Reprogramming toward Heart Regeneration: Stem Cells and Beyond
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Transcript of Reprogramming toward Heart Regeneration: Stem Cells and Beyond
Cell Stem Cell
Review
Reprogramming toward Heart Regeneration:Stem Cells and Beyond
Aitor Aguirre,1 Ignacio Sancho-Martinez,1,2 and Juan Carlos Izpisua Belmonte1,2,*1Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA2Center of Regenerative Medicine in Barcelona, Dr. Aiguader, 88, 08003 Barcelona, Spain*Correspondence: [email protected]://dx.doi.org/10.1016/j.stem.2013.02.008
Finding a cure for cardiovascular disease remains a major unmet medical need. Recent investigations havestarted to unveil the mechanisms of mammalian heart regeneration. The study of the regenerative mecha-nisms in lower vertebrate and mammalian animal models has provided clues for the experimental activationof proregenerative responses in the heart. In parallel, the use of endogenous adult stem cell populationsalongside the recent application of reprogramming technologies has created major expectations for thedevelopment of therapies targeting heart disease. Together, these new approaches are bringing us closerto more successful strategies for the treatment of heart disease.
Heart disease remains one of the leading causes of human
mortality in the developed world. In the United States alone, 80
million people suffer from some kind of heart disease, and every
year it kills more Americans than any other illness. Furthermore,
statistics show a pessimistic trend worldwide in which heart
disease is expected to be the leading cause of death in
the world by 2020 (data from The Heart Foundation, http://
www.theheartfoundation.org/heart-disease-facts/heart-disease-
statistics; Kelly et al., 2012). Heart injuries, such as acutemyocar-
dial infarction (MI), can lead to immediate death, due to loss of
oxygenation in ventricular muscle, usually by occlusion of
a coronary artery, which quickly results in ischemia/reperfusion
injury and necrosis of the tissue. In the best of cases, living
patients face progressive deterioration of their condition over
several years, ultimately resulting in heart failure. This failure
stems from a lack of intrinsic regenerative responses able to
replenish the vast amounts of lost cardiomyocytes and results
in scar formation and rapidly compromised heart function in
mammals. Indeed, upon loss of contractile function, the heart
elicits a major hypertrophic response resulting in larger, but not
necessarily a greater number of, cardiomyocytes. As a result,
a larger heart size does not translate into increased contractility
and this feedback loop continues in an ever-worsening direc-
tion that further debilitates the muscle and augments suscepti-
bility to future infarctions (Laflamme and Murry, 2011), eventually
leading to death.
Whereas a few reports have previously indicated the possi-
bility for endogenous heart repair and cardiomyocyte genera-
tion, the adult mammalian heart has nevertheless been generally
considered a terminally differentiated organ (Beltrami et al.,
2001). Cardiomyocytes were assumed to be fully postmitotic
cells. Indeed, cardiomyocytes exhibit a phenotype that seems,
a priori, incompatible with mitotic division. First, they possess
a very complex and well-developed cytoskeleton consisting of
hundreds of sarcomeres, which physically impede mitotic
spindle formation and tight cell-cell junctions necessary for
synchronous beating (e.g., GAP junctions). Second, adult mam-
malian cardiomyocytes are often multinucleated and polyploid.
Despite these characteristics, an elegant study by Beltrami
et al. (2001) shook this dogma of modern cardiac biology
by demonstrating that the adult human heart possessed popula-
tions of cardiomyocytes with proliferative potential. More re-
cently, an innovative approach based on the integration of
carbon-14 showed that adult human cardiomyocytes renew,
albeit very slowly, with decreasing numbers as we age (�1%
at age 25) (Bergmann et al., 2009). Sadly, this pool of cells is
clearly insufficient to heal the heart after myocardial infarction
and seemed to be more relevant for long-term homeostatic
maintenance of heart function (Figure 1). These observations
have given impetus and increased momentum to efforts focused
on the finding and activation of cardiac-resident stem cells, the
transplantation of in vitro stem cell-derived cardiomyocytes
(Loffredo et al., 2011; Moretti et al., 2006), and/or the administra-
tion of compounds, such as growth factors and/or cytokines,
aimed at improving vascularization, preventing scar formation,
and even inducing cardiomyocyte proliferation (Bersell et al.,
2009; Marsano et al., 2013; van Rooij et al., 2008).
Unfortunately, the efficiency of most of these approaches
remains controversial. Interestingly, findings from certain non-
mammalian vertebrates showing that heart regeneration occurs
by partial dedifferentiation and subsequent proliferation of
cardiomyocytes and/or mobilization of epicardial cells (Jopling
et al., 2010; Kikuchi et al., 2010, 2011; Smart et al., 2011) have
raised the question of whether similar dormant mechanisms
are present in the mammalian heart, and if so, whether experi-
mental manipulation could serve to stimulate heart regeneration
in mammals. As of now, different and somewhat conflicting lines
of evidence have emerged in the heart field. While the existence
of marginal proliferative cell populations indicative of attempts at
regenerating the damaged mammalian heart have been demon-
strated, the precise identity of these populations, and how and to
what degree they are involved in heart repair remains unclear.
