfarcted heart typically shows cavitary dilation, wall
thinning, decreased systolic ventricular pressure, and an
increase in diastolic and systolic wall stress (29).
Conversely, pressure overload hypertrophy induced
by systemic hypertension is characterized by the preservation of chamber volume with thickening of the wall and
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hypertrophic decompensated heart with new, better functioning myocytes and vascular structures, and, perhaps,
to reverse ventricular dilation and wall thinning, restoring
the physiological and anatomical characteristics of the
normal heart. This hypothesis is supported by the identification of CSCs in the human heart (47, 497, 498). Additionally, CSCs injected locally in the infarcted myocardium of immunodeficient animals repair the necrotic tissue and improve ventricular function (47, 308). These
observations have formed the basis of a new paradigm in
which multipotent CSCs are implicated in the normal
turnover of myocytes, endothelial cells, smooth muscle
cells, and fibroblasts. Understanding the mechanisms of
cardiac homeostasis would offer the extraordinary opportunity to potentiate this naturally occurring process and
promote myocardial regeneration following tissue injury.
Hypothetically, stimulation of CSCs is unlikely to induce
malignant neoplasms since they are an extremely rare
form of cardiac pathology (414). CSCs should be more
effective in making new myocardium than progenitor
cells from other organs. CSCs are programmed to create
heart muscle and, upon activation, can rapidly engender
parenchymal cells and coronary vessels possibly rescuing
the failing heart (115, 391, 497, 498).
However, the field of regenerative cardiology is in its
infancy, and great caution has to be exercised in the
implementation of this form of cellular therapy in humans
before we have obtained the basic information concerning the ability of CSCs to migrate, divide, and differentiate. Similarly, ischemic and nonischemic pathological
states may have profound and distinct implications for the
function of the CSC pool (199, 498) including growth and
lineage commitment. Unfortunately, there is no good animal model that can be employed to collect the information that is of tremendous importance in the application
of CSC treatment to the patient population. In an attempt
to develop strategies relevant to the future treatment of
patients, new hypotheses have to be raised and tested to
move the field in a direction that defines CSC therapy on
an individual basis. This would maximize the efficacy of
CSC administration in heart failure.
Novel approaches have to be developed to impact on
the future directions of regenerative cardiology. Negative
experimental findings resulting from differences in protocols, methodological shortcomings, and suboptimal techniques for the analysis of myocardial repair have muddied
the arena of regenerative cardiology. Therefore, the promoters of the field of stem cell therapy have to acknowledge these recent condemnations, repeat some of the
work, and provide unequivocal answers to the criticisms.
But mostly they have to continue to search for new and
more innovative strategies for the reconstitution of the
severely damaged pathological heart.
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LERI, KAJSTURA, AND ANVERSA
an increase in wall thickness-to-chamber radius ratio. The
hypertensive heart is faced initially by an increased systolic load only, because diastolic dysfunction is a minor
alteration promoted by the increase in myocardial stiffness (176, 177, 551). The late stages of hypertensive hypertrophy when cardiac decompensation develops are
characterized by an abnormal elevation in LV end-diastolic pressure, relative thinning of the wall, and expansion in chamber volume (29, 30). Myocyte death is scattered throughout the ventricle and frequently affects clusters of myocytes leading to the formation of foci of
replacement fibrosis across the wall (20). Defects in coronary blood flow become apparent and involve increases
in minimal coronary vascular resistance and reductions in
coronary flow reserve (42). Abnormalities in myocardial
perfusion are more prominent in hypertensive hypertroPhysiol Rev • VOL
phy than in the postinfarcted heart (42, 230). Thus differences exist between these two forms of cardiac pathology, which may impact differently on the CSC pool.
Ischemic heart disease, hypertension, idiopathic dilated cardiomyopathy, and the unsuccessful repair of a
valvular defect with persistence of ventricular dysfunction and myocardial hypertrophy lead with time to severe
ventricular decompensation. Although cavitary dilation is
a common characteristic of these pathological states, the
phenotypic architecture and loading of the heart vary
significantly among these conditions and among patients
in the same group. If we consider the evolution of the
postinfarcted heart, the size of the infarct is a predictor of
the short-, mid-, and long-term outcome of the disease in
all patients (Fig. 11). But remodeling and accumulation of
damage in the viable myocardium is the critical determi-
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FIG. 10. Myocardial regeneration in the human
heart. A: an area of spontaneous formation of myocytes (␣-sarcomeric actin, red) is present in proximity of dead, infarcted myocardium and a region of
inflammatory reaction. B and C: metaphase chromosomes (PI, blue) are apparent in two small dividing myocytes (cardiac myosin heavy chain, red)
in the heart of patients affected by chronic aortic
stenosis. [Modified from Urbanek et al. (497).]
