Review

Regeneration of the heart

Review Series
Cardiovascular Diseases

Regeneration of the heart

Matthew L. Steinhauser1*, Richard T. Lee1,2**

Keywords: cardiovascular disease; cardiac myocyte; heart failure; regeneration; stem cell

DOI 10.1002/emmm.201100175

Received June 6, 2011 / Revised July 3, 2011 / Accepted August 1, 2011

The death of cardiac myocytes diminishes the heart’s pump function and is a major
cause of heart failure, one of the dominant causes of death worldwide. Other than
transplantation, there are no therapies that directly address the loss of cardiac
Introduction myocytes, which explains the current excitement in cardiac regeneration. The field is
evolving in two important directions. First, although endogenous mammalian
Heart failure is often the consequence
of cardiac myocyte loss after insult,
cardiac regeneration clearly seems to decline rapidly after birth, it may still persist
such as myocardial infarction. Patients in adulthood. The careful elucidation of the cellular and molecular mechanisms of
hospitalized with an index episode of endogenous heart regeneration may therefore provide an opportunity for develop-
heart failure have a prognosis as low as ing therapeutic interventions that amplify this process. Second, recent break-
patients with common malignancies throughs have enabled reprogramming of cells that were apparently terminally
(Stewart et al, 2001). With the excep- differentiated, either by dedifferentiation into pluripotent stem cells or by trans-
tion of heart transplantation and
differentiation into cardiac myocytes. These achievements challenge our con-
implantation of mechanical ventricular
ceptions of what is possible in terms of heart regeneration. In this review, we
assist devices, current therapies do
not address the central problem of discuss the current status of research on cardiac regeneration, with a focus on the
decreased pumping capacity owing to challenges that hold back therapeutic development.
a depleted pool of cardiac myocytes.
Thus, there has been enormous interest in the prospect of including endothelial cells, smooth muscle cells and cardiac
cardiac regeneration rather than slowing the inexorable decline myocytes.
of heart function. If progenitor cells residing in the adult are capable of
Until recently, the prospect of cardiac regeneration was producing new heart cells, the therapeutic delivery of such
largely a subject of science fiction. The longstanding paradigm progenitors might facilitate the generation of de novo functional
held that the mammalian heart is a terminally differentiated myocardium. In this context, cell-based therapies for the heart
organ, incapable of replenishing any myocyte attrition. During have been rapidly translated into the clinic to treat heart disease,
the past decade, however, studies revealed not only that but randomized clinical trials with bone marrow progenitors
mammalian cardiac myocytes retain some capacity for division have shown at best modest improvements in ventricular
(Beltrami et al, 2001), but also identified endogenous cardiac function (Martin-Rendon et al, 2008). In short, the promise of
progenitor cells in the heart (Beltrami et al, 2003) or bone complete cardiac regeneration has not yet been realized.
marrow (Orlic et al, 2001). These cells retain some potential for In light of mixed results from clinical trials, it is worth
differentiation into the cellular components of the heart, revisiting both the foundations of cardiac regeneration and
highlight recent advances that may portend future directions in
the field. We will first define the problem, that is elucidating
(1) Division of Cardiovascular Medicine, Department of Medicine, Brigham the scope of endogenous mammalian regeneration and, by
and Women’s Hospital, Harvard Medical School, Partners Research extension, the scale of the regenerative deficit. We will then
Building, Cambridge, MA, USA
summarize current regenerative approaches, including both
(2) Harvard Stem Cell Institute, Cambridge, MA, USA
cell-based therapies and pharmacoregenerative strategies. In
*Corresponding author: Tel: þ1-617-768-8702; Fax: þ1-617-768-8280;
E-mail: msteinhauser@partners.org this context, we will summarize the many challenges that stand
**Corresponding author: Tel: þ1-617-768-8282; Fax: þ1-617-768-8280; in the way of cardiac regeneration, both endogenous repair
E-mail: rlee@partners.org processes and exogenous regenerative therapies.