Furthermore, the question of which cell population and/or
approach is most suitable for experimental manipulation and
development of therapeutic alternatives for the treatment of
heart disease remains amajor topic of discussion in the scientific
community. Interestingly, recent reports on the establishment in
the laboratory of new regenerative animal models addressing the
Cell Stem Cell 12, March 7, 2013 ª2013 Elsevier Inc. 275
Figure 1. Endogenous Regeneration in the Neonatal Mammalian HeartNeonatal mice (P1–P7) are able to regenerate the lost muscle by activating a cardiomyocyte-mediated proliferative organ-wide response (Porrello et al., 2011b)that restores complete function and leaves no scarring. These cardiomyocytes exhibit typical traits of dedifferentiation such as those observed in the zebrafish(Jopling et al., 2010). However, adult mice fail to regenerate the myocardial mass.
Cell Stem Cell
Review
basic biology underlying heart regeneration have unveiled novel
molecular mechanisms and identified avenues toward inducing
heart repair.
In this Review, we will discuss several recent findings that
have further clarified potential sources of regeneration in the
mammalian heart as well as provide a contextual overview of
the different stem cell strategies targeting heart injury. Together,
these reports provide a fresh look at heart regeneration and open
new doors to exciting therapeutic opportunities based onmanip-
ulating cardiomyocyte proliferation, epicardial cell activation,
and both endogenous and engineered stem cells.
Understanding Heart Regeneration in Animal Models:From Adult to Neonatal RegenerationUnlike the mammalian heart, the zebrafish heart undergoes
minimal scarring and exhibits a robust regenerative response
upon injury. After amputation of �20% of the ventricular apex,
a transient fibrin clot forms, followed by a robust and sustained
regenerative response that replenishes the lost cardiac muscle
in a period of 30 to 60 days (Poss et al., 2002; Raya et al.,
2003) thus, demonstrating a general rather than local
response that involves cardiomyocyte migration through the
heart (Jopling et al., 2010; Itou et al., 2012). The whole phenom-
enon is partially mediated by reactivation of dormant develop-
mental pathways important for heart formation, such as the
Notch pathway (Raya et al., 2003). But where do these cardio-
276 Cell Stem Cell 12, March 7, 2013 ª2013 Elsevier Inc.
myocytes originate? The current line of evidence indicates that
the new muscle originates not from differentiating stem cells,
as suggested by others (Lepilina et al., 2006), but from mature
cardiomyocytes that undergo dedifferentiation and subse-
quent proliferation, as independently demonstrated by genetic
lineage-tracing approaches by Kikuchi and colleagues and our
laboratory (Jopling et al., 2010; Kikuchi et al., 2010). Dedif-
ferentiated, proliferating cardiomyocytes are characterized by
re-expression of the cardiac progenitor marker GATA4, downre-
gulation and disassembly of sarcomeres, and acquisition of an
infiltrative mesenchymal-like phenotype (Kikuchi et al., 2010;
Jopling et al., 2010; Itou et al., 2012). Once dedifferentiation
and proliferation has finished, dedifferentiated cardiomyocytes
undergo a subsequent maturation step and return to their basal,
quiescent state, completely restoring cardiac function.
While progress has been steady in the zebrafish, endogenous
mammalian cardiomyocyte dedifferentiation has remained elu-
sive and questionable until very recently. In 2011, Porrello and
colleagues observed a remarkably similar regenerative response
in neonatal murine hearts (Porrello et al., 2011b). Mice subjected
to 10%–15% ventricle amputation were able to elicit a regenera-
tive response during the early days of life (up to 7 days). Murine
neonatal heart regeneration was characterized by proliferating
cardiomyocytes (Porrello et al., 2011b). These newly generated
cardiomyocytes arose from pre-existing cardiomyocytes, as
demonstrated by Cre/lox genetic lineage-tracing approaches,
Cell Stem Cell
Review
and restored complete heart function after approximately
30 days. Fibrotic scarring and hypertrophy, both typical hall-
marks of the postinjured mammalian heart, were completely
absent (Figure 1). Even though no specific dedifferentiation
experiments were performed, the authors of the study noted
the characteristic disassembly of sarcomeric structures indica-
tive of a dedifferentiated phenotype (Porrello et al., 2011b).
More recently, Porrello et al. have established a model of
ischemic MI in postnatal mice and demonstrated that the
neonatal heart mounted a robust regenerative response leading
to proliferation of pre-existing cardiomyocytes and functional
recovery within 21 days. Interestingly, the authors demonstrated
that inhibition of the miR-15 family increased cardiomyocyte
proliferation in the adult heart and improved ventricular systolic
function after adult MI (Porrello et al., 2013). Together, these
reports demonstrate that an efficient regenerative response
can be elicited in mammalian hearts and that the regenerative
responses are amenable to experimental manipulation.
In spite of these unexpected similarities, some important
differences exist between the zebrafish and the neonatal
mouse model that must be taken into account. The adult
zebrafish heart is a postmitotic tissue, with extremely low
levels of proliferative cardiomyocytes in uninjured conditions,
whereas the neonatal mouse heart is a growing organ, with car-
diomyocytes entering the cell cycle for several days after birth.