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ADULT STEM CELLS
FIG. 11. Myocardial infarcts incompatible with survival. A and B: large
transverse myocardial sections of paraffin-embedded tissue illustrating the left
ventricle (LV), interventricular septum
(IS), and right ventricle (RV). Tissue necrosis is present in a large portion of the
IS and in some areas of the anterior aspect of LV (arrowheads). Foci of myocardial scarring are also detected (*). [Modified from Olivetti et al. (351).]
It is remarkable that there is a persistent view that
the formation of vessels alone can result in a significant
restoration of performance of the failing heart (120). Minimal amounts of hibernating or stunned myocardium may
be rescued by regional improvement of coronary blood
flow, but this modest reinstitution of contractile activity
in viable tissue cannot be confused with actual cardiac
repair or myocardial regeneration (497, 498). The objective of cellular therapy is the creation of cardiomyocytes
and vascular structures, which together replace dead or
scarred myocardium, mending the broken heart. Whether
healed infarcts or foci of replacement fibrosis contain a
few vessels or a large number of vessels, ventricular
function does not change. The increases in ejection fraction demonstrated in several clinical trials may very well
reflect regeneration of myocytes and coronary vasculature, mimicking the results obtained experimentally (27,
51, 228, 258, 277, 539). There is a tight relationship between myocytes and the coronary vasculature, particularly the capillary network, with myocardial growth (31,
102, 393). This occurs during fetal and postnatal maturation of the heart as well as in conditions of accelerated
growth induced by physiological overloads in the adult,
such as dynamic exercise (32). The increased work demand may be counteracted by the activation of CSCs,
which could generate new tissue with optimal quantities
of myocytes and vessels.
FIG. 12. Ventricular remodeling after myocardial infarction. Large transverse myocardial sections of paraffin-embedded tissue illustrating a healed myocardial infarct (HMI) with thinning of the
wall (A) and multiple sites of replacement fibrosis (RF) in the noninfarcted
viable tissue (B). [Modified from Beltrami et al. (52).]
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nant of the onset of cardiac dysfunction and its progression to terminal failure (Fig. 12).
The number of acute events varies in the patient
population and, by the nature of the damage, segmental
losses of myocardium necessitate approaches that are by
far more complex than those required by minute areas of
tissue injury or scattered myocyte death across the ventricular wall. This notion is based on observations obtained in humans, acutely after infarction (54, 498), or
sustained pressure overload (497), and in animal models
(51, 115, 227). The degree of differentiation that newly
formed parenchymal cells acquire when they are clustered together in a large area of lost tissue markedly
differs from that of regenerated myocytes scattered
throughout the viable myocardium in close proximity to
mature preexisting cells. In the former case, myocytes are
small and the myofibrils are rare and occupy only a minimal portion of the cytoplasm resembling fetal-neonatal
cells in humans (Fig. 10) and animals (Fig. 13). In the
latter case, the regenerated myocytes are indistinguishable from the adjacent muscle cells and exhibit an adult
phenotype (Fig. 13). This attractive biological problem
raises interesting questions about the microenvironment
and the cross-talk between differentiated and developing
myocytes. Similarly, from a clinical standpoint, the reconstitution of infarcted myocardium with properties mimicking the structural organization of the normal heart is
critical for the complete recovery of cardiac function.
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LERI, KAJSTURA, AND ANVERSA
CSCs, progenitors, and precursors express c-Met,
insulin-like growth factor I receptors (IGF-IR), and
ANG II AT1 receptor subtype. Hepatocyte growth factor
(HGF) activates c-Met promoting cell migration,
growth, and survival (277). IGF-I has no influence on
locomotion in vitro and in vivo but enhances cell proliferation, differentiation, and viability (277). ANG II
induces multiplication of vascular cells and fibroblasts
and hypertrophy of cardiomyocytes (148, 267, 278, 408,
409, 418). The HGF-cMet and IGF-I/IGF-IR systems oppose apoptotic and necrotic death signals while ANG II,
through the AT1 receptor effector pathway, leads to the
formation of reactive oxygen species (ROS) (93, 265).
