Regeneration Of Heart
Cardiac Stem Cell Therapy and the Promise of Heart
Regeneration
By: - Deepak Kumar & Tarun Kumar
Stem
cell therapy for cardiac disease is an exciting but highly controversial
research area. Strategies such as cell transplantation and reprogramming have
demonstrated both intriguing and sobering results. Yet as clinical trials
proceed, our incomplete understanding of stem cell behavior is made evident by
numerous unresolved matters, such as the mechanisms of cardiomyocyte turnover
or the optimal therapeutic strategies to achieve clinical efficacy. In this
Perspective, we consider how cardiac stem cell biology has led us into clinical
trials, and we suggest that achieving true cardiac regeneration in patients may
ultimately require resolution of critical controversies in experimental cardiac
regeneration.
Introduction
The race is on: throughout the
world, basic and clinical investigators want to be the first to identify new
approaches to regenerate cardiac tissue and to prove the effects of these
therapies in patients with heart disease. Despite substantial progress in
treating many types of heart disease, the worldwide heart failure burden will
remain enormous through this century. The potential of stem cells and the scope
of the heart failure problem have fueled a stampede to be the first to achieve
human heart regeneration. Cell transplantation approaches are attractive given
their relative ease of use and good safety profile to date, but reproducible
results endorsing a specific strategy for routine patient care are lacking.
Meanwhile, cellular reprogramming strategies are appealing because they
potentially allow precise control over cellular behavior, but much work remains
before the safety of reprogramming allows clinical testing. Current clinical
trials focus largely on injection of cells with cardiomyogenic potential into
the heart; however, given the limitations of this approach, we wonder: is this
the path to take right now?
As we consider the current state of the heart regeneration
field, it is worth pausing to reflect on the 1960s, when heart transplantation
emerged. Initial excitement over heart transplantation led to over 100 heart
transplantations worldwide in 1967 and 1968. However, disappointing results
soon followed, with only a quarter of the patients surviving more than a few
months (Kantrowitz, 1998). Renowned
cardiologist Helen Taussig expressed concern in 1969 that it was not yet time
for human trials, warning, ‘‘.our hope should be that physicians and surgeons
will proceed with extreme caution until such time as a cardiac transplant will
not announce the imminence of death but offer the patient the probability of a
return to a useful life for a number of years’’ (Taussig,
1969). During the 1970s, few human heart transplants occurred as the
number of surgeons willing to perform heart transplants dwindled due to high
mortality in the first year after transplants (Kantrowitz,
1998). Only after rigorous research in organ rejection and
immunosuppression in the 1980s did heart transplantation become the accepted
medical practice that it is today (Kantrowitz, 1998).
Unfortunately, limitations in organ supply and other issues allow
transplantation in only a minority of patients with heart failure, and
transplantation will not be a solution for the growing problem of heart
disease.
Half a century after the first human heart transplant, we
are now confronted with the new challenge of regenerating damaged hearts in the
growing number of patients with heart failure. Will we be following a similar
path to that of cardiac transplantation? Despite the enormous potential, it is
not clear whether we know enough fundamentals to move forward clinically or how
fast we should go. Some investigators contend that we know all we need to know
to move forward, while others are less confident. In this Perspective, we
consider both established principles and ongoing controversies that guide
cardiac regeneration research.
Established Principles
We believe that
three fundamental principles of cardiac regenerative biology have now been
established. First, multipotent cardiac progenitor cells (CPCs) exist in the
embryonic mammalian heart (Moretti et al., 2006;
Wu et al., 2006); second, there is creation
of a limited number of new heart cells after birth in mammals (Beltrami et al., 2003; Bergmann
et al., 2009; Malliaras et al., 2013;
Mollova et al., 2013; Senyo et al., 2013); and third, some vertebrates,
such as newts (Oberpriller and Oberpriller, 1974),
zebrafish (Jopling et al., 2010; Poss et al., 2002), and neonatal mice (Porrello et al., 2011), can regenerate myocardium
following experimental injury. In an often-controversial field, the
establishment of these three principles from different lines of evidence by
different laboratories represents seminal progress.