These and other differences most likely reflect the intrinsic
developmental status of the heart and/or developmental differ-
ences present in the surrounding niche. Whereas regeneration
in the fish involves the reactivation of a proliferative program
reminiscent of development—with all the associated reprog-
ramming events that this might involve—in neonatal mice, the
regenerating heart is a partially immature tissue that is still
undergoing development, albeit at its latest stages. Despite
these unknowns, it is remarkable that neonatal mouse cardio-
myocytes can respond to the injury in such an efficient and
similar manner to that seen in the zebrafish. Together, these
observations suggest that pathways leading to cardiomyocyte
proliferation, and eventually heart regeneration, are much
more conserved than we might have expected before and
positions the neonatal murine model as a powerful tool for
the elucidation of strategies restoring adult heart function in
pathological conditions.
Regeneration by Cardiomyocyte Dedifferentiationand ProliferationWhile these and other studies indicate that activation of prorege-
nerative responses is indeed possible in mammalian hearts, the
mechanisms at play remain largely unknown. Does dedifferenti-
ation occur in response to injury conditions in adult mammals? If
so, how do cardiomyocytes dedifferentiate? What are the epige-
netic modifications governing mammalian cardiomyocyte dedif-
ferentiation and/or proliferation? Which molecules are activating
and controlling this process?
By combining traditional genetic fate-mapping approaches
with multi-isotope imaging mass spectrometry (MIMS), Senyo
and colleagues recently provided compelling evidence impli-
cating mature cardiomyocytes as the source of new cardio-
myocytes in aging mice (Senyo et al., 2013). To determine
whether cardiomyocytes proliferate, the authors first focused
on measuring the incorporation ratio between15N-labeled
thymidine—a stable natural nitrogen isotope—and normal14N-thymidine, and further compared it against the normal
endogenous occurring values (0.37%). This method has several
advantages over traditional halogen-substituted nucleotide
analogs, such as BrdU, namely, the lack of toxicity as well as
the high imaging resolutions that can be attained. Whereas
employing this methodology did not directly address prolifera-
tion per se, as DNA synthesis can occur in the absence of cell
division, they further validated the existence of newly generated
cardiomyocytes by lineage-tracing experiments using double-
transgenic MerCreMer/ZEG mice. The reported findings are in
agreement with previous studies in humans (Bergmann et al.,
2009) and yield very similar rates of proliferation under homeo-
static conditions with a 0.76% turnover rate for young mice
versus 1% for young humans. The authors also found that a
population of mononucleated proliferating cardiomyocytes
appeared in the peri-infarct region in adult mice subjected to
experimental myocardial infarction, raising the rate of pro-
liferating cardiomyocytes over the observed basal levels. Inter-
estingly, they also identified hypertrophic cardiomyocytes
undergoing rounds of karyokinesis without mitosis (by detecting
increased DNA content), suggesting that at least two indepen-
dent populations of cardiomyocytes with different proliferative
capacities might exist (Senyo et al., 2013). Although the
response observed is insufficient to regenerate the damaged
muscle, the fact that the adult mammalian heart is able to mount
a cardiomyocyte-mediated regenerative response, which so
closely resembled that seen in neonatal mice and adult zebra-
fish, is a remarkable finding with exciting implications (Figure 2).
Most interestingly, the authors were unable to observe prolifera-
tion of endogenous progenitor cells, indicating that they did
not contribute to the regenerating heart and/or that pools of
progenitor cells were quickly exhausted upon injury (Senyo
et al., 2013).
Taking a different approach, researchers have recently looked
at molecules expressed during dilated cardiomyopathy (DCM).
Under DCM, the heart undergoes remodeling leading to hyper-
trophy, ventricular dilation, and dysfunction, factors which
together contribute to heart failure. Cardiomyocytes in these
circumstances show certain signs of dedifferentiation, having
disorganized sarcomeric structures and generally possessing
a phenotype similar to that of fetal or embryonic cardiomyocytes
(Kubin et al., 2011). By analyzing samples of patients with DCM,
it was found that oncostatin M (OSM), an inflammatory cyto-
kine belonging to the interleukin-6 family, as well as its cognate
receptors, were significantly overexpressed (Kubin et al.,
2011). Interestingly, treatment of adult cardiomyocytes with
OSM induced in vitro dedifferentiation, activation of the Ras/
Raf/MEK/ERK cascade, reactivation of cardiac progenitor cell
marker expression (particularly c-Kit), and finally re-entry into
the cell cycle. When the same treatment was applied in vivo to
normal mice, cardiomyocyte dedifferentiation was observed. In
this setting, dedifferentiated cardiomyocytes seemed unable to
proliferate and instead led to reduced contractile strength and
pathologic remodeling (Kubin et al., 2011). These findings high-
light the fact that cardiomyocyte dedifferentiation is not always
synonymous with proliferation and improvement of heart func-
tion (Figure 2). One possible explanation for this apparent
Cell Stem Cell 12, March 7, 2013 ª2013 Elsevier Inc. 277
Figure 2. Regeneration in the Adult Mammalian HeartMammalian cardiomyocytes possess a very limited proliferative capacity. Under normal physiological conditions, two potential sources of cells contribute to theturnover of cardiomyocytes: cardiac progenitor cells andmature cardiomyocytes. This turnover is very low and decreases significantly with age (Bergmann et al.,2009; Senyo et al., 2013). Upon myocardial injury, the mammalian heart initiates an endogenous regenerative response, the extent of which is age dependent.An endogenous, cardiomyocytic hyperplasic response is observed but is very limited and restricted to the peri-infarct region (Senyo et al., 2013). Thesecardiomyocytes exhibit partially dedifferentiated traits. However, prolonged dedifferentiation can lead to hypertrophic cardiomyocytes and further contribute toheart failure (Kubin et al., 2011). In addition to cardiomyocytes, epicardial-derived cells might contribute to the myocardial mass and activation of inflammatorysignals (Smart et al., 2011; Huang et al., 2012).