ROS, in turn, stimulates DNA damage and, ultimately,
cell death (148). Stem cell surface antigens and growth
factor receptor systems may operate together, condiPhysiol Rev • VOL
tioning growth, death, and commitment of CSCs to
distinct cell lineages.
A critical component of CSCs is related to the ability
of these cells to translocate upon stimulation. Three fundamental properties of CSCs regulate the cell turnover of
the heart: migration, division, and differentiation. The fact
that the myocardium is not a static organ and cell renewal
is not restricted to endothelial cells, vascular smooth
muscle cells, and fibroblasts but includes parenchymal
cells, offers a reinterpretation of the biology of the heart
and mechanisms of cardiac aging. Aging may promote
alterations in the degree of CSC proliferation, maturation,
and locomotion, attenuating cell replacement and the
substitution of damaged cells and vascular structures.
Although the acceptance of this concept finds resistance
among traditional cardiologists and cardiovascular scien-
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FIG. 13. Myocardial regeneration after infarction in rats. A–F: the local intramyocardial injection of EGFP-tagged
CSCs in syngeneic rats acutely after permanent coronary artery occlusion results
in the formation of new myocardium.
A–C: in the infarcted region, the regenerated myocytes are small (A; ␣-sarcomeric
actin, red) and consistently show EGFP
in the cytoplasm (B; green). C: the merge
of A and B. The regenerated myocytes
express both ␣-sarcomeric actin and
EGFP (yellow-green). These myocytes
are ⬃700 ␮m3 in volume and, because of
their size, cannot be the product of cell
fusion. Nuclei are labeled by PI (blue).
The localization of laminin at the periphery of the developing myocytes is illustrated by white lines. D–F: two newly
formed myocytes (arrows) are detected
within the surviving myocardium of the
border zone. They are positive for ␣-sarcomeric actin (D; red) and EGFP (E;
green). F: the merge of D and E. The new
myocytes are indistinguishable from the
adjacent myocytes, ⬃20,000 ␮m3 in
volume.
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ADULT STEM CELLS
tists, recent information strongly supports this new view
of the heart (484).
B. Cell Fusion and Myocardial Regeneration
Physiol Rev • VOL
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The notion of cell fusion, which was extremely popular in the 1980s, has recently been reintroduced successfully in the field of regenerative medicine (58, 60, 61, 368).
Originally, cell fusion referred to the joining of two fully
differentiated cells (60, 61). This phenomenon was frequently observed in the skeletal muscle and liver that are
typical fusogenic organs (180, 182, 190, 454). The concept
of cell fusion has evolved and, currently, indicates the
coalescence of a stem cell and a differentiated cell (90,
111, 205, 501, 507, 508, 510). The merge of an adult stem
cell and a committed cell results in the formation of a
binucleated heterokaryon or a mononucleated hyperploid
synkarion (58, 60, 61, 236, 368). The growth of the binucleated heterokaryon depends on the nucleus of the primitive cell that dominates the nucleus of the somatic cell by
transferring its replication properties. However, the destiny of the heterokaryon is regulated by the nucleus of the
differentiated cell (58, 60, 61, 153, 368, 384). When cell
fusion is accompanied by nuclear fusion, a mononucleated hybrid cell with a hyperploid DNA content is formed.
In this case, the bulky burden of the high nuclear DNA
content leads to genetic instability and reduced or abrogated replicative potential (70, 130, 182, 190, 292, 384,
454). Cell fusion in mononucleated cells with 2C DNA
content has been proposed and justified on the basis of
reductive mitosis (510). Theoretically, diploid cells can
result from hybrid cells that underwent reductive cell
division converting the hyperploid cell into a diploid
karyotype, which concealed fusion history (91, 376, 437,
526). Reductive cell division of hybrid cells in vivo has
only been suggested in hepatocytes generated under stringent selection pressure that conferred survival and
growth advantage on them (510). These “pseudo-diploid”
cells accumulate slowly and might be present exclusively
when a large number of fused cells are generated.