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Regeneration of the heart

Heart regeneration in lower organisms after birth; apical excision results in myocyte proliferation and
structural restoration similar to regeneration in zebrafish
The regenerative deficit of the mammalian heart is obvious and newts (Porrello et al, 2011). Afterwards, however, injured
when compared with organisms such as zebrafish and newts, myocardium is largely replaced by fibrosis and scarring.
which demonstrate a remarkable survival capacity after Distinguishing whether the adult mammalian heart is incapable
removal of up to 20% of the heart by transection of the of cardiac myocyte replacement or whether it retains a low-level
ventricular apex (Oberpriller & Oberpriller, 1974; Poss et al, capacity for repair is therefore fundamentally important because
2002). Pre-existing cardiac myocytes adjacent to the site appear even low-level myocyte turnover raises the specter of amplifying
to undergo a process of dedifferentiation, characterized by that response for therapeutic ends. Thus, it is worth reviewing
dissolution of sarcomeric structures. This is followed by both the evidence supporting the evolving view of a more plastic
incorporation of deoxyribonucleic acid (DNA) synthesis mammalian heart, while closely considering potential artefacts
markers (e.g. nucleotide analogues) consistent with prolifera- that may affect the interpretation of these results.
tion (Jopling et al, 2010; Kikuchi et al, 2010; Laube et al, 2006). The first arguments against the paradigm of the terminally
Ultimately, newly generated cardiac myocytes are functionally differentiated quiescent cardiac myocyte date back decades; the
integrated with the preexisting myocardium. The heart is left increasing force of these arguments reflects the emergence
with little residual evidence of the injury, thus providing a of new data made possible by methodological innovations. Early
natural example of complete myocardial regeneration. evidence supporting cardiac myocyte plasticity relied on
mathematical modelling of the myocyte population based
on cytometric indices. For example, the measured average
Evidence for heart regeneration in mammals volume increase of cardiac myocytes was calculated to fall
short of the increase predicted by the observed volumetric
During embryonic development and the early post-natal period, changes in the whole heart (Astorri et al, 1971). These data
mice also demonstrate a remarkable regenerative capacity. and similar analyses based on DNA content asserted that changes
Embryos heterozygous for a cardiac myocyte-specific null in heart volume could not be explained by hypertrophy alone,
mutation in the x-linked holocytochrome c synthase (Hccs) and that cardiac myocyte hyperplasia (increase in cell number)
gene demonstrate cardiac myocyte replacement during foetal contributed to changes in heart mass. However, these studies
development (Drenckhahn et al, 2008): when one of two were not widely accepted because their conclusions relied on a
X-chromosomes is randomly inactivated in each female somatic number of assumptions about myocyte size and DNA content.
cell, approximately 50% of the cardiac myocytes are rendered Modern microscopic analyses to detect cell cycle markers
Hccs-null and hence dysfunctional. Proliferative functional such as Ki67 or the incorporation of nucleotide analogues (e.g.
Hccs-expressing cardiac myocytes compensate for dysfunctional iododeoxyuridine or 3H-thymidine) into newly synthesized
Hccs-null myocytes, such that, at birth, 90% of the heart is DNA further support the notion that the mammalian heart may
derived from myocytes containing one functional Hccs allele. generate new myocytes both during normal homeostasis and
Similar plasticity in post-natal mice is confined to the first week after injury. Multiple studies have demonstrated that murine or

Glossary Polyploid
Cells or organisms that contain one or more extra sets of chromo-
Cardiac myocytes somes.
Heart muscle cells.
Progenitor—ancestor
Fibrosis A cell that can differentiate into a specific cell type.
Development of excessive fibrous connective tissue in an organ.
Sarcomeric
Gastrulation Pertaining to the sarcomere. Sarcomere is one of the segments into which a
Early phase of embryonic development during which the germ layers form. striated muscle is divided.
Heart failure
Condition in which the heart is not able to pump enough blood into the rest Stromal cells
of the body. Also called congestive heart failure. Cells of the connective tissue of any organ.

Ki67 Teratomas
Marker protein for cell proliferation. Congenital, encapsulated tumours, which contain one or more germ
layers.
Mesodermal
Pertaining to the mesoderm. Mesoderm is one of the three germ layers, which Transcription factors
forms skeletal muscle, the skeleton, the dermis of skin, connective tissue, the Proteins, which are involved in regulation of gene expression after binding
urogenital system, the heart, blood (lymph cells), the kidney and the spleen. to a specific DNA sequence.
Nucleotide analogues
Nucleotides, which contain chemical modifications and are incorporated Transdifferentiation
The conversion of one differentiated cell to another cell type.
into the DNA during synthesis and cell proliferation. They can thus be
easily detected.

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 Review
Matthew L. Steinhauser and Richard T. Lee