Cell Stem Cell
Review
discrepancy could be the lack of efficient redifferentiation;
however, these questions await further investigation. Most
intriguingly, these results raised the question of whether it is
possible at all to manipulate the mechanisms of cardiomyocyte
proliferation in an adult mammal without negative conse-
quences, such as hypertrophy and loss of contractile function.
Altogether, these observations pinpoint the need to delve deeper
into the mechanisms underlying dedifferentiation and prolifera-
tion before we can safely manipulate this regenerative process
in humans.
A recent report by Giacca and colleagues suggests that it is
indeed feasible to experimentally drive adult mammalian cardio-
myocytes toward a proliferative state (Eulalio et al., 2012) and,
furthermore, that induction of proliferation can be used toward
therapeutic applications. In this study, in vivo proliferation of
mature cardiomyocytes was achieved by manipulation of
microRNA expression. The authors employed high-throughput
analysis and high-content microscopy to screen neonatal prolif-
eration-competent cardiomyocytes with a library of more than
800microRNAmimics. The study focused on findingmicroRNAs
that promote the expression of proliferative markers (Ki67,
H3S10ph) and incorporation of DNA analogs indicative of DNA
278 Cell Stem Cell 12, March 7, 2013 ª2013 Elsevier Inc.
synthesis. The authors found 204 microRNAs that enhance
proliferation more than 2-fold in rat and mouse cardiomyocytes
and chose two highly efficient microRNAs, namely miR-199a
and miR-590, to be delivered in uninjured and infarcted rats via
adeno-associated virus (AAV) transduction. The results showed
a spectacular increase in the number of proliferating cardiomyo-
cytes in both conditions, leading to a significant improvement of
cardiac function after infarction. Along this line, Porrello et al.
(2011a) revealed that miR-195, a member of the miR-15 family,
is a critical regulator of cardiomyocyte proliferation. miR-195 is
more highly expressed in postnatal day 10 (P10) mice compared
to P1, and its precocious overexpression leads to hypoplasia.
Since P1 mice hearts exhibited remarkable cardiomyocyte-
mediated regenerative capacity in the heart, the authors hypoth-
esized that experimental miR-195 downregulation in older
animals could eliminate a major roadblock and facilitate cardio-
myocyte proliferation. As expected, miR-195 downregulation
in adult mouse hearts resulted in significant cardiomyocyte
proliferation (Porrello et al., 2011a).
Although both of these experiments show that experimental
manipulation of cardiomyocyte proliferation is possible andcould
be effective toward recovery from heart failure, dedifferentiation
Figure 3. Therapeutic Strategies to Improve Cardiac Function In VivoCardioprotective agents aiming at reducing and/or preventing the early deleterious effects of myocardial infarction (e.g., ischemia/reperfusion, inflammation, andfibrotic scarring) may constitute the gold standard approach toward repairing damage directly in the patient’s body. However, with this approach there is noreplenishment of lost cardiac mass. Alternatively, the heart regeneration approaches recently described (Porrello et al., 2011a, 2011b; Eulalio et al., 2012; Smartet al., 2011; Zhou et al., 2008) aim to restore the cardiac tissue lost after the infarction by increasing the number of cardiomyocytes, either bymobilizing epicardialprogenitors with cardiomyocyte differentiation potential or by activating endogenous regenerative pathways leading to dedifferentiation and proliferation ofmature tissue-resident cardiomyocytes. In vivo reprogramming by lineage conversion of cardiac fibroblasts into cardiomyocytes is another attractive possibilityto replenishmyocardial tissue loss (Song et al., 2012; Qian et al., 2012). In this approach, cardiac fibroblasts can be reprogrammed to become cardiomyocytes byforced expression of GATA4, TBX5, MEF2C, and HAND2.
Cell Stem Cell
Review
of cardiomyocytes before or during proliferation has not yet been
assessed, and the underlying cellular andmolecularmechanisms
leading to cardiomyocyte re-entry into the cell cycle remain
unknown (Figure 3).