The notion of myocardial regeneration by spontaneous or induced activation of CSCs as well as by transplantation or mobilization of progenitor cells from the bone
marrow has been questioned, and the possibility that
cardiac repair is not a primary event but a secondary
process has been alleged (151). Fusion of a resident primitive cell or BMC with a preexisting myocyte that subsequently reenters the cell cycle and leads to the formation
of a myocyte progeny has been proposed as an alternative
mechanism of growth in the adult heart (14, 45, 151, 324,
336, 338) or other organs (14, 79, 80, 248, 301, 332, 501,
507, 508, 510, 522, 530, 545). If this were the case, spontaneous myocardial regeneration by activation of CSCs
would be limited in scope and BMCs would not be able to
differentiate across lineage boundaries and reconstitute
damaged myocardium. Moreover, the turnover of cardiac
cells would not be modulated by a stem cell compartment
but by a complex biological process, that does not replace
old dying myocytes or vascular smooth muscle cells and
endothelial cells. Old cells would persist and somehow
would constitute a necessary requirement for cell division
to occur. Essentially, the engrained paradigm of the heart
as a postmitotic organ comes back in a different manner,
postulating once more that the life of myocytes corresponds to the life of the organ and the organism. The
intrinsic capacity of the heart to heal itself would be
negligible and of no biological and clinical importance.
The wave of enthusiasm for cell fusion was prompted
by two reports in 2002 that demonstrated the “extremely”
rare occurrence of cell fusion under unusual in vitro
settings that have no in vivo counterpart (469, 537). Bone
marrow progenitor cells (469) or neural progenitors (537)
were plated with ESCs. These coculture systems involved
not only two types of undifferentiated cells but also highly
stringent conditions favoring the occurrence of fusion
events (135). In spite of this peculiar environment, cell
fusion was occasional at best: 2–11 ⫻ 106 cells (469). The
experimental design largely precluded the survival of
ESCs that may have fused with the adult progenitor cells
to acquire the antibiotic resistance gene necessary to
remain viable under the selective in vitro protocol.
Whether the formation of hybrid cells was dictated by the
properties of the tissue-derived adult progenitor cells or
ESCs was not determined (135). Surprisingly, this little
evidence of cell fusion was used by skeptics of stem cell
plasticity who questioned the “unexpected” regeneration
of adult tissues including the brain, the liver, and the heart
and proposed cell fusion as the only form of repair in
adult organs (14, 79, 80, 248, 301, 332, 336, 338, 501, 507,
508, 510, 522).
One-year later, mutant mice with an enzymatic defect
in liver parenchymal cells were rescued by the injection of
BMCs (501, 510), and the beneficial effect was totally
attributed to fusion of BMCs with hepatocytes lacking the
fumarylacetoacetate hydrolase (FAH). Four issues have
been overlooked. First, the normal adult liver is a highly
fusogenic organ (130, 182, 183, 454), such as the skeletal
muscle (90, 130, 190, 205), and pathological states enhance the generation of polyploidy in both tissues (70,
173, 180, 182, 241, 433, 463). Second, the FAH mutant
mouse has an unusually large number of hepatocytes with
an abnormal karyotype, which facilitates cell fusion (135,
510). Third, 6 –15% of hepatocytes with normal DNA content were of donor origin, showing a female phenotype in
male recipients (510). This finding is consistent with BMC
transdifferentiation more than the complex, unproven
suggestion of reductive mitosis. Fourth, the engraftment
of BMCs and the incidence of cell fusion is very low in the
liver of FAH mice, 1/150,000 cells (501, 510). However,
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LERI, KAJSTURA, AND ANVERSA
Physiol Rev • VOL
role of progenitor cells. It is also unlikely that resident
progenitor cells follow a growth pathway distinct from
the intrinsic hierarchical process of commitment and differentiation. Additionally, the discovery that nanotubular
highways are formed between cells could explain not only
the translocation of enzymes between adjacent cells but
also the migration of entire organelles from one cell to the
neighboring cell (404). These nanotubules consist of Factin-positive structures that protrude from one cell to
establish connection with another cell (164, 550). However, this phenomenon has been demonstrated in vitro,
and its functional significance in vivo is unclear.