human cardiac myocytes can reenter the cell cycle, but the a timeframe that is practical to directly measure the generation
described rates of this phenomenon differ by more than one of cardiac myocytes, the number of newborn cells (numerator)
order of magnitude (Beltrami et al, 2001; Kajstura et al, 2010a,b; is much smaller than the total number of cells (denominator). As
Soonpaa & Field, 1997). a result, small errors may magnify projections of absolute yearly
Recent dating experiments, made possible by nuclear arms or lifetime myocyte turnover. Another source of error is the
testing in the middle of the 20th century, provide the most occasional mis-identification of cellular components, which is an
convincing evidence for post-natal human cardiac myocyte inevitable consequence of analyzing a three-dimensional organ
turnover. Since the Partial Test Ban Treaty of 1963, the marked with two-dimensional tissue sections, particularly when relying
spike in atmospheric 14C levels has dissipated as the biosphere on light microscopy which has limited fidelity of resolving
absorbed the carbon. Thus, the period of nuclear testing serves cellular borders in complex tissues like the heart (Laflamme &
as a historical DNA labelling pulse, and the period after the test Murry, 2005). The problem is further exacerbated by the
ban treaty serves as a chase. The genomic DNA of cells autofluorescence of myocardium, which complicates any method
generated during either the pulse or the chase reflect the earth’s that relies on the detection of a fluorescent signal, including the
atmospheric 14C concentration at that point in time, which detection of nucleotide analogues, Ki67 or fluorescent labels as
allows investigators to date the age of cardiac myocytes by used in genetic lineage-mapping experiments.
measuring the concentration of 14C in their nuclei (Bergmann et Such potential confounders could also affect the 14C dating
al, 2009). It showed that the adult heart contains some cardiac method, because it requires the isolation of cardiac myocyte
myocytes generated during the human life-span; their model nuclei by digestion and flow cytometric sorting. Any impurities
predicted an approximate turnover rate of 1% per year at age 25, in the isolated nuclear preparation could introduce bias to the
declining to a rate of 0.45% by the age of 75. analysis; preferential isolation of senescent myocytes could
result in an underestimate of myocyte turnover, whereas an
impure nuclear isolate that includes non-myocyte nuclei might
Confounding factors in the quantitation of cardiac result in an overestimate.
myocyte turnover The heart also presents a unique challenge compared to other
organs owing to the propensity of cardiac myocytes to
The data generated by these diverse methodologies support the synthesize DNA during S-phase without completing either
notion that the mammalian heart has conserved some capacity mitosis and/or cytokinesis (Fig 1). During early post-natal
for cardiac myocyte turnover though there is lingering con- development, for example, the majority of rodent cardiac
troversy about the scope of the mammalian heart’s endogenous myocytes (Li et al, 1996) and an estimated 25–57% of human
regenerative capacity. Studies relying on the identification mitotic cardiac myocytes (Olivetti et al, 1996; Schmid & Pfitzer, 1985)
cardiac myocytes (Kajstura et al, 1998), immuno-detection of cell become binucleated. By adulthood, most cardiac myocyte nuclei
cycle markers such as Ki67 (Beltrami et al, 2001) or of IdU have also become polyploid with at least one (4n: tetraploid)
incorporation into newly synthesized DNA (Kajstura et al, 2010b) or two (8n: octoploid) additional rounds of chromosomal
suggest that nearly 30% of the heart can be replaced within 1 year replication (Bergmann et al, 2010). Although some studies
during normal homeostasis, a rate that increases 50-fold after demonstrated that the polyploidy rate in the cardiac myocyte pool
injury. These remarkably high rates imply that the entire heart is is not affected by ageing or injury (Olivetti et al, 1996), others
replaced approximately every 3 years during normal home- suggested that the ploidy state is more dynamic. The ploidy state
ostasis, and that all cardiac myocytes lost to an infarction could be of cardiac myocytes may increase with myocardial hypertrophy
replaced within 3 weeks. In contrast, data from nuclear fallout or injury (Adler & Friedburg, 1986), which could be mistaken for
studies (Bergmann et al, 2009) and quantitative studies of DNA myocyte division. Conversely, hearts that have been unloaded by
synthesis using autoradiographic measurement of 3H-thymidine implantation of a ventricular assist device may have a lower
incorporation (Soonpaa & Field, 1997) indicate turnover rates of percentage of polyploid myocytes, because more 2n cardiac
1% or less per year. myocytes are being generated (Wohlschlaeger et al, 2010). These
The actual scale of the endogenous regenerative response aspects of cardiac myocyte biology inevitably represent potential
has important implications for rational therapeutic design. If the confounders that must be considered in any quantification of
turnover rate is relatively low, therapeutic interventions should cardiac myocyte formation. As with any controversial hypothesis,
focus on replacing lost or dysfunctional myocytes. If the achieving consensus regarding adult mammalian cardiac
mammalian heart indeed has a robust intrinsic capacity for myocyte turnover will likely require multiple lines of evidence
myocyte repletion, the therapeutic focus should shift to promoting using multiple different methodologies.
the assembly of endogenously generated myocytes into functional
myocardium. Thus, in the context of discrepant evidence about
the true scale of endogenous mammalian regeneration, it is Defining the cellular source of new cardiac
worth reviewing potential sources of bias inherent to all existent myocytes
methodologies that are used to study cell fate.
The difficulty of measuring cardiac myocyte turnover in the The majority of reports suggest some endogenous capacity for
heart is largely a result of the slow rate of myocyte turnover cardiac myocyte renewal, which has generated a broad focus on
relative to adjacent stromal cells (Bergmann et al, 2009). Within finding the cellular source of newly generated cardiac myocytes.