Regeneration by Epicardial Cell ActivationCells of the epicardium are another potential source of regener-
ating cells besides differentiated cardiomyocytes. Epicardial
cells have been consistently described in the literature and
identified to play a role in regenerating mammalian hearts. The
epicardium is a mesodermal-derived layered structure covering
the myocardium and has been reported to be a source of
progenitor cells. During development, the epicardium contrib-
utes to several heart populations, including fibroblasts, smooth
muscle cells, and pericytes. Previous reports have attributed
the capacity to form cardiomyocytes de novo to epicardial cells
(Zhou et al., 2008), although some contradictory results highlight
the need for further investigation. For example, genetic fate-
mapping approaches based on regulatory sequences from
Tbx18 and Wilm’s tumor 1 (Wt1) have been problematic since
their expression is not restricted to epicardial progenitors alone
(Zhou et al., 2008; Kikuchi et al., 2011; Smart et al., 2011). Using
Tcf21 as a more specific epicardial cell marker in combination
with genetic lineage fate mapping, it was found that epicardial
cells do not contribute to myocardial lineages in the zebrafish
(Kikuchi et al., 2011). However, in the last 2 years, two indepen-
dent groups have described epicardial progenitor cell-mediated
effects in cardiac recovery after MI (Smart et al., 2011; Huang
et al., 2012). The findings by Smart et al. describe a population of
epicardial progenitors that are activated to re-express Wt1 upon
thymosin b4 treatment, a peptide known to promote vascular
regeneration after injury (Smart et al., 2011). To assess that
labeling of Wt1 cells was restricted to epicardial-derived progen-
itors, the authors used a double-labeling approach, consisting
Cell Stem Cell 12, March 7, 2013 ª2013 Elsevier Inc. 279
Cell Stem Cell
Review
of constitutive GFP expression by means of Wt1GFP/Cre+ and
pulsed expression of YFP driven by Wt1CreERT2/YFP+ in adult
animals. Treatment of these mice with Tb4 resulted in an expan-
sion of Wt1 cells, which were able to contribute to myocardial
mass, as determined by genetic lineage tracing (GFP/YFP+ car-
diomyocytes). After experimental myocardial infarction, animals
treated with the compound exhibited better recovery than
control animals, although in general the rate of cardiomyocyte
generation from Wt1 cells was modest. A different study
provided a fresh look into how epicardial cells might contribute
to recovery after MI (Huang et al., 2012). By analyzing conserved
regions in epicardial-conserved genes in vertebrates, the
authors identified enhancer elements that exhibit activity in the
embryonic epicardium in organ culture systems. Interestingly,
all of the investigated genes had common enhancer elements
responsive to the CCAAT/enhancer binding proteins (C/EBPs),
a family of basic-leucine zipper transcription factors. Since the
epicardium is significantly activated in the adult mammalian
heart after MI and ischemia/reperfusion (IR) injury, it was
reasoned that C/EBP inhibition might contribute to enhance
heart tissue repair by modulating the inflammatory response.
Supporting this hypothesis, C/EBP inhibition impaired epicardial
activity and resulted in improved cardiac function and dimin-
ished infarct size. Furthermore, inflammatory cell infiltration in
the heart was substantially reduced, demonstrating the cardio-
protective effects of this treatment.
Altogether, it seems clear that the epicardium is able to
mediate signals leading to the healing of the heart, albeit by
poorly understood mechanisms (Figures 2 and 3). The recent
reports by Huang and Smart might at first seem contradictory,
as Smart et al. rely on epicardial activation, whereas Huang
et al. rely on epicardial inhibition, for tissue repair. Yet, it is impor-
tant to consider that epicardial activation might be distinct
depending on the stimuli and/or the degree of injury employed
and that the overall specific type of response in heart recovery
might be different.Tb4 treatment led to recovery paralleled by
marginal de novo cardiomyocyte formation from progenitor
cells, possibly accompanied by proangiogenic effects, whereas
the results observed by Huang et al. seem to be the conse-
quence of a reduced inflammatory response with cardioprotec-
tive effects hampering scar formation in a similar way to that
observed upon spinal cord injury (Letellier et al., 2010). Epicardial
activation might then be a double-edged sword, both necessary
for myocardial muscle regeneration mediated by epicardial
progenitors and deleterious during the inflammatory phase of
injury due to an exacerbated inflammatory and immune
response. Studies comparing the beneficial effects of both
strategies may help to determine the most advantageous path
for heart function recovery. In summary, while the detailed role
of epicardial cells and their responses awaits further investiga-
tion, it is evident that precise manipulation of the beneficial
effects while controlling the inflammatory responses elicited by
the epicardiummay contribute to overall improved heart function
(Figure 3).
Stem Cell-Based Approaches for Heart RepairTaking into account the major need for therapeutic strategies
targeting heart injury, it is not surprising that stem cell-based
approaches have emerged as a major opportunity for cardiovas-
280 Cell Stem Cell 12, March 7, 2013 ª2013 Elsevier Inc.
cular applications. Indeed, the fact that adult organisms
possess populations of multipotent stem cells with differentia-
tion potential, alongside the recent discovery of reprogramming
approaches for the induction of pluripotency in somatic cell
lineages, has led to the development of stem cell-based strate-
gies including bone marrow stromal cells (BMSCs), endogenous
cardiac progenitor cells (CPCs), as well as pluripotent stem cells
(PSCs), comprising both embryonic stem cells (ESCs) and
induced pluripotent stem cells (iPSCs). All these stem cell-based
approaches constitute a body of promising complementary tools
for the repair of damaged heart tissue, by mobilization of endog-
enous progenitor populations and/or by cell transplantation.
Since this Review is geared toward regeneration rather than
cell transplantation, we have focused on describing in more
detail those strategies allowing for the promotion of heart regen-
eration in vivo while more briefly exploring the use of PSCs
and refer the reader to other excellent Reviews discussing
promising research avenues for cell transplantation-based
therapies (Passier et al., 2008; Braam et al., 2009; Passier and
Mummery, 2010; Laflamme and Murry, 2011).