Whether the twist in fate of a stem cell occurs by
transdifferentiation or fusion, reprogramming of chromatin configuration is required (153, 237, 270, 271). The
reorganization of chromatin is mediated by an alternate
turning on and off of transcription factors to drive the
adult stem cell towards the creation of a specific progeny
(237). This process is slow and limited in efficiency (145,
150, 487). If cell fusion is involved, the replicative potential of the fused cell is at best transient (173, 180, 241). In
organs such as the brain and the heart, cell fusion may
lead only to a temporary rescue of an old preexisting
terminally differentiated parenchymal cell. Heterokaryon
formation in the Purkinje cells of the brain results in a
short-lived rejuvenation of old or damaged cells, which is
not associated with the acquisition of replicative potential
(515). Nuclear reprogramming of transdifferentiated cells
or synkaryons involves epigenetic modifications of chromatin (210, 394). Extrinsic signals present in the new
environment drive chromatin remodeling. The principal
mechanisms by which the expression pattern of tissuespecific genes or global gene silencing is established or
maintained involve chromatin modification. This includes
DNA methylation as well as histone acetylation, phosphorylation, methylation, and ubiquitinylation (161, 270).
Dynamic changes in these epigenetic modifications underlie chromatin remodeling that is responsible for cell
transdifferentiation or nuclear fusion (161, 210).
To assess whether cell fusion plays a role in cardiac
repair, myocardial regeneration mediated by the delivery
of BMCs in mice (228, 356) and following CSC implantation in rats was analyzed (51). Since female rat clonogenic
CSCs were injected in female infarcted rats and the probe
for the rat X-chromosome was not available, we measured
the number of chromosome 12 in the newly formed myocytes. In all cases, at most two chromosomes 12 were
found excluding cell fusion (Fig. 14). Moreover, the DNA
content was measured in regenerating myocytes and surviving myocytes. In all cases, a 2C DNA content was
identified, and higher levels of DNA content reflected
cycling myocytes. The absence of cell fusion was consistent with the number and size of newly formed myocytes
within the band of regenerating myocardium (51). Importantly, the injection of male mouse BMCs in infarcted
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this small number of fused cells grows rapidly under the
selective pressure of the enzymatic deficiency, making
this a particularly favorable situation for uncommon hepatocyte replacement (135, 510).
The cre-lox genetic system is frequently used to detect cell fusion. Cre is a recombinase enzyme that cuts
DNA segments flanked by binding sequences termed
LoxP sites (6, 96, 403, 487). The cre-lox system can be
generated by cross-breeding two transgenic mice or by
implanting cells from a Cre mouse into a LacZ or EGFP
mouse (6, 96, 260, 338, 403, 487). Importantly, a tissuespecific promoter drives the Cre recombinase. The Cre
mouse is bred with a mouse carrying an inactive LacZ or
EGFP gene placed under the control of a ubiquitous promoter in which a stop codon flanked by LoxP sites is
inserted between the promoter and the LacZ or EGFP
gene. The fusion of cells carrying the Cre recombinase
with cells carrying the LacZ or EGFP gene leads to the
binding of Cre to the LoxP sites. In the event of cell
fusion, the Cre recombinase excises the stop codon or
inhibitory segment resulting in the activation of LacZ or
EGFP gene. Therefore, fused cells are labeled by ␤-Gal or
EGFP (487). More recently, a conditional Cre mouse has
been developed in which Cre is regulated by the presence
of the mouse estrogen receptor (mer-cre-mer). In this
mouse, the activity of Cre is dependent on the administration of tamoxifen (260, 375), since mer is not activated
by the endogenous 17-estradiol.
A few studies have employed this approach to determine whether the injection of cardiac progenitor cells (260,
338) or BMCs (336) results in myocardial regeneration after
infarction and whether tissue reconstitution is mediated by
cell fusion. Myocyte formation was demonstrated with Sca1-positive cardiac progenitors while BMCs apparently failed
to repair the damaged heart. In the former case, nearly 50%
of new myocytes were considered the product of fusion
while the remaining 50% were derived from differentiation
of cardiac precursors (338). Moreover, evidence of fusion of
BMCs with resident cardiomyocytes has been found in
healthy hearts (336). However, the cre-lox system is not
perfect. It is surprising that the possibility of metabolic
cooperation was not considered, since this phenomenon
may account for some of these observations (455). By metabolic cooperation, a cell may acquire the Cre recombinase
from a neighboring cell and undergo excision of the floxflanked DNA segment in the absence of cell fusion. This
exchange of the enzyme between the donor cell and the
recipient cell occurs through intercellular junctions (366,
478, 503, 504).