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Regeneration of the heart

a proliferative phase before redifferentiating into mature cardiac

Cardiac myocytes.
myocyte
Newly generated adult mammalian cardiac myocytes may
arise from an endogenous pool of progenitor cells after injury.
DNA synthesis Our laboratory developed a genetic lineage mapping approach
to quantify progenitor-dependent cardiac myocyte turnover
(Fig 2) (Hsieh et al, 2007). In the bitransgenic MerCreMer/ZEG
Mitosis Mitosis Mitosis inducible cardiac myocyte reporter mouse, mature cardiac
Cytokinesis Cytokinesis Cytokinesis myocytes undergo an irreversible genetic switch from
constitutive b-galactosidase expression to green fluorescent
Binucleation Division Polyploidy
protein (GFP) expression upon tamoxifen pulse. During a
chase period, we evaluated the effect of myocardial injury on
the proportion of GFPþ or b-galþ cardiac myocytes. Pressure
overload or myocardial infarction resulted in a significant
reduction in the percentage of GFPþ cardiac myocytes and a
corresponding increase in the percentage of B-galþ cardiac
myocytes, consistent with repletion of the myocyte pool
by B-gal
expressing progenitors. Though this approach
theoretically captures the sum progenitor contribution to
myocyte turnover, it cannot directly identify the molecular
identity or anatomic location of the progenitor pool.
25– 27% 66% > 4n
binucleated or more One approach to characterizing the molecular phenotype of
cardiac progenitors is to study cardiac embryologic develop-
ment, guided by the assumption that developmental paradigms
Figure 1. The majority of post-natal human DNA synthesis in the heart
are recapitulated during post-natal repair. When examined
does not lead to new myocyte formation. Cardiac myocytes can complete
S-phase, followed by mitosis and cytokinesis (centre) resulting in myocyte
through a developmental lens, an increasingly detailed picture
doubling. Cardiac myocytes can also complete mitosis without cytokinesis emerges of the soluble and transcriptional signals that guide the
(left), resulting in a binucleated cell. Cardiac myocytes can also undergo cardiogenic programme from gastrulation (formation of distinct
chromosomal replication without completing either mitosis or cytokinesis germ layers) through the ultimate maturation of cardiac
(right), resulting in polyploidy nuclei. By the completion of post-natal myocytes (Mercola et al, 2011; Yi et al, 2010). The induction
development, the majority of human myocyte nuclei contain 4n
of mesoderm posterior (MESP)-1 expression by brachyury-
chromosomal copies.
expressing primitive mesodermal cells is a proximal require-
ment for the ultimate production of differentiated heart cells
(Bondue et al, 2008; David et al, 2008; Lindsley et al, 2008). As
Three main pathways for new cardiac myocyte generation have the developing embryo grows beyond the germ layer phase, its
been proposed: division of preexisting mature cardiac myocytes developing heart receives cells from distinct anatomic progeni-
(Kajstura et al, 1998), amplification of dedifferentiated cardiac tor sources: the 1st and 2nd heart fields provide the majority of
myocytes (Jopling et al, 2010; Kikuchi et al, 2010) and the myocardium, with some contribution from epicardial
differentiation of progenitor cells (Hsieh et al, 2007). Evidence progenitors. Certain fields may be preferentially marked by
exists for all three processes in different species and under specific transcription factors; for example, the first heart field by
different conditions. It may be possible that more than one of T-box transcription factor 5 (Tbx5) (Takeuchi et al, 2003), the
these mechanisms can operate within a given species at different second heart field by Lim-homeodomain protein Islet1 (Isl1)
developmental or pathological stages. (Cai et al, 2003) and epicardial progenitors by Wilms tumour-1
Although there are many reports of mature cardiac myocyte (WT1) or T-box transcription factor 18 (Tbx18) (Cai et al, 2008;
division in adult mammals, the majority of those reports suggest Zhou et al, 2008). Other cardiogenic factors identified by
that it is a rare event. This is not surprising, given the densely embryonic lineage tracing or analysis of gene silencing include
packed and highly organized sarcomeric contractile apparatus. homeobox protein nkx2.5 (Wu et al, 2006), the myocyte
In fact, cardiac myocyte replication after injury in the neonatal enhancer factor 2C (Mef2c) (Lin et al, 1997) and GATA4
mouse, newts and zebrafish is preceded by molecular and/or (Molkentin et al, 1997).
cytoskeletal evidence of dedifferentiation (Jopling et al, 2010; In contrast to the relatively well-defined pre-natal develop-
Kikuchi et al, 2010; Oberpriller & Oberpriller, 1974; Porrello ment, there is no consensus yet about the molecular identity of
et al, 2011). Cardiac myocytes near the site of injury can post-natal mammalian cardiac progenitor cells or ‘adult cardiac
reexpress gene markers of immaturity, like the zinc-finger stem cells’. A number of laboratories have identified cell
transcription factor GATA binding protein-4 (GATA4), together populations within the post-natal mouse, which fulfil some
with disassembly of sarcomeric structures that is evident criteria of cardiac progenitors. The common approach of such
by electron microscopy. This population of dedifferentiated studies has been to enzymatically digest myocardial tissue and
cardiac myocytes expresses markers of DNA synthesis during to isolate specific cell populations based on transcriptional

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 Review
Matthew L. Steinhauser and Richard T. Lee

Progenitor lineage mapping Differentiated cell lineage mapping

Candidates for inducible genetic labeling: MerCreMer-ZEG mouse:

c-kit, sca-1, nkx2.5, mef2c, other? α-MHC drives labeling
Cells are unlabeled at baseline Constitutive β-galactosidase in all cells

Progenitor Cardiac Progenitor Cardiac

niche myocytes niche myocytes

Labeling
pulse
OH-tamoxifen
pulse
~80% GFP +
Progenitors are Progenitors remain
~20% β-gal+
labeled GFP –

Myocardial Myocardial
infarction infarction

Chase Chase
Myocyte Myocyte
differentiation differentiation
~65% GFP +
~35% β-gal+

Myocyte repletion by Myocyte repletion by

positively-labeled progenitors GFP –/β-gal+ progenitors

Figure 2. Lineage-mapping in the adult heart. Left: Theoretical progenitor lineage-mapping is depicted. Progenitors would be selectively marked by fluorescent
protein expression. After injury, the appearance of fluorescently labelled cardiac myocytes would support the concept that these progenitors were contributing to
new myocyte formation. Right: Differentiated cell (cardiac myocyte) lineage-mapping. Upon treatment of the MerCreMer-ZEG mouse with OH-tamoxifen,
approximately 80% of the cardiac myocytes undergo a permanent switch from b-galactosidase to GFP expression. The dilution of the GFPþ cardiac myocyte pool
after injury is consistent with repletion by b-galþ progenitors.

