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: belmonte@salk.eduhttp://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.

REFERENCES

Andersen, D.C., Andersen, P., Schneider, M., Jensen, H.B., and Sheikh, S.P.(2009). Murine ‘‘cardiospheres’’ are not a source of stem cells with cardiomyo-genic potential. Stem Cells 27, 1571–1581.

Beltrami, A.P., Urbanek, K., Kajstura, J., Yan, S.M., Finato, N., Bussani, R.,Nadal-Ginard, B., Silvestri, F., Leri, A., Beltrami, C.A., and Anversa, P.(2001). Evidence that human cardiac myocytes divide after myocardial infarc-tion. N. Engl. J. Med. 344, 1750–1757.

Beltrami, A.P., Barlucchi, L., Torella, D., Baker,M., Limana, F., Chimenti, S., Ka-sahara, H., Rota, M., Musso, E., Urbanek, K., et al. (2003). Adult cardiac stemcells are multipotent and support myocardial regeneration. Cell 114, 763–776.

Bergmann, O., Bhardwaj, R.D., Bernard, S., Zdunek, S., Barnabe-Heider, F.,Walsh, S., Zupicich, J., Alkass, K., Buchholz, B.A., Druid, H., et al. (2009).Evidence for cardiomyocyte renewal in humans. Science 324, 98–102.

Bersell, K., Arab, S., Haring, B., and Kuhn, B. (2009). Neuregulin1/ErbB4signaling induces cardiomyocyte proliferation and repair of heart injury. Cell138, 257–270.

Braam, S.R., Passier, R., and Mummery, C.L. (2009). Cardiomyocytes fromhuman pluripotent stem cells in regenerative medicine and drug discovery.Trends Pharmacol. Sci. 30, 536–545.

Brockes, J.P., and Kumar, A. (2008). Comparative aspects of animal regener-ation. Annu. Rev. Cell Dev. Biol. 24, 525–549.

Burridge, P.W., Keller, G., Gold, J.D., and Wu, J.C. (2012). Production of denovo cardiomyocytes: human pluripotent stem cell differentiation and directreprogramming. Cell Stem Cell 10, 16–28.

Cherry, A.B.C., and Daley, G.Q. (2013). Reprogrammed cells for diseasemodeling and regenerative medicine. Annu. Rev. Med. 64, 277–290.

Daley, G.Q. (2012). The promise and perils of stem cell therapeutics. Cell StemCell 10, 740–749.

Ellison, G.M., Torella, D., Karakikes, I., and Nadal-Ginard, B. (2007). Myocytedeath and renewal: modern concepts of cardiac cellular homeostasis. Nat.Clin. Pract. Cardiovasc. Med. 4(Suppl 1 ), S52–S59.

Eulalio, A., Mano, M., Dal Ferro, M., Zentilin, L., Sinagra, G., Zacchigna, S., andGiacca, M. (2012). Functional screening identifies miRNAs inducing cardiacregeneration. Nature 492, 376–381.

Guan, K., and Hasenfuss, G. (2007). Do stem cells in the heart truly differentiateinto cardiomyocytes? J. Mol. Cell. Cardiol. 43, 377–387.

Huang, G.N., Thatcher, J.E., McAnally, J., Kong, Y., Qi, X., Tan, W., DiMaio,J.M., Amatruda, J.F., Gerard, R.D., Hill, J.A., et al. (2012). C/EBP transcriptionfactors mediate epicardial activation during heart development and injury.Science 338, 1599–1603.

Ieda, M., Fu, J.-D., Delgado-Olguin, P., Vedantham, V., Hayashi, Y., Bruneau,B.G., and Srivastava, D. (2010). Direct reprogramming of fibroblasts into func-tional cardiomyocytes by defined factors. Cell 142, 375–386.

Cell Stem Cell 12, March 7, 2013 ª2013 Elsevier Inc. 283

Cell Stem Cell

Review

Itou, J., Oishi, I., Kawakami, H., Glass, T.J., Richter, J., Johnson, A., Lund, T.C.,and Kawakami, Y. (2012). Migration of cardiomyocytes is essential for heartregeneration in zebrafish. Development 139, 4133–4142.

Jayawardena, T.M., Egemnazarov, B., Finch, E.A., Zhang, L., Payne, J.A.,Pandya, K., Zhang, Z., Rosenberg, P., Mirotsou, M., and Dzau, V.J. (2012).MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiacfibroblasts to cardiomyocytes. Circ. Res. 110, 1465–1473.

Jopling, C., Sleep, E., Raya, M., Martı, M., Raya, A., and Izpisua Belmonte, J.C.(2010). Zebrafish heart regeneration occurs by cardiomyocyte dedifferentia-tion and proliferation. Nature 464, 606–609.

Kelly, B.B., Narula, J., and Fuster, V. (2012). Recognizing global burden ofcardiovascular disease and related chronic diseases. Mt. Sinai J. Med. 79,632–640.