Possibly the most-studied approach for heart repair involves
the use of adult stem cells (BMSCs and CPCs). Over the last
few years, evidence of the existence of adult cardiac progenitor
cell populations (CPCs) in the heart has been reported (Guan and
Hasenfuss, 2007). CPCs were first characterized by expression
of c-Kit+ cells and their ability to participate in the repair of the
left ventricle of the heart. c-Kit+ cells not only contributed to
the pool of newly regenerated cardiomyocytes but also to
other cell types including smooth muscle cells and endothelial
cells (Beltrami et al., 2003). Based on Islet-1 expression and
lineage-tracing experiments, another population of cells able to
differentiate into endothelial, smooth muscle, and cardiomyo-
cytes was identified during cardiogenesis (Laugwitz et al.,
2005; Moretti et al., 2006). Further analysis has indicated that
rather than fully distinct populations, the observed differences
in marker expression and overall cellular phenotype could be
due to the analysis of subpopulations and/or progenitor popula-
tions at different stages rather than the existence of distinct indi-
vidual cardiac progenitor cell populations. Interestingly, even the
progenitor nature of certain putative CPC populations has been
questioned (Andersen et al., 2009). Moreover, the recent report
by Senyo et al. suggests that the contribution of CPCs to
cardiac regeneration is negligible (Senyo et al., 2013). Similar
to CPCs, BMSCs have been reported to bear differentiation
potential toward the vascular and cardiac muscle lineages
in vitro, and their transplantation into injured hearts has resulted
in improved ventricular function. While it was initially suggested
that the mechanisms by which BMSCs could repair the injured
heart involved transdifferentiation of BMSCs into cardiac line-
ages, later reports do not support this conclusion (Braam
et al., 2009). Nowadays, the prevailing theory is that transplanted
BMSCs may exert their beneficial effect indirectly by improving
heart function through paracrine mechanisms protecting the
myocardium, rather than increasing the number of contractile
cardiomyocytes (Ranganath et al., 2012). Together, and despite
the existence of controversy surrounding both populations of
adult stem cells, it is clear that understanding the precise nature
of CPC populations, as well as the mechanisms by which
BMSCs protect the injured myocardium, open the possibility
Figure 4. Heart Repair Approaches toImprove Functional Recovery in CardiacDiseaseThese strategies rely on cell therapy to provide asource of cardiac progenitors capable of restoringthe damaged tissue. Typically, cardiac progenitorcells and PSCs have been proposed for thispurpose after expansion in culture (Moretti et al.,2006; Laugwitz et al., 2005). With the adventof reprogramming technologies, production ofcardiomyocyte-like cells for therapy can also beachieved from other unrelated cell types bylineage conversion and/or from patient-specificiPSCs (Ieda et al., 2010). After expansion andgeneration of a sufficient number of cells, they aretransplanted into the area of interest.
Cell Stem Cell
Review
for developing strategies aimed at the activation and mobiliza-
tion of endogenous adult stem cell pools (Figure 4), as well as
the tailoring of specific differentiation protocols for the directed
differentiation of PSCs to CPCs (Passier et al., 2008; Laflamme
and Murry, 2011). One very recent example of this latter
approach was used by the Mercola group, who applied a small
molecule TGFbR2 inhibitor to promote cardiac fate in both
mouse and human ESCs (Willems et al., 2012).
Strategies to use stem cells have become more apparent and
realistic with the advent of iPSC technologies (Figure 4). Reprog-
ramming methodologies have not only opened the door for the
unlimited generation of cardiac cells but also bring about the
possibility of patient-specific therapies (Cherry and Daley, 2013;
Okano et al., 2013; Daley, 2012; Tiscornia et al., 2011). The use
Cell Stem Cell
of iPSCs has additionally provided a reli-
able platform for the understanding of
developmental and genetic components
of disease while avoiding the ethical
concerns arising from the use of ESCs.
Two different approaches for the reprog-
ramming of somatic cells have been
used in the cardiac field: (1) reprogram-
ming to pluripotency and subsequent
differentiation to cardiomyocytes and (2)
the lineage conversion of fibroblasts to
cardiomyocytes (Burridge et al., 2012).
Whereas both avenues have inherent
advantages and disadvantages, it is the
second strategy that seems to be gaining
more interestafter the reportson the invivo
lineage conversion into functional cardio-
myocytes in relevant animal models of
heart injury (Jayawardena et al., 2012;
Song et al., 2012; Qian et al., 2012).
Lineage conversion methodologies
allowing for the generation of specific cell
populations while bypassing reprogram-
ming to pluripotency (Sancho-Martinez
et al., 2012; Vierbuchen and Wernig,
2011) present the inherent benefit of elim-
inating the risk of teratoma formation and
potentially allow for in vivo reprogramming
of tissue-resident cells into another
lineage of interest. In this sense, cardiac fibroblasts are an attrac-
tive cell population, since they constitute one of the most abun-
dant cell types in theheart andareproliferationcompetent. Inprin-
ciple, lineageconversionof cardiac fibroblasts to cardiomyocytes
would allow replenishment of myocardial muscle from a starting
population of cells that can proliferate and thus self-renew.