expression of a developmentally important gene (isl-1(Laugwitz also expressed by non-cardiovascular cells/progenitors or

et al, 2005)), specific cell surface receptor profile (c-kit (Beltrami because they may be part of the stress-induced reactivation
et al, 2003) or sca-1 (Oh et al, 2003)), the physiologic capacity to of ‘foetal gene programmes’, a phenomenon that is well
actively exclude Hoechst dye (so-called side population cells documented in adult cardiac myocytes (Bisping et al, 2006;
(Martin et al, 2004)) or based on the outgrowth of typical Kolodziejczyk et al, 1999; Saadane et al, 1999).
spherical colonies in tissue culture (Messina et al, 2004; Smith et
al, 2007) (Fig 3). In general, the label of ‘cardiac stem cell’
results from the observation of self-propagation and cardiac Moving towards a regenerative therapy
myocyte transdifferentiation when exposed to cardiogenic
conditions in vitro or when delivered in vivo after injury. The plasticity of the mammalian heart and the identification of
It seems unlikely that an organ with such limited regenerative cell preparations that demonstrate potency for cardiac differ-
capacity would harbour so many biologically distinct progeni- entiation has sparked efforts to develop regenerative therapeutic
tors (Laflamme & Murry, 2011; Mercola et al, 2011). Ultimately, strategies. The therapeutic challenge is considerable: a typical
the field will benefit from careful in vivo lineage tracing large myocardial infarction that leads to heart failure will kill
studies—without ex vivo culture steps—to study if and how a around 1 billion cardiac myocytes (Laflamme & Murry, 2005),
given cell type contributes to cardiac myocyte replenishment roughly a quarter of the heart’s myocytes (Fig 4). A possible
during either normal homeostasis or after injury (Fig 2). The therapeutic approach would coax an endogenous stem cell
lack of such publications to date owes in part to the lack of population or an exogenously delivered cell-based therapy to
specificity of many stem cell markers, either because they are replace lost cardiac myocytes in a coordinated fashion with

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Regeneration of the heart

Multipotent Committed
cardiovascular cardiac myocyte
progenitor progenitor

Mesodermal
Mature
precursor
cardiomyocyte

MESP1+
Developmental markers of
cardiomyocyte progenitors Troponin T +
brachyury+
Sarcomeric actin+
CSC prep Sca-1 SP c-kit Isl-1 GATA4 nkx2.5 Mef2C
Mature sarcomeres
Isl-1+ – – – + + +
c-kit+ + + + +
Sca1+ + Low – + – +
SP cells + + + + +
Cardiosphere Low Low +

Figure 3. Possible recapitulation of developmental paradigms by endogenous post-natal cardiac stem cells. Between mesodermal development and the
emergence of cardiac myocytes, cardiovascular progenitors express a number of markers that have also been detected in the various post-natal cardiac stem cell
(CSC) preparations. Expression as measured by messenger RNA (mRNA) or protein expression is denoted with (þ). Absent expression is denoted by (
).
Blank ¼ untested.

long-term functional integration. Amongst the myriad of other contemporaneous studies suggested that the source of
potential cell-based therapies, no clear winning strategy has circulating progenitors could be the bone marrow. In both cases,
so far emerged (Segers & Lee, 2008). a bone marrow cell population with a higher density of the cell
surface receptor c-kit, showed repopulation of murine cardiac
myocytes after experimental myocardial infarction (Jackson et al,
Bone marrow derived progenitors 2001; Orlic et al, 2001). Since these initial reports, a number of
studies failed to demonstrate similar rates of chimerism in
A series of nearly simultaneous landmark studies sparked transplanted hearts (Deb et al, 2003; Hocht-Zeisberg et al, 2004;
excitement about a possible adult cardiogenic progenitor Laflamme et al, 2002; Muller et al, 2002) or potency of bone
originating outside of the heart. A post-mortem examination marrow stem cells (Balsam et al, 2004; Murry et al, 2004) leading
of male heart transplant patients who had received female donor some to postulate that cardiac myocyte transdifferentiation was
hearts found that approximately 10% of a-sarcomeric actin- overestimated owing to an artefact (Laflamme & Murry, 2005).
positive cardiac myocytes had Y-chromosomes. These cells Nonetheless, some therapeutic effect was observed even in
obviously came from the male patient and from a circulating studies with no detectable transdifferentiation (Balsam et al,
progenitor outside of the heart graft (Quaini et al, 2002). Two 2004). Cell-based therapy using autologous bone marrow

O=
O
O=O

Exogenous Ischemia
cell-based O O=O
O=
O= O
therapy

Endogenous
stem cell
niche
Myocyte deficit
Inflammation ~ 1 billion cells
Homing Proliferation Differentiation

Paracrine
Fibrosis
D

factors vi
i

sio
n

Figure 4. The challenge of regenerating the heart. Both exogenously delivered cell therapies and progenitors in the endogenous niche encounter a similar
hostile environment after myocardial injury, often including inadequate blood supply (ischemia), inflammation and fibrosis/scarring. Regenerative pathways may
be activated by as yet unknown paracrine pathways, responsible for recruiting progenitors from the niche, stimulating proliferation and coaxing differentiation.