Kikuchi, K., Holdway, J.E.,Werdich, A.A., Anderson, R.M., Fang, Y., Egnaczyk,G.F., Evans, T., Macrae, C.A., Stainier, D.Y., and Poss, K.D. (2010). Primarycontribution to zebrafish heart regeneration by gata4(+) cardiomyocytes.Nature 464, 601–605.

Kikuchi, K., Gupta, V., Wang, J., Holdway, J.E., Wills, A.A., Fang, Y., and Poss,K.D. (2011). tcf21+ epicardial cells adopt non-myocardial fates during zebra-fish heart development and regeneration. Development 138, 2895–2902.

Kubin, T., Poling, J., Kostin, S., Gajawada, P., Hein, S., Rees, W., Wietelmann,A., Tanaka, M., Lorchner, H., Schimanski, S., et al. (2011). Oncostatin M isa major mediator of cardiomyocyte dedifferentiation and remodeling. CellStem Cell 9, 420–432.

Laflamme, M.A., and Murry, C.E. (2011). Heart regeneration. Nature 473,326–335.

Laugwitz, K.-L., Moretti, A., Lam, J., Gruber, P., Chen, Y., Woodard, S., Lin,L.-Z., Cai, C.-L., Lu, M.M., Reth, M., et al. (2005). Postnatal isl1+ cardioblastsenter fully differentiated cardiomyocyte lineages. Nature 433, 647–653.

Lepilina, A., Coon, A.N., Kikuchi, K., Holdway, J.E., Roberts, R.W., Burns,C.G., and Poss, K.D. (2006). A dynamic epicardial injury response supportsprogenitor cell activity during zebrafish heart regeneration. Cell 127, 607–619.

Letellier, E., Kumar, S., Sancho-Martinez, I., Krauth, S., Funke-Kaiser, A.,Laudenklos, S., Konecki, K., Klussmann, S., Corsini, N.S., Kleber, S., et al.(2010). CD95-ligand on peripheral myeloid cells activates Syk kinase to triggertheir recruitment to the inflammatory site. Immunity 32, 240–252.

Loffredo, F.S., Steinhauser, M.L., Gannon, J., and Lee, R.T. (2011). Bonemarrow-derived cell therapy stimulates endogenous cardiomyocyte progeni-tors and promotes cardiac repair. Cell Stem Cell 8, 389–398.

Marsano, A., Maidhof, R., Luo, J., Fujikara, K., Konofagou, E.E., Banfi, A., andVunjak-Novakovic, G. (2013). The effect of controlled expression of VEGF bytransduced myoblasts in a cardiac patch on vascularization in a mouse modelof myocardial infarction. Biomaterials 34, 393–401.

Miyagawa, S., Roth, M., Saito, A., Sawa, Y., and Kostin, S. (2011). Tissue-engineered cardiac constructs for cardiac repair. The Annals of ThoracicSurgery 91, 320–329.

Moretti, A., Caron, L., Nakano, A., Lam, J.T., Bernshausen, A., Chen, Y.,Qyang, Y., Bu, L., Sasaki, M., Martin-Puig, S., et al. (2006). Multipotent embry-onic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial celldiversification. Cell 127, 1151–1165.

Okano, H., Nakamura, M., Yoshida, K., Okada, Y., Tsuji, O., Nori, S., Ikeda, E.,Yamanaka, S., and Miura, K. (2013). Steps toward safe cell therapy usinginduced pluripotent stem cells. Circ. Res. 112, 523–533.

Panopoulos, A.D., Ruiz, S., and Izpisua Belmonte, J.C. (2011). iPSCs: inducedback to controversy. Cell Stem Cell 8, 347–348.

Passier, R., and Mummery, C. (2010). Getting to the heart of the matter: directreprogramming to cardiomyocytes. Cell Stem Cell 7, 139–141.

Passier, R., van Laake, L.W., and Mummery, C.L. (2008). Stem-cell-basedtherapy and lessons from the heart. Nature 453, 322–329.

Porrello, E.R., Johnson, B.A., Aurora, A.B., Simpson, E., Nam, Y.J., Matkovich,S.J., Dorn, G.W., 2nd, van Rooij, E., and Olson, E.N. (2011a). MiR-15 familyregulates postnatal mitotic arrest of cardiomyocytes. Circ. Res. 109, 670–679.

284 Cell Stem Cell 12, March 7, 2013 ª2013 Elsevier Inc.

Porrello, E.R., Mahmoud, A.I., Simpson, E., Hill, J.A., Richardson, J.A., Olson,E.N., and Sadek, H.A. (2011b). Transient regenerative potential of the neonatalmouse heart. Science 331, 1078–1080.

Porrello, E.R., Mahmoud, A.I., Simpson, E., Johnson, B.A., Grinsfelder, D.,Canseco, D., Mammen, P.P., Rothermel, B.A., Olson, E.N., and Sadek, H.A.(2013). Regulation of neonatal and adult mammalian heart regeneration bythe miR-15 family. Proc. Natl. Acad. Sci. USA 110, 187–192.