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Annual Rate of Cardiomyocyte
Renewal Species Method Reference
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Multipotent CPCs Exist in the Mammalian Embryo During
embryonic development, CPCs arise from a subpopulation of mesodermal precursors
that can be modeled from in vitro differentiated embryonic stem cells (ESCs) (Kouskoff et al., 2005). The expression of FLK1
marks a panmesodermal cell population that can give rise to cells in both the
primary and secondary heart fields (Kattman et al.,
2006) as well as skeletal muscles in the head, neck, and trunk (Motoike et al., 2003). For the primary heart
field, a population of bipotential KIT+ (also referred to as c-kit+)/NKX2.5+
progenitor cells gives rise to myocardial and smooth muscle cells (Wu et al., 2006). For the secondary heart field,
ISL1+ progenitor cells have been described to undergo multilineage differentiation
into myocardial, smooth muscle, and endothelial cells (Moretti
et al., 2006). Taken together, these studies provide unequivocal
evidence for the existence of multipotent progenitor cells in the developing
embryo heart. Understanding the mechanisms of embryonic development—in
particular, identifying the signals that initiate and terminate heart
development—will be crucial to establishing therapeutic regenerative approaches
that utilize similar molecular pathways.
Postnatal Cardiomyocyte Renewal
Occurs in Mammals,
Including Humans
The classic 20th
century teaching was that mammalian cardiomyocytes cease replication soon
after birth, with subsequent growth of the heart attributed to cardiomyocyte
hypertrophy rather than hyperplasia. In the 1990s, the Anversa laboratory
provided crucial evidence that mammalian cardiomyocytes not only enter the cell
cycle in adulthood, but can also subsequently undergo karyokinesis and
cytokinesis (Kajstura et al., 1998; Quaini et al., 1994). Recent studies definitively
demonstrate that cardiomyocyte turnover occurs throughout life in mammals,
including humans, although estimates of the rate of cardiomyocyte turnover vary
dramatically.
Perhaps the most stunning
evidence for cardiomyocyte regeneration in humans was revealed by retrospective
isotope dating studies. Taking advantage of the dramatic spike and decline of
worldwide atmospheric carbon-14 (14C) levels during the 1950s to
1960s due to above ground nuclear bomb testing, Frisen and colleagues developed
an ingenious approach to determine the birth date of cardiomyocytes in humans
by measuring nuclear 14C content (Bergmann
et al., 2009). Their data showed that new cardiomyocytes form in human
myocardium at a rate of approximately 1.5% per year at age 25 years, decreasing
substantially in the latter half of life (Bergmann
et al., 2009).
Using the 14C method
developed by the Frisen group, Anversa and colleagues arrived at much higher
values for cardiomyocyte turnover in humans (7%–23% per year); in addition,
they reported the surprising finding that cardiogenesis increases with age (Kajstura et al., 2012). Mathematical modeling
assumptions in the 14C method could explain some of the differences
in the
14 C studies.
Multiple additional lines of
evidence support a low rate of mammalian cardiogenesis and that the rate
declines further with age (Table 1). Earlier
studies using [3H]thymidine in adult mice estimated an annual
renewal rate of approximately 1% per year (Soonpaa
and Field, 1997), almost identical to the rates of cardiogenesis
estimated by more recent mouse studies (Malliaras
et al., 2013; Senyo et al., 2013). A
similar rate of cardiogenesis in young human adults was recently confirmed
(1.9% at 20 years) using an imaged-based assay in tissue samples procured from
donor hearts prior to transplantation (Mollova et
al., 2013). Thus, while all studies reveal cardiomyocyte renewal in
postnatal mammals, the majority of studies indicate that this rate is very low,
on the order of 1% per year, and that the rate declines with age.
Myocardial Regeneration Occurs
after Injury in Certain
Vertebrates
Critical insight into how we might
regenerate human hearts has arisen from vertebrates that can indisputably
regenerate myocardium following injury. Urodele amphibians such as newts can
survive after amputation of the apical myocardium and demonstrate cardiomyocyte
regeneration by 30 days postamputation (Oberpriller
and Oberpriller, 1974). Similarly, in zebrafish, amputation of the apex
of the heart leads to complete regeneration (Poss
et al., 2002). This dramatic regeneration in urodele amphibians and
zebrafish is thought to be due to limited dedifferentiation of mature
cardiomyocytes and reentry into the cell cycle (Laube
et al., 2006). This is supported by evidence of sarcomere disassembly (Jopling et al., 2010) as well as expression of
Gata4, a transcription factor that is normally expressed during embryonic
development to regulate myocardial formation (Kikuchi
et al., 2010).