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Matthew L. Steinhauser and Richard T. Lee

progenitors was rapidly translated into the clinic to treat human and cardiosphere-derived cells (Andersen et al, 2009), both of
ischemic heart disease. A number of randomized trials, using which are being used in clinical trials. It is possible that ongoing
bone marrow mononuclear cells have been performed and most trials with these putative endogenous cardiac progenitors will
studies demonstrated modest cell therapy-mediated improve- demonstrate benefits to cardiac function, but this may not
ments in ventricular function. In the largest study to date, the necessarily indicate differentiation into cardiac myocytes.
reinfusion of enriched progenitor cells and infarct remodeling in
AMI (REPAIR-AMI) study (Schachinger et al, 2006), 204
randomized patients with acute myocardial infarctions received Pluripotent stem cells
intracoronary delivery of bone marrow cells or vehicle control.
Follow-up after 1 year demonstrated significant improvements Embryonic stem (ES) cells represent the prototypical stem cells
in both cardiac function and in a combined clinical endpoint with the hallmarks of clonogenicity, self-renewal and pluripo-
consisting of death, recurrent myocardial infarct or any tency. The potency of these cells also represents a real safety
revascularization procedure. In contrast, analysis of 18-month concern, given their tendency to form teratomas (Nussbaum et al,
follow-up data from the similarly conducted bone marrow transfer 2007), even several years after therapeutic delivery (Amariglio
to enhance ST-elevation infarct regeneration (BOOST) study et al, 2009). One approach to overcoming this prohibitive safety
(Meyer et al, 2006), suggested a waning of early improvements in problem has been to generate pluripotent-derived progenitors that
ventricular function. However, it is worth noting that although have already committed to a cardiogenic pathway. As a proof-of-
improvements in ventricular function have been modest, no principle example of such a strategy, cells with an expression
pattern of adverse events has emerged from these trials. profile of Oct4, stage-specific embryonic antigen 1 (SSEA-1) and
Bone marrow-derived mesenchymal stem cells have also MESP1 demonstrated some regenerative potency when delivered
been the subject of clinical translation research. These cells are therapeutically in a primate infarct model, without detectable
generated from bone marrow samples that are serially passaged teratoma formation (Blin et al, in press). One could envision a
and cultured on plastic. Though traditionally defined by their similar strategy using cardiogenic intermediates that express any
capacity for trilineage differentiation into osteoblasts, chon- of the previously mentioned transcription factors associated with
drocytes and adipocytes, mesenchymal stem cells have also cardiac progenitors or cell surface markers such as the receptor for
demonstrated evidence for cardiac myocyte differentiation both vascular endothelial growth factor (Flk1/KDR) (Yang et al, 2008).
in vitro in some culture conditions and when delivered after Yet, such a strategy should still demonstrate both substantial
myocardial infarction to mice (Toma et al, 2002). Given their preclinical efficacy without tumorigenicity before human
reported tendency to evade rejection owing to ‘immune translation. If such criteria are met, ES-derived therapies have
privilege’, these cells have been administered to humans as the potential of providing ‘off-the-shelf’ cardiac myocytes to treat
an allogeneic treatment after myocardial infarct (Hare et al, acute myocardial infarctions or chronic heart failure.
2009). The initial clinical experience is consistent with trials A second approach, which may also obviate the risk of
using bone marrow mononuclear cells: patients experience teratomas, is to generate a pure population of ES-derived cardiac
modest improvements in ventricular function with subtle myocytes for therapeutic delivery either as a cell suspension or
improvements in heart failure symptoms. Overall, the experi- after ex vivo tissue engineering. There has already been
ence with cell-based therapies in humans does not support wide- enormous progress during the past decade in defining the
spread adoption at this time given their modest benefits, factors and transcription signals to differentiate cardiac
insufficient safety data and the anticipated quality control costs myocytes from ES-cells. As discussed in greater detail (Noseda
of bringing these therapies to the bedside. et al, 2011), cardiac myocyte development is dictated by the time
and dose-dependent exposure to a series of growth factors from
the wingless-type MMTV integration site (Wnt), fibroblast
Endogenous cardiac progenitors growth factor (FGF), bone morphogenetic protein (BMP) and
nodal families. Guided by these mechanistic insights, several
Since the initial suggestion that the mammalian heart may laboratories have successfully generated ES-derived prepara-
contain an endogenous population of progenitor cells (Beltrami tions with more than 50% of functional cardiac myocytes
et al, 2003), there has been an intense discussion about the (Laflamme et al, 2007; Yang et al, 2008). Because of the inherent
therapeutic potential of these cells. There are a number of technical challenge of delivering a sufficient number of
theoretical advantages to this approach. Autologous transfer differentiated cardiac myocytes—not to mention ensuring their
may be feasible, even if an ex vivo expansion stage would survival and electromechanical integration—the most realistic
likely be necessary given the small number of stem cells in future for such technical advances may be as an unlimited
heart biopsy specimens. Furthermore, other than cells isolated source of cardiac myocytes for engineering myocardial grafts.
from non-cardiac tissue beds, progenitors derived from the The generation of induced pluripotent stem (iPS) cells may
heart itself are theoretically partially pre-programmed to overcome two important limitations of ES cells: ethical concerns
differentiate into heart cells. However, some reports actually about harvesting ES cells from embryos and graft rejection by
question the intrinsic stem-like properties of some of the the patient’s immune system, because iPS cells can be custom-
endogenous adult progenitor preparations, including the engineered from a patient’s stromal cells for autologous
‘stemness’ of adult cardiac c-kitþ cells (Tallini et al, 2009) transplantation. A recent demonstration of heightened