Proof-of-principle of this idea has been accomplished by the
reprogramming field in recent years (Figure 4). Ieda et al. evalu-
ated up to 14 different transcription factors for the direct lineage
conversionofmousefibroblasts intocardiomyocytesanddemon-
strated that overexpression of a minimal set of three transcription
factors (GATA4, Mef2c, and Tbx5) sufficed for the conversion of
up to 20% mouse cardiac fibroblasts into cardiomyocyte-like
cells (Ieda et al., 2010). More recently, the Srivastava and Olson
12, March 7, 2013 ª2013 Elsevier Inc. 281
Cell Stem Cell
Review
laboratories further demonstrated the suitability of direct lineage
conversion strategies for the reprogramming of cardiac fibro-
blasts into cardiomyocytes in vivo (Qian et al., 2012; Song et al.,
2012). Qian et al. used retroviruses containing the transcription
factors of interest and injected them in the peri-infarct area. Since
retroviruses need actively dividing cells for efficient transduction,
targeting of cardiac fibroblasts is achieved efficiently and effort-
lessly. Cardiac fibroblasts converted intomature cardiomyocytes
with beating properties indicating electrical coupling. The study
by Song et al. found that a combination of GATA4, MEF2C, and
TBX5 with HAND2 significantly increased conversion efficiencies
(Song et al., 2012). Similarly, simultaneous overexpression of
miRNAs enriched in cardiac cells, namely miRs-1/133/208/499,
hasalsobeenshown to allow for the lineageconversionof cardiac
fibroblasts into functional cardiomyocyte-like cells in vivo (Jaya-
wardena et al., 2012). All of these reports showed significant
recovery after experimental myocardial infarction, indicating
that in vivo direct lineage conversion is indeed a sound strategy
with potential clinical applications for restoring heart function.
Although all these approaches offer interesting avenues
for research and clinical applications in the future, in vitro
iPSC-derived cardiomyocytes suffer from some of the same
drawbacks as their ESC-derived counterparts, namely, mixed
subpopulations of different cardiomyocytic cell types and lack
of a definitive mature phenotype (Tohyama et al., 2013). On the
other hand, in vivo direct lineage conversion of cardiac fibro-
blasts into cardiomyocytes might have unexpected outcomes
when transplanted into a therapeutic setting. For example,
what would be the consequences of depleting cardiac fibro-
blasts to replenish the cardiomyocyte pool and would that fibro-
blast pool provide sufficient numbers of cells for significant
improvement in a large mammal? This and other related ques-
tions demand further studies to determine the viability of the
approaches here described.
Conclusions and Future ProspectsRegeneration is a process that has fascinated humanity for centu-
ries, from Aristotle, who observed that lizards could regrow their
lost tails, tomore recent timeswith the studies in planarians, earth-
worms, and hydras carried out by Spallanzani andBonnet, among
others, in the 18th and 19th centuries. Nowadays, new models of
regeneration such as the zebrafish (Poss et al., 2002; Raya et al.,
2003; Brockes and Kumar, 2008; Sanchez Alvarado, 2000),
neonatal mouse (Porrello et al., 2011b), and others (Seifert et al.,
2012) are bringing refreshing perspectives into the field and
changingour viewof how regenerationoccurs inmammals (Senyo
et al., 2013). Thus, it has recently become clear that the adult
mammalian heart possesses intrinsic repair and regenerative
potential, at least to some degree. In spite of this capacity, endog-
enous regenerative responseselicitedupon injury fall short of func-
tionally repairing the injured heart, mostly due to the paucity of
newly generated cardiomyocytes compared to the large number
of cells lost upon injurious trauma. Thecurrent trendsof cardiovas-
cular researchare focusedon (1) theuseofendogenousadult stem
cells, (2) the application of reprogramming approaches for the
in vitro differentiation of PSCs, and the in vivo lineage conversion
of cardiac fibroblasts into functional cardiomyocytes, and (3) the
more recent strategies based on the experimental activation of
dormant regenerativemechanismspromotingmammaliancardiac
282 Cell Stem Cell 12, March 7, 2013 ª2013 Elsevier Inc.
repair. Table 1 briefly summarizes the application of these different
strategies to in vivomodelsof heart disease, aswell as their effects
on the recovery of heart function.
The observations that endogenous epicardial or cardiac pro-
genitor cells can lead to pleiotropic activities improving cardiac
function has set the stage for the application of compounds
and/or modulation of genetic networks aimed at not only
replenishing lost cardiomyocytes but also inducing the neovas-
cularization of ischemic areas (Smart et al., 2011; Ellison et al.,
2007). The finding that, in neonatal mouse, cardiomyocytes are
able to enter a proliferative state through dedifferentiation in
process towhat has been described in the adult zebrafish, repre-
sents an unprecedented model for the identification of novel
factors promoting adult heart regeneration. Indeed, naturally
occurring proregenerative pathways identified in the murine
neonatal model have already led to the establishment of
miRNA-based strategies for the induction of cardiomyocyte
proliferation and subsequent heart regeneration (Eulalio et al.,
2012; Porrello et al., 2011a).
Alternatively, the cardiac progenitor cell and reprogramming
approaches to repair heart damage are promising avenues for
stimulating endogenous regeneration, but they also face their
own share of problems. The existence of cardiac progenitor cells
hasbeenknown formore thanadecade; however, reports conflict
with regard to the significance of their contribution to heart repair.