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demonstrated some efficacy. Fourth, we must identify the best therapeutic approach for clinical
cardiac regeneration. Finally, we must determine the ideal method to promote
stable differentiation of nonmyocytes into cardiac myocytes. What Is the Source of Regenerated Cardiomyocytes?
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Two theories emerged over
the past decade to explain the origin of new cardiomyocytes in adult mammals:
(1) a progenitor or stem cell gives rise to new cardiomyocytes, or (2) mature
cardiomyocytes reenter the mitotic cell cycle to give rise to new
cardiomyocytes (Figure 1). There are data
to support both of these hypotheses: putative adult progenitor cells in the
myocardium have been identified by multiple markers, including c-kit (Beltrami et al., 2003; Fransioli
et al., 2008), SCA1 (Oh et al., 2003),
and the so-called ‘‘side population’’ cells (Pfister
et al., 2005) (more extensively reviewed by Bollini
et al., 2011). However, other data suggest that the dominant mechanism
of cardiomyocyte generation is not from progenitor cells, but instead from
preexisting cardiomyocytes (SenyoStudies
investigating mammalian cardiomyocyte mitosis after injury can be found as
early as the 1970s (Rumyantsev, 1974),
although more definitive evidence for the potential of embryonic and neonatal
mammalian myocardium to regenerate has recently emerged. Using an elegant
mouse model to effectively damage 50% of the developing cardiac tissue by
inactivating the gene encoding holocytochrome c synthase, Cox and colleagues
demonstrated that lost myocardium is replaced by healthy tissue during fetal
development, resulting in only 10% of the cardiac volume occupied by diseased
tissue at birth (Drenckhahn et al., 2008).
Furthermore, Sadek and colleagues showed that the 1-day-old neonatal mouse
heart is capable of regeneration after resection of approximately 15% of the
ventricle at the apex (Porrello et al., 2011).
This neonatal mouse heart regeneration appears to occur as a result of
dedifferentiation followed by proliferation of preexisting cardiomyocytes.
However, the ability to regenerate myocardium is rapidly lost by 7 days after
birth; instead, the heart develops fibrotic scars similar to the response
observed following myocardial injury in adult mice and humans (Porrello et al., 2011). These experiments raise
the critical question of what prevents mouse heart regeneration after the
first days of life, and point to this first week of life as a crucial period
for understanding inherent regenerative mechanisms in mammals.
Unresolved Questions
Though not all encompassing, here
we discuss five substantial controversies that will require resolution as we
push forward to achieve true cardiac regeneration in a clinical setting.
First, we must understand the source of regenerated cardiomyocytes during
aging and injury. Second, we must establish the ideal cell source for cell
transplantation. Third, we must describe the mechanism by which cell
transplantation clinical trials have et al., 2013).
Although these hypotheses are not mutually exclusive, it is likely that one
mechanism will ultimately prove dominant in the uninjured mammalian heart.
It is possible that theories
of cardiomyocyte refreshment will parallel those of other fields influenced
by the explosion of stem cell science, where early reports of adult stem
cells as the source of renewal were not supported by later lineage mapping experiments.
For example, pancreatic beta cells were thought to arise from progenitor
cells, but rigorous lineage mapping studies revealed that beta cells
themselves are the dominant source of new beta cells (Dor et al., 2004). Lineage mapping experiments using several
markers for putative cardiac progenitors are now underway in many
laboratories, and it is likely that these experiments in aggregate will
reveal or exclude an important role for adult CPCs in mammals.
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The mechanism for cardiomyocyte
homeostasis in normal mammalian myocardium is potentially different from
regeneration after injury, which could trigger a cascade of signals that
activate dormant progenitor cells or induce proliferation of existing
cardiomyocytes (Figure 2). There is
growing evidence for dedifferentiation of existing cardiomyocytes as the
primary pathway for cell renewal both in injury models and during aging (Porrello et al., 2011; Senyo
et al., 2013), while the magnitude of response is perhaps related to
signals activated after injury.
In addition, activation of the
surrounding epicardium, the thin layer of connective tissue and nonmyocytes
on the outer surface of the heart, may contribute to myocardial repair after
injury (Huang et al., 2012). Epicardial
cells that demonstrate an epithelial-to-mesenchymal transition may lead to
myocardial revascularization and perhaps to cardiomyocyte formation as well (Lepilina et al., 2006; Zhou
et al., 2008a). Pretreatment of mice with thymosin beta-4 appears to
enhance the formation of new cardiomyocytes derived from epicardial
progenitor cells (Smart et al., 2011).