Poss, K.D., Wilson, L.G., and Keating, M.T. (2002). Heart regeneration inzebrafish. Science 298, 2188–2190.

Qian, L., Huang, Y., Spencer, C.I., Foley, A., Vedantham, V., Liu, L., Conway,S.J., Fu, J.D., and Srivastava, D. (2012). In vivo reprogramming of murinecardiac fibroblasts into induced cardiomyocytes. Nature 485, 593–598.

Ranganath, S.H., Levy, O., Inamdar, M.S., and Karp, J.M. (2012). Harnessingthe mesenchymal stem cell secretome for the treatment of cardiovasculardisease. Cell Stem Cell 10, 244–258.

Raya, A., Koth, C.M., Buscher, D., Kawakami, Y., Itoh, T., Raya, R.M., Sternik,G., Tsai, H.-J., Rodrıguez-Esteban, C., and Izpisua-Belmonte, J.C. (2003).Activation of Notch signaling pathway precedes heart regeneration in zebra-fish. Proc. Natl. Acad. Sci. USA 100(Suppl 1 ), 11889–11895.

Sanchez Alvarado, A. (2000). Regeneration in the metazoans: why does ithappen? Bioessays 22, 578–590.

Sancho-Martinez, I., Baek, S.H., and Izpisua Belmonte, J.C. (2012). Lineageconversion methodologies meet the reprogramming toolbox. Nat. Cell Biol.14, 892–899.

Seifert, A.W., Kiama, S.G., Seifert, M.G., Goheen, J.R., Palmer, T.M., andMaden, M. (2012). Skin shedding and tissue regeneration in African spinymice (Acomys). Nature 489, 561–565.

Seif-Naraghi, S.B., Singelyn, J.M., Salvatore, M.A., Osborn, K.G., Wang, J.J.,Sampat, U., Kwan, O.L., Strachan, G.M.,Wong, J., Schup-Magoffin, P.J., et al.(2013). Safety and Efficacy of an Injectable Extracellular Matrix Hydrogel forTreating Myocardial Infarction. Science Translational Medicine 5, 173ra25–173ra25.

Senyo, S.E., Steinhauser, M.L., Pizzimenti, C.L., Yang, V.K., Cai, L., Wang, M.,Wu, T.-D., Guerquin-Kern, J.-L., Lechene, C.P., and Lee, R.T. (2013). Mam-malian heart renewal by pre-existing cardiomyocytes. Nature 493, 433–436.

Smart, N., Bollini, S., Dube, K.N., Vieira, J.M., Zhou, B., Davidson, S., Yellon,D., Riegler, J., Price, A.N., Lythgoe, M.F., et al. (2011). De novo cardiomyo-cytes from within the activated adult heart after injury. Nature 474, 640–644.

Soldner, F., and Jaenisch, R. (2012). Medicine. iPSC disease modeling.Science 338, 1155–1156.

Song, K., Nam, Y.-J., Luo, X., Qi, X., Tan, W., Huang, G.N., Acharya, A., Smith,C.L., Tallquist, M.D., Neilson, E.G., et al. (2012). Heart repair by reprogram-ming non-myocytes with cardiac transcription factors. Nature 485, 599–604.

Tiscornia, G., Vivas, E.L., and Izpisua Belmonte, J.C. (2011). Diseases in a dish:modeling human genetic disorders using induced pluripotent cells. Nat. Med.17, 1570–1576.

Tohyama, S., Hattori, F., Sano, M., Hishiki, T., Nagahata, Y., Matsuura, T.,Hashimoto, H., Suzuki, T., Yamashita, H., Satoh, Y., et al. (2013). Distinctmetabolic flow enables large-scale purification of mouse and human pluripo-tent stem cell-derived cardiomyocytes. Cell Stem Cell 12, 127–137.

van Rooij, E., Sutherland, L.B., Thatcher, J.E., DiMaio, J.M., Naseem, R.H.,Marshall, W.S., Hill, J.A., and Olson, E.N. (2008). Dysregulation of microRNAsafter myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc.Natl. Acad. Sci. USA 105, 13027–13032.

Vierbuchen, T., and Wernig, M. (2011). Direct lineage conversions: unnaturalbut useful? Nat. Biotechnol. 29, 892–907.

Willems, E.,Cabral-Teixeira, J., Schade,D.,Cai,W., Reeves, P., Bushway, P.J.,Lanier, M., Walsh, C., Kirchhausen, T., Izpisua Belmonte, J.C., et al. (2012).Small molecule-mediated TGF-b type II receptor degradation promotes cardi-omyogenesis in embryonic stem cells. Cell Stem Cell 11, 242–252.

Zhou, B., Ma, Q., Rajagopal, S., Wu, S.M., Domian, I., Rivera-Feliciano, J.,Jiang, D., von Gise, A., Ikeda, S., Chien, K.R., and Pu, W.T. (2008). Epicardialprogenitors contribute to the cardiomyocyte lineage in the developing heart.Nature 454, 109–113.