More recent studies highlight the fact that their rolemight be insig-
nificant in an injury setting, and it is still unclear whether experi-
mental activation of endogenous CPC pools could be accom-
plished in an efficient manner that would ultimately contribute to
heart repair. Similarly, in vitro generation of pure and fully mature
populations of cardiomyocytes from PSCs is a daunting task.
Improvingcultureprotocols throughabetterunderstandingofcar-
diomyocyte lineage specification is necessary if we are ever going
to employ these cells in a clinical setting. Even if in vitro protocols
for producing pure cardiomyocytes are sufficiently developed,
additional challenges to direct cell transplantation remain, such
as those posed by rapid clearing and low grafting efficiencies of
the transplanted cells. To address these issues, innovative strate-
giesmustbedevelopedwith newapproaches that combine tissue
engineering with cell transplantation representing a potentially
promising way forward (Seif-Naraghi et al., 2013; Miyagawa
et al., 2011). Altogether, stem cell-based approaches offer
multiple opportunities for cell transplantation or even endogenous
reprogramming into functional cell populations; however, their
therapeutic promise still hinges on future clarification of some
safety issues (Daley, 2012; Panopoulos et al., 2011; Sancho-Mar-
tinez et al., 2012; Soldner and Jaenisch, 2012).
Overall, the recent advances in the field of cardiac regeneration
suggest that the regenerative pathways leading to cardiomyo-
cyte-mediated heart regeneration are greatly conserved across
vertebrates at the cellular and tissue level and that they can be
experimentally activated in mammals. In retrospect, we may be
closer to achieving heart regeneration than previously thought,
with regenerating animal models offering more insight into the
cellular and molecular mechanisms facilitating regeneration that
we had previously anticipated. Although this is an exciting time,
there are still several unanswered questions. If the mechanisms
of regeneration are conserved and can be forcefully induced in
adult mammals, then why are they so consistently silenced
Table 1. Heart Regeneration and Repair Strategies in Models of Myocardial Infarction
Strategy
Target
Cell Type Species Mechanism of Action
Functional
Improvement Follow-up References
Cardioprotection
Oncostatin M CM Mouse Resistance to hypoxia? EF, ESV, CO 1 month Kubin et al. (2011)
C/EBP inhibition EPC Mouse Inhibition of proinflammantory
C/EBP signaling
EF 3 months Huang et al. (2012)
BMSC transplantation BMSC Mouse Activation of endogenous CPCs EF 2 months Loffredo et al. (2011)
Cardiomyocyte dedifferentiation/proliferation
miR-15 family inhibitors CM Mouse Dedifferentiation and proliferation
of CMs
FS 21 days Porrello et al. (2011a, 2013)
miR-590/199a mimics CM Mouse Proliferation of CMs (dedifferentiation
not determined)
EF, FS, LVAWT 2 months Eulalio et al. (2012)
Epicardial-derived progenitor cell
Thymosin b4 EPDC Mouse EPDC expansion and differentiation
to CMs
EF, DSV 28 days Smart et al. (2011)
In vivo reprogramming of somatic heart cells
GATA4, Mef2c,
Tbx5, Hand2
CF Mouse Lineage conversion of CFs into CMs EF, SV 3 months Song et al. (2012)
GATA4, Mef2c, Tbx5 CF Mouse Lineage conversion of CFs into CMs EF, SV, CO 3 months Qian et al. (2012)
miR-1/133/208/499 CF Mouse Lineage conversion of CFs into CMs Not determined 1 month Jayawardena et al. (2012)
CM, cardiomyocyte; EPC, epicardial cell; EPDC, epicardial-derived progenitor cell; CF, cardiac fibroblast; BMSC, bone marrow stromal cell; CPC,
cardiac progenitor cell; PSC, pluripotent stem cell; EF, ejection fraction; SV, stroke volume; CO, cardiac output; DSV, diastolic/systolic volume; FS,
fractional shortening; LWAT, left ventricle anterior-wall thickness; ESV, end-systolic volume.
Cell Stem Cell
Review
even in traumatic situations in which their activation would be an
advantage for the organism?Whereas from a theoretical point of
view this remains one of themost interesting questions regarding
regeneration in mammals, from a practical point of view the
results described here unlock unprecedented opportunities for
the treatment of cardiovascular disease. Therefore, the identifica-
tion and locale and/or systemic administration of drugs, nucleic
acids, and/or other molecules, such as growth factors, that are
capable of activating the latent regenerative potential ‘‘hidden’’
in the mammalian heart may provide an alternative to cell trans-
plantation in addition to providing a complementary and prom-
ising approach toward the treatment of heart injury in humans.
ACKNOWLEDGMENTS
We would first like to apologize to all those authors whose outstanding workcould not be cited due to space constraints. We thank all members of theIzpisua Belmonte laboratory for critical discussion. I.S.-M. was partiallysupported by a Nomis Foundation Fellowship. Work in the laboratory ofJ.C.I.B. was supported by TERCEL-ISCIII-MINECO,CIBER, FundacionCellex,G. Harold and Leila Y. Mathers Charitable Foundation, and The Leona M. andHarry B. Helmsley Charitable Trust.
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