However, a subsequent study in which mice were treated with thymosin beta-4
after myocardial infarction showed that injury led to epicardial activation,
which resulted in angiogenesis, but not cardiogenesis (Zhou et al., 2012). Whether the epicardium in
the mammalian heart is able to give rise to cardiomyocytes is a topic that
remains actively discussed. What Is the Ideal
Cell Type for Cell Transplantation Approaches?
The majority of cardiac
regenerative approaches in clinical trials to date have involved
transplantation or infusion of cells with potential progenitor features into
infarcted myocardium. Types of stem cells considered for exogenous delivery
include embryonic, inducible pluripotent, and adult progenitor (including
cardiac, bone marrow, and skeletal myoblast) stem cells. While there are
encouraging signals of benefit in some very rigorously designed and
well-performed studies, there is no consensus on the ideal cell type to use
for cell transplantation (or whether it might be advantageous to use a
combination of cell types, for example, to facilitate both vasculogenesis and
cardiomyogenesis). Ultimately, selection of a cell type that allows for
autologous transplantation, rapid expansion in vitro, and specific
differentiation into cardiomyocytes is desired.
ESCs. Since the first isolation of human ESCs in 1998 (Thomson et al., 1998), the possibility of an
unlimited supply of cardiomyocytes has driven progress in deriving
cardiomyocytes in vitro from human ESCs. When human ESCs are exposed to
activin A and bone morphogenic protein 4, one can generate a highly purified
population of human ESC-derived cardiomyocytes that, when subsequently
transplanted in a prosurvival cocktail, demonstrate enhanced survival
properties in vivo (Laflamme et al., 2007).
Furthermore, by sorting cells based on differences in glucose and lactate
metabolism, cardiomyocyte populations of up to 99% purity have been isolated
from human ESC precursors (Tohyama et al., 2013).
Human ESC-derived cardiomyocytes can also electromechanically couple with
host cells to allow synchronous contraction between the grafted cells and the
host tissue (Shiba et al., 2012). While
human ESC transplantation into human myocardium has not yet been studied,
teratoma formation was observed when incompletely purified human ESC-derived
cardiomyocytes were transplanted into immunosuppressed Rhesus monkeys (Blin et al., 2010). Ultimately, ethical concerns
may prevent the use of human ESCs for clinical cardiac regeneration; however,
human ESCs remain an important laboratory tool for understanding
differentiation and pluripotency in the cardiogenesis process.
Induced Pluripotent Stem Cells (iPSCs). The discovery that
embryonic and mature mouse fibroblasts (Takahashi
and Yamanaka, 2006) can be induced to become pluripotent stem cells by
retroviral transduction of four transcription factors, OCT3/4, SOX2, c-MYC,
and KLF4, revolutionized regenerative biology. Creation of iPSCs from human
fibroblasts (Takahashi et al., 2007; Yu et al., 2007) heightened clinical appeal and
led to rapid implementation of iPSCs as a source of cardiomyocytes (Davis et al., 2012; Nelson
et al., 2009). Like ESCs, iPSCs are multipotent and clonogenic.
However, iPSCs circumvent many of the ethical issues surrounding ESCs, and
the ability to create autologous iPSCs from a skin biopsy, hair follicle
cells, or blood (Aasen and Izpisu´ a Belmonte,
2010) allows potential disease modeling as well as the generation of
large numbers of autologous cardiomyocytes. However, developing procedures to
efficiently and cost-effectively produce sufficient quantities of autologous
cells for transplantation within a therapeutic time frame remains a
challenge. Different types of cardiomyocytes, including atrial-,
ventricular-, and nodal-like cells, can form by differentiation of iPSCs with
distributions similar to that seen with ESC-derived cardiomyocytes (Zhang et al., 2009). Alternative methods to
create iPSCs that avoid the use of viral vectors have been developed to
address tumorigenicity concerns (Okita et al.,
2008). An important issue concerning cardiogenesis with iPSCs is
achieving the long-term stability and integration into the myocardium, as
many cell types derived from iPSCs are incompletely differentiated compared
to the mature cell.
Skeletal Myoblasts. Skeletal myoblasts were among the
first cells tested for cardiac cell therapy applications. However, the MAGIC
clinical trial had disappointing efficacy results and an increased incidence
of arrhythmias in patients who received intramyocardial injection of
autologous skeletal myoblasts obtained via thigh muscle biopsy (Leobon et al., 2003). Because of these
discouraging results, combined with the recent availability of more
attractive cell sources, skeletal myoblast studies have declined in recent
years.
Bone-Marrow-Derived Stem Cells.
Bone-marrow-derived cells are able to differentiate in vitro into a wide
variety of cells, including cardiomyocytes and vascular endothelial cells (Ohnishi et al., 2007). They can also be
harvested for autologous transplantation and have shown relatively safe
profiles in animal and early clinical trials (Amado
et al., 2005; Hare et al., 2012). A
meta-analysis of 33 randomized controlled trials studying transplantation of
adult bone-marrow-derived cells to improve cardiac function after myocardial
infarction revealed substantial heterogeneity between trials, but a
statistically significant improvement in left ventricular ejection fraction
(LVEF) in response to progenitor cell therapy that was not associated with
significant improvements in morbidity or mortality (Clifford
et al., 2012).
In a well-done randomized and
blinded clinical trial, autologous bone marrow cells led to improved outcomes
and ventricular function in patients after myocardial infarction at 2 years
posttransplantation (Assmus et al., 2010)
(REPAIR-AMI trial). However, two recent clinical trials evaluating the safety
and efficacy of bone-marrow-derived cell therapies have been somewhat
discouraging (Marba´ n and Malliaras, 2012).
The TIME trial did not show any improvement in ventricular function after
intracoronary delivery of autologous bone marrow cells (Traverse et al., 2012). Similarly, the POSEIDON
trial, while demonstrating a reassuring safety profile, did not show an
improvement in global ventricular function (as determined by LVEF) after
transendocardial delivery of bone-marrow-derived cells in patients with
ischemic cardiomyopathy (Hare et al., 2012).
Whether bone marrow cells can reduce mortality after myocardial infarction is
now being studied in a large multinational trial in Europe (BAMI trial).
CPCs. Many reports have
described CPCs as multipotent, clonogenic cells that can differentiate into
cardiomyocytes and vascular cells (Beltrami et
al., 2003; Messina et al., 2004).
In some publications, the presence of the c-kit marker is used as a
definition of CPCs (Bearzi et al., 2007; Bolli et al., 2011). These putative progenitors
can be isolated from cardiac tissue obtained during heart surgery or
endocardial biopsy and then expanded in culture for use in autologous
transplantation (Smith et al., 2007). The
use of a single marker to isolate CPCs from adult mammalian myocardium is
problematic and highly susceptible to contamination from nonprogenitor cells.
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delivered to
promote wound healing via cardiomyocyte proliferation or angiogenesis.
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effects from injected cells
has become popular despite only indirect evidence for this theory (Govaert et al., 2009; Loffredo
et al., 2011). In addition to the modulation of the extracellular
milieu in vitro (Baffour et al., 2006),
the effect of transplanted bone-marrow-derived cells on improving cardiac
function may be due primarily to a paracrine effect (Gnecchi
et al., 2005; Iso et al., 2007; Loffredo et al., 2011; Williams
and Hare, 2011).Two prominent clinical trials have reported early
results after transplantation of autologous cells with human progenitor
characteristics. The SCIPIO phase 1 trial demonstrated a 12.3% improvement in
LVEF in patients 1 year after intracoronary injection with autologous c-kit+,
lineage– CPCs following myocardial infarction (Bolli
et al., 2011). In the CADUCEUS phase 1 trial, patients 2–4 weeks
postmyocardial infarction were randomized to receive an intracoronary
injection of cardiosphere-derived autologous stem cells or standard of care (Makkar et al., 2012). While there was no
significant difference between the two groups in measures of global function,
such as LVEF, there was a reduction of the scar mass and an increase of
viable tissue and regional contractility when evaluated by cardiac magnetic
resonance imaging (MRI) at 6 months (Makkar et
al., 2012). No adverse events related to cell transplantation were
reported in either study at 1 year (SCIPIO) or 6 months (CADUCEUS). To date,
no single cell type has proven itself to meet sufficient criteria for
widespread use in clinical applications, a fact that may ultimately hinder
progress in cell transplantation approaches.