Metabolic cooperation is important in a tissue that is
functionally a syncytium like the myocardium. The creation of myocytes by fusion of a cardiac progenitor with
a fully mature myocyte cannot trigger the division of the
recipient terminally differentiated cell. The hybrid cell
loses the ability to proliferate abrogating the fundamental
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ADULT STEM CELLS
female mice resulted in the generation of male myocytes
with at most one X and one Y chromosome with a 2C DNA
content (228, 356). Moreover, at 5 days, the regenerating
band contained myocytes, which varied in size from 80 to
1,200 ␮m3, with an average cell volume of 360 ␮m3. Together, 2.7 million new myocytes were formed in 5 days after
infarction. At 10 days, the regenerating band contained myocytes, which varied in size from 110 to 2,000 ␮m3 with an
average cell volume of 610 ␮m3. Together, 4.3 million new
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FIG. 14. Myocyte regeneration without cell fusion. A–C: formation
of new myocytes following the injection of CSCs in an infarcted heart is
identified by the expression of cardiac myosin heavy chain (A, red).
Additionally, the number of chromosome 12 in the nuclei is determined
by FISH (B; yellow dots). In all cases, at most two chromosomes 12 are
identified in each nucleus, excluding cell fusion. C: the merge of A and
B. The regenerated myocytes express ␣-sarcomeric actin and have one
or two chromosomes 12 in their nuclei. Nuclei are labeled by PI (blue).
The localization of laminin at the periphery of the developing myocytes
is illustrated by white lines.
myocytes were formed in 10 days after infarction. If cell
fusion were the mechanism of myocyte regeneration, the
formed cells should have a volume of at least 20,000 ␮m3 or
larger because the volume of resident myocytes is ⬃20,000
␮m3. Moreover, fusion of a primitive cell with a terminally
differentiated myocyte would not be able to activate the cell
cycle. In this regard, the number of myocytes formed markedly exceeded the number of myocytes remaining after infarction, further excluding cell fusion. Finally, the short interval between the injection of BMCs and the generation of
diploid male cells makes reductive mitosis an unlikely possibility (190, 228, 356).
When myocardial regeneration was induced by injection of clonogenic CSCs in infarcted mice or male BMCs
were implanted in female infarcted mice, a question persisted. Permanent coronary artery occlusion in rodents
leads to transmural infarcts leaving no viable partner cells
for fusion (51). For this reason, a model of ischemiareperfusion injury in the rat was implemented, and EGFPpositive clonogenic CSCs were delivered through the coronary circulation (115). Again, a diploid DNA content was
found and the new myocytes expressed at most two chromosomes 12. Thus cell fusion can be considered a negligible mechanism of cardiac repair whether CSCs or BMCs
are employed as therapeutic tools (51, 115, 228, 258, 356).
The occurrence of cell fusion in vivo appears to be extremely limited. Consistent with the observations in the
heart, cell fusion in other organs including the skin, the
lung, epithelial lining (190), and the brain (14, 204, 515) is
restricted to a few cells, which, by inference, have no
physiological consequences on baseline function or on
tissue regeneration in pathological states.
The discovery that resident progenitor cells exist in
the adult myocardium overcomes the need for the complex and time-consuming process of nuclear reprogramming (384) and cell fusion (135). This is because primitive
cells nested in the cardiac microenvironment are predetermined to evolve into cardiac cell lineages, i.e., myocytes and vascular smooth muscle and endothelial cells.
Moreover, the heart possesses an intrinsic growth reserve
capable of responding to the physiological and pathological demands of the myocardium. Importantly, resident
stem cells may be more efficient and powerful for tissue
reconstitution. These results introduce a new strategy for
myocardial reconstitution expanding the notion that injection of exogenous cells into the damaged heart is required for the replacement of lost myocytes. The demonstration that the heart harbors stem cells capable of
creating functional myocardium explains earlier observations of a robust regenerative response in the acute
postinfarcted heart in humans (54), but raises the question of why this regenerative response stops before the
repair process is completed. However, cardiac stem cells
may be coaxed in vivo to home to the damaged region of
the heart and promote the formation of functionally com-
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LERI, KAJSTURA, AND ANVERSA
petent myocardium (277). This possibility offers an alternative or complementary therapeutic approach to exogenous cells.
