stem cell stimulation of endogenous myocyte regeneration

Clinical Science (2013) 125, 109–119 (Printed in Great Britain) doi: 10.1042/CS20120641

Stem cell stimulation of endogenous myocyteregenerationBrian R. WEIL∗1 and John M. CANTY, Jr∗†‡§

∗Center for Research in Cardiovascular Medicine, Department of Medicine, University at Buffalo, NY 14203, U.S.A.†Department of Physiology and Biophysics, University at Buffalo, NY 14214, U.S.A.‡Department of Biomedical Engineering, University at Buffalo, NY 14260, U.S.A.§VA Western New York Health Care System, Buffalo, NY 14215, U.S.A.

AbstractCell-based therapy has emerged as a promising approach to combat the myocyte loss and cardiac remodelling thatcharacterize the progression of left ventricular dysfunction to heart failure. Several clinical trials conducted over thepast decade have shown that a variety of autologous bone-marrow- and peripheral-blood-derived stem andprogenitor cell populations can be safely administered to patients with ischaemic heart disease and yield modestimprovements in cardiac function. Concurrently, rapid progress has been made at the pre-clinical level to identifynovel therapeutic cell populations, delineate the mechanisms underlying cell-mediated cardiac repair and optimizecell-based approaches for clinical use. The following review summarizes the progress that has been made in thisrapidly evolving field over the past decade and examines how our current understanding of the mechanisms involvedin successful cardiac regeneration should direct future investigation in this area. Particular emphasis is placed ondiscussion of the general hypothesis that the benefits of cell therapy primarily result from stimulation ofendogenous cardiac repair processes that have only recently been identified in the adult mammalian heart, ratherthan direct differentiation of exogenous cells. Continued scientific investigation in this area will guide theoptimization of cell-based approaches for myocardial regeneration, with the ultimate goal of clinical implementationand substantial improvement in our ability to restore cardiac function in ischaemic heart disease patients.

Key words: cardiac regeneration, cardiovascular disease, cell therapy, myocardial repair, stem cell

INTRODUCTION

Despite significant advances in the understanding and treatmentof cardiovascular diseases, the development of heart failure sec-ondary to coronary artery disease remains a significant causeof morbidity and mortality worldwide [1]. The evolution of LV(left ventricular) dysfunction to heart failure is characterizedby chronic myocyte loss that is inadequately compensated for bycellular hypertrophy of remaining myocytes, ultimately leadingto deterioration of cardiac contractile performance [2]. Unfortu-nately, the majority of available treatment options only delay theprogression of disease without addressing the fundamental prob-lem of cardiomyocyte loss, while the lone exception of organtransplantation is hampered by the limited availability of donor

Abbreviations: BrdU, bromodeoxyuridine; CDC, cardiosphere-derived cell; CSC, cardiac stem cell; CXCR, CXC chemokine receptor; EPC, endothelial progenitor cell; HGF, hepatocytegrowth factor; HNA, human nuclear antigen; IGF, insulin-like growth factor; JAK, Janus kinase; LAD, left anterior descending; LV, left ventricular; MSC, mesenchymal stem cell; pHH3,phospho-histone H3; SDF, stromal-cell-derived factor; STAT, signal transducer and activator of transcription; VEGF, vascular endothelial growth factor.1Winner of the Cardiovascular Section Clinical Science Young Investigator Award at Experimental Biology 2012, held in San Diego, CA, U.S.A., from 21–25 April 2012.

Correspondence: Dr Brian R. Weil (email [email protected]).

hearts. As a result, there is a critical need for the development oftherapeutic strategies aimed at reversing myocyte loss, restoringcardiac function and preventing the progression to clinical heartfailure.

With this goal in mind, the administration of stem and progen-itor cells has emerged as an innovative approach to replace lostmyocytes and rejuvenate damaged and dysfunctional myocar-dium. Given the tremendous clinical potential of such efforts, itis not surprising that this field has evolved at a rapid pace sincethe publication of initial studies demonstrating that transplanta-tion of skeletal myoblasts to infarcted myocardium could improvecardiac contractile performance [3,4]. Indeed, the past decade haswitnessed major advances in the development of cell-based ap-proaches for cardiac repair, including the completion of several

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large clinical trials confirming the safety and efficacy of variousautologous peripheral-blood- and bone-marrow-derived cell pop-ulations in patients with ischaemic heart disease. For example, arecent meta-analysis of 50 studies including over 2000 patientstreated with autologous bone-marrow-derived cells concludedthat cell therapy leads to small, but significant, improvementsin LV ejection fraction (∼4 %) and reduces infarct size (∼4 %),LV end-systolic volume (∼9 ml) and LV end-diastolic volume(∼5 ml) [5]. Moreover, minimal adverse events have been re-ported, and several trials have shown beneficial effects of celltherapy on indices of patient performance such as exercise ca-pacity and 6-min walk distance [6,7]. These encouraging resultshave fuelled interest in optimizing cell-based approaches to buildupon the modest improvements observed in the first generationof clinical trials of cell therapy for cardiac repair.

Concurrent with the initial stages of clinical translation, rapidprogress has been made at the pre-clinical level to improve ourunderstanding of the mechanisms involved in successful cardiacregeneration after injury. Although the heart has traditionallybeen considered a terminally differentiated post-mitotic organ in-capable of cellular turnover, a growing body of evidence suggeststhat this is not the case [8]. Several studies utilizing various tech-niques of cellular dating have shown that considerable myocyteturnover occurs throughout life in the healthy and diseased heart