What Is the Mechanism of Action
by which Cell
Transplantation Demonstrates Clinical Efficacy? The
mechanism by which exogenous administration of autologous progenitor cells
contributes to improving cardiac function remains unclear. It is possible
that these autologous cells are leading to regeneration, but it is also
plausible that paracrine effects or changes in the myocardial response to
injury are responsible. The available technology for imaging cell fate and
myocardium does not allow determination of true regeneration; therefore we
must rely on surrogate measures of efficacy.
Prominent claims that bone
marrow cells can become cardiomyocytes after transplantation into myocardium
(Orlic et al., 2001) have not been
replicated by other laboratories (Loffredo et
al., 2011; Murry et al., 2004; Wagers et al., 2002). This conflict is responsible
for some of the ongoing confusion in the field (Limbourg
and Drexler, 2005). The use of bone marrow cells for prevention and
treatment of heart failure has had varied clinical success to date but
remains under intense clinical investigation as described above.
Extensive data indicate that
most cells transplanted into the heart do not survive long-term, and thus the
concept of paracrine Even in the case of human cardiosphere-derived cells,
which are derived from human myocardium, the benefits of cell therapy may be
paracrine (Li et al., 2012). The factors
secreted or released from injected cells that benefit cardiac function remain
to be identified. If there is a specific combination of multiple factors from
a defined population of cells, then unraveling the paracrine cocktail may be
very challenging. Furthermore, as improved methods to enhance cell survival
and engraftment are developed, distinguishing between independent cell
effects and paracrine effects will become even more difficult.
A major challenge in cell therapy approaches is how to
improve engraftment. An excellent review by Terrovitis and colleagues
describes methods to both evaluate and optimize engraftment (Terrovitis et al., 2010). Methods to quantify
engraftment remain controversial, and correlation of engraftment to
improvements in morbidity and mortality remain unclear. Surrogate measures of
success such as global heart function with LVEF may not provide adequate
resolution, although cardiac MRI may facilitate both local and global assessment.
Finally, introducing cells into a hostile, diseased environment such as
ischemic myocardium likely hinders engraftment, and without the
reestablishment of adequate vascularization, it is unlikely that
transplantation of cardiomyocytes alone will achieve success.
What Is the Ideal Approach for
Clinical Cardiac Regeneration?
Multiple approaches are under
investigation for human cardiac regeneration (Figure
3). As described above, significant progress has been made in cell
transplantation approaches; however, these methods are challenged by poor
cell survival and engraftment and may lack true regeneration. Alternatively,
reprogramming of endogenous nonmyocytes into cardiomyocytes may allow in situ
transdifferentiation, although these methods require further validation
before they will be ready for clinical trials.
Despite the lack of evidence for true regeneration with
cell therapy approaches, clinical success will ultimately depend on evidence
of clinical efficacy, and some cell therapy methods have shown limited
improvement in cardiac function as described above. Importantly, cardiac cell
therapy has been surprisingly safe to date. No report of tumor formation has
occurred in over 1,500 patients involved in bone marrow cell cardiac trials (Clifford et al., 2012). Teratoma formation has
been seen in monkeys injected with unpurified human ESC-derived
cardiomyocytes (Blin et al., 2010);
however, adequate purification of cardiac populations prior to
transplantation may prevent tumor formation (Blin
et al., 2010; Tohyama et al., 2013).
No consensus has been reached about the optimal delivery
method for transplanted cells. Intravenous, intracoronary, and
intramyocardial injection methods have all been proposed, although all are
limited by poor local retention (Dib et al., 2011).
Tissue engineering approaches combine cells with biomaterials to address
logistical challenges. Use of injectable hydrogels has been studied with both
natural and synthetic biomaterials to try to improve local retention (Ye et al., 2011). Biodegradable scaffolds seeded
with cells can be used to form well-defined architectures as in valve tissue
engineering (Schmidt et al., 2007).
Finally, placement of a cardiac patch formed with stem cells can provide both
structural and paracrine support after myocardial injury (Wei et al., 2008). While tissue engineering
approaches are still in development, these approaches will likely augment the
behavior, and ultimately the success, of transplanted cells.
Cellular reprogramming approaches aim to modify the
phenotype of native cells to induce cardiomyocyte renewal via delivery of
small molecules in vivo. Cellular reprogramming strategies may ultimately win
over cell transplantation because of the challenges of timely production of
sufficient quantities of autologous cells that meet all criteria necessary
for safe and efficacious transplantation. However, much work remains before
the safety and efficacy of reprogramming allows clinical testing. Aguirre and
colleagues (Aguirre et al., 2013) recently
provided an excellent review on animal models for cardiac reprogramming, and
this topic is discussed further in the following section. How Can We Promote Stable Differentiation of
Nonmyocytes into Cardiac Phenotypes?
The possibility of skipping the
multipotent state and directly reprogramming cells in vivo from one
differentiated phenotype to another was demonstrated in pancreatic cells by
Melton and colleagues (Zhou et al., 2008b).
The Srivastava group devised a method to directly reprogram fibroblasts to
cardiomyocyte-like cells using a combination of three transcription factors
(GATA4, MEF2C, and TBX5) (Ieda et al., 2010).
Using a retroviral system to deliver GATA4, MEF2C, and TBX5 to 2-month-old
male mice in vivo via intramyocardial delivery, the same group found that
cardiomyocyte-like cells were formed from the resident fibroblast population,
and this intervention resulted in improved myocardial function after
infarction (Qian et al., 2012). Similarly,
four transcription factors (GATA4, HAND2, MEF2C, and TBX5) were used to
reprogram mouse tail-tip and cardiac fibroblasts into functional
cardiomyocyte-like cells in vivo (Song et al.,
2012).
Subsequent studies have demonstrated direct
reprogramming using microRNA (Jayawardena et al.,
2012) or alternative transcription factors such as ETS2 and MESP1 (Islas et al., 2012). However, these methods
exhibit low efficiency and incomplete efficacy in reprogramming fibroblasts
into cardiomyocyte-like cells (Chen et al., 2012),
and further investigation is required to better understand the mechanisms by
which transdifferentiation occurs. If, as suggested by Srivistava and
colleagues (Qian et al., 2012), maturation
of reprogrammed cells can occur in vivo, then it is conceivable that
long-term stable integration of reprogrammed cardiomyocytes may be possible.
It remains unclear if delivery of transcription factors may have effects on
noncardiac tissues in the event of poorly localized delivery, or if
uncontrolled cardiomyocyte reprogramming has adverse effects such as rhythm
disturbances. Prior to clinical translation of cellular reprogramming
methods, we must achieve a deeper understanding of the molecular mechanisms
of regeneration.
Conclusions
Stem cell
biology holds significant promise for heart diseases. Because autologous
cardiac cell therapy appears to be safe and possibly effective, investigators
are aggressively advancing this clinical approach. At this early stage, these
efforts must undergo rigorous study, preferably with randomization and
blinded outcome assessment. We believe that cardiac cell therapy outside of
such carefully designed and monitored trials is currently unethical. As is
apparent to most investigators in the field, the current published data on
cardiac regeneration and cardiac stem cells conflict in important ways. While
confusion is to be expected in early days of an exciting field, this is
especially true when new technologies are coming out rapidly and when
clinical trials have begun, as investigators feel even more invested in the
‘‘established’’ premises underlying their work. But as the enthusiasm for
cardiac regeneration charges ahead toward clinical translation, it is crucial
for all investigators to maintain objectivity and seek new and complementary
approaches to resolve apparent controversies.
Are we on the right path?
Although it is possible that current cardiac cell therapy trials in humans
are causing true regeneration, we suggest that the overall evidence is most
consistent with the concept that cardiac cell therapy is regulating an
endogenous repair process and not leading to true regeneration. Nonetheless,
patients who achieve improved recovery will not care if we call it
‘‘regeneration’’ or ‘‘repair,’’ so enhancing heart function through cell
transplantation is a worthy goal, even if it turns out not to be through true
regeneration.
Ultimately, though, we must understand the dramatic
differences between cardiac regeneration in experimental models like zebrafish
and neonatal mice and the profound postnatal loss of cardiac regenerative
potential in adult mammals like mice and humans. Is this due to intrinsic
properties of cardiomyocytes or due to failure of stem/progenitor
populations? Is it due to noncardiomyocytes, such as activated fibroblasts
creating scarring that blocks regeneration? As in regeneration of many
different mammalian organs, the core issues in cardiac regeneration remain
mysterious, and we have yet to understand what signals start the regenerative
process, how regeneration is guided, and finally, how regeneration is
terminated.
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