V. FUTURE DIRECTIONS
Several fundamental areas of stem cell research have
to be addressed in the near future. An important question
involves whether distinct classes of human stem cells
condition the efficacy of myocardial regeneration.
Whether the expression of a single stem cell antigen or a
combination of epitopes is linked to the formation of a
preferential cardiac cell progeny is of paramount relevance for management of human disease. This recognition may allow us to develop powerful and novel strategies for the regeneration of myocytes and coronary vasculature and the restoration of cardiac performance in the
failing heart. According to need, a new tool may become
available for the predominant formation of myocytes
and/or coronary vessels. Similarly, the impact of age,
gender, type, and duration of the overload on stem cell
proliferation and lineage commitment is unknown. Aging
effects on stem cells may be comparable to those induced
by a prolonged and sustained overload on the myocardium, and strategies may have to be developed to modify
genetically the cells before treatment. A similar relevant
question is whether stem cells in women are less susceptible to senescence and death signals, possessing an inherent ability to preserve their growth potential and to
counteract the activation of the cell death pathway. In
fact, myocyte death with aging (347, 350) and heart failure
(179, 498) is reduced in women who have a higher nuclear
Physiol Rev • VOL
accumulation of the survival kinase phospho-Akt473 (81).
These observations strengthen the notion that age, gender, and type and duration of the cardiac disease have a
common effect on stem cells, i.e., senescence and death.
Of relevance, culture conditions may promote aging of
primitive and progenitor/precursor cells (424, 425, 428),
pointing to the significance of the problem at hand for
cellular therapy in humans. The same phenomenon may
be operative in committed cells impinging on their regenerative growth. However, these negative modulators of
the stem cell pool may affect only a subset of stem cells,
while a fraction of cells retains its youth and, thereby, an
intact ability to grow and give rise to a highly dividing
committed progeny.
Identical questions apply to CSCs and their potential
future clinical application. In this regard, there is a relevant issue to be acknowledged, i.e., whether the use of
undifferentiated CSCs is superior, inferior, or equal to the
utilization of CSCs together with highly dividing early
committed cells. These cells already express nuclear
and/or cytoplasmic markers of the myocyte, endothelial,
and smooth muscle cell lineages (51, 277, 497, 498). A pool
of CSCs and partially differentiated cells may engender a
greater and faster regenerative response. A mixture of
cells, with clearly defined properties and with high potential for proliferation, may be the most effective in reconstituting dead myocardium after infarction. Additionally,
this composite cell population may actively replace
poorly contracting myocytes in the chronically failing
senescent heart, with younger better functioning cells and
vascular supply. Under this setting, coronary reserve is
impaired, and minimal coronary vascular resistance is
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FIG. 15. Tissue engineering and
stem cells. The injection of a self-assembling bioengineered scaffold loaded with
adult CSCs and selective growth factors
may enhance myocardial regeneration
and orderly organization of parenchymal
cells and vascular structures. Moreover,
the presence of growth factors may favor
the recruitment of endogenous progenitor cells in the area of injury.
1403
ADULT STEM CELLS
Physiol Rev • VOL
324). It might be relevant to reiterate that the current
treatment of choice in heart failure consists of angiotensin converting enzyme (ACE) inhibitors and ␤-blockers.
These drugs improve quality of life and significantly decrease mortality. ACE inhibitors have been used for ⬃20
years (378 –380), and ␤-blockers have been around for
⬃40 years (494). ␤-Blockers were considered detrimental
for the decompensated heart. Contrary to expectation,
␤-blockers are the most powerful agent available to patients with congestive heart failure (468). But, how ACE
inhibitors and ␤-blockers interfere with the progression
of heart failure remains to be identified.
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence: P.
Anversa, Cardiovascular Research Institute, New York Medical
College, Vosburgh Pavilion, Rm. 303, Valhalla, NY 10595 (E-mail:
[email protected]).
GRANTS
This work was supported by National Institutes of Health
Grants HL-38132, AG-15756, HL-65577, HL-66923, HL-65573, AG17042, AG-023071, HL-43023, HL-50142, AG-026107, and HL-081737.
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