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 Review
Regeneration of the heart

immunogenecity in syngeneically transplanted iPS cells, how- Perhaps the most stunning proof-of-principle approach to
ever, suggests that the immune system cannot yet be discounted generating new cardiac myocytes from endogenous cells is the
in the development of iPS-based therapies (Zhao et al, 2011). recent transformation of resident cardiac fibroblasts into cardiac
The initial protocols for iPS cell generation involved retro- myocytes (Ieda et al, 2010). Similar to protocols for generating
viral-mediated expression of four stem-cell genes (Takahashi & iPS cells, the forced expression in cardiac fibroblasts of three
Yamanaka, 2006; Yu et al, 2007). As virally reprogrammed cells genes—GATA4, MEF2C and TBX5—resulted in the expression
may harbour an associated risk of neoplastic conversion, of the cardiac myocyte-specific myosin heavy chain 6 (Myh6)
alternative reprogramming strategies, such as the use of small promoter in approximately 20% of the cells and spontaneous
molecules (Shi et al, 2008) or non-viral gene modifying beating in approximately 1% of reprogrammed cells. When
approaches (Warren et al, 2010) will probably be a necessary isolated cardiac fibroblasts were infected with viruses carrying
component of any future therapeutic strategies. However, the the genes encoding the three reprogramming factors and
most important lesson from these landmark studies may be the reintroduced to the heart within 24 h, some transformed directly
remarkable plasticity of even the most terminally differentiated into cardiac myocytes. Although this approach is still in its
cells when exposed to the right combination of signals. infancy, it suggests the exciting possibility that the fibroblast
rich post-infarct scar could be transformed into functional
myocardium via reprogramming.
Tissue engineering A more realistic short-term goal may be to exploit paracrine
signalling to amplify the existent endogenous regenerative
With stem cells as a potential unlimited source of cardiac response. Recent cell transplantation experiments conducted in
myocytes, engineering ex vivo myocardial grafts could, in our laboratory, using an inducible cre-based genetic lineage
theory, provide another therapeutic approach. The most mapping approach, tested the hypothesis that cell-based
clinically advanced tissue engineering to date involves thin, therapies might exert proregenerative effects via a paracrine
simple, relatively avascular tissues, such as tracheas (Macchiar- mechanism (Loffredo et al, 2011) (Fig 5). Consistent with some
ini et al, 2008). Owing to the complexity and physiological prior studies (Balsam et al, 2004; Murry et al, 2004), we found
requirements of functional myocardial tissue, an abundant no evidence for transdifferentiation of exogenously delivered
source of cardiac myocytes alone is not enough to build a bone marrow cells into cardiac myocytes. However, we did find
clinically useful graft. Historically, the greatest challenge in increased generation of cardiac myocytes from endogenous
tissue engineering has been an adequate supply of oxygen and progenitors in mice, which were administered c-kitþ bone
nutrients for metabolically active tissues such as the heart. One marrow cells but not mesenchymal stem cells. This finding
approach has been to engineer thin cardiac sheets, which can suggests paracrine signalling between exogenously delivered
then be stacked for in vivo delivery (Shimizu et al, 2002). cells and endogenous resident progenitors. It provides a
Although these layered sheets demonstrate some degree of rationale for therapeutic interventions aimed at activating
electromechanical coordination and neovascularization in vivo, progenitors or recruiting them from their niche to the injury
it is not clear yet if such an approach can be optimized to yield site (Steinhauser & Lee, 2009). One such approach involves the
full-thickness myocardium with an adequate blood supply. The administration of the peptide thymosin b4, which appears to
addition of non-myocyte cellular components, such as fibro- induce WT-1-expressing epicardial cells to differentiate into
blasts and endothelial cells, leads to the formation of primitive de novo cardiac myocytes (Smart et al, 2011). Interestingly, the
vascular structures within engineered grafts, but the electro- WT-1þ cells do not appear to differentiate into cardiac myocytes
mechanical properties are not sufficient for normal functionality in the adult mouse without being primed by thymosin b4, which
(Stevens et al, 2009). Ultimately, success in the engineering suggests the involvement of a reprogramming mechanism.
of functional, full-thickness myocardium will likely only An alternative to myocyte generation by pharmacologic
be possible if the problem of vascularization is solved means involves stimulating cell-cycle reentry of preexisting
(Zimmermann et al, 2006). cardiac myocytes. Inhibition of p38 MAP kinase increases
mitotic activity in cardiac myocytes (Engel et al, 2005), although
concomitant growth factor therapy (FGF1) was necessary to
Circumventing cell-based therapy with achieve a noticeable improvement in cardiac function (Engel et
pharmacoregeneration? al, 2006). The administration of the growth factor neuregulin-1
stimulates mature cardiac myocyte division in vitro, which
It is hard to imagine that a single pharmacologic intervention translated into a modest increase in the division of the
could lead to complete cardiac repair, but a number of recent mononucleated cardiac myocytes in vivo after systemic delivery
discoveries lend credence to the underlying concept that of neuregulin (Bersell et al, 2009). Similar stimulation of cardiac
paracrine signals may activate repair mechanisms in the heart. myocyte mitosis was observed after prolonged local delivery of
In general, the development of small molecule or protein-based periostin (Kuhn et al, 2007), an extracellular matrix signalling
therapies raises relatively modest problems with quality control protein; however, the effect was not replicated in a transgenic
compared to cell-based therapies. Therefore, pharmacoregen- model of periostin overexpression (Lorts et al, 2009).
erative approaches may be a more practical near-term approach Although the regenerative cardiology field has primarily
to promote cardiac repair. focused on progenitor cells, these data suggest that controlling

708 ß 2011 EMBO Molecular Medicine EMBO Mol Med 3, 701–712 www.embomolmed.org
 Review
Matthew L. Steinhauser and Richard T. Lee

Exogenous
stem cell
therapy
Differentiation
Smooth muscle
cells
Immunomodulation

Angiogenesis Endothelial
Paracrine cells
factors

Cardioprotection

Endogenous
progenitors
Cardiac myocytes
Differentiation

Figure 5. Proposed mechanisms of action for cell-based therapies. In theory, exogenously delivered cells may directly differentiate into endothelial cells,
smooth muscle cells and cardiac myocytes. They may also release paracrine factors which may result in non-regenerative effects, such as immunomodulation,
angiogenesis or cardioprotection. Recent work from our laboratory suggests that a dominant mechanism achieved with bone marrow progenitor therapy may be
via the activation of endogenous progenitor recruitment (Loffredo et al, 2011).

the mitotic activity of mononucleated cardiac myocytes may Future directions

provide an alternative approach to replenishing cardiac
myocytes. A major concern with systemic growth factor In this review, we have described the current status of
therapy, however, is the potential for mitogenic effects that research on cardiac regeneration, highlighting important
may impact other organs. Thus, the future of pharmacologic recent discoveries and ongoing controversies. The initial
regeneration may lie in the local delivery of engineered proteins hope that a cell progenitor would emerge with the capacity
and small molecules that target specific survival, growth and to regenerate the injured mammalian heart in the same
differentiation pathways. manner that bone marrow may be reconstituted has not
been realized in animal studies or randomized clinical
studies. Even if some studies have questioned the
Pending issues regenerative potency of various endogenous progenitor
Dissect the mechanistic differences between adult mammals with
populations, it is clearly feasible to reprogramme many cell
limited regenerative capacity and organisms, such as neonatal mice, types to differentiate into cardiomyocytes. Interestingly,
zebrafish and newts, that demonstrate unambiguous cardiac
myocyte regeneration. Understanding these differences may reveal evolving notions of cellular plasticity may ultimately help to
new pathways that can be therapeutically targeted to achieve more explain the frustration that many in the field have experienced
robust regeneration.
when attempting to replicate the findings of landmark studies or
Complete molecular and functional characterization of endogenous when trying to demonstrate progenitor activity in vivo. It may
cardiac myocyte progenitors. Multiple laboratories have isolated
progenitors from the heart with different molecular characteristics. well turn out that ex vivo progenitor cultures undergo epigenetic
What are the in vivo functional roles of these progenitors? Do the changes that affect cell fate, and that such modifications could
observed molecular differences between these isolated cells
represent functionally distinct cell types? account for some of the stem-like properties attributed to
endogenous progenitors.
Identify paracrine signalling pathways responsible for activation and
recruitment of endogenous cardiac myocyte progenitors. This may There is reason for optimism for a regenerative medicine
facilitate a pharmacoregenerative therapy, in which treatment with approach to heart failure, given the intense research
a protein or small molecule would hold the promise of amplifying
endogenous regeneration. efforts and the capacity of higher organisms, including
the neonatal mouse, to regenerate myocardium. Perhaps
Refine reprogramming strategies to more efficiently produce mature
cardiac myocytes, both in vitro and in vivo. The ultimate the most important issue in this field is identifying which
bioengineering goal is to produce a pure population of mature, fully findings are consistently supported by multiple experimental
functional cardiac myocytes for ex vivo tissue engineering (or) to
control the proliferation and differentiation of endogenous cell approaches. Ultimately, the findings that are easily repro-
populations or exogenously delivered cell therapies such that scar
tissue is replaced by myocardium. These different strategies are duced by most or all laboratories will most likely benefit
unified by an underlying requirement to understand the funda- patients.
mental pathways involved in cardiac myocyte differentiation and
maturation.
The authors declare that they have no conflict of interest.

www.embomolmed.org EMBO Mol Med 3, 701–712 ß 2011 EMBO Molecular Medicine 709
 Review
Regeneration of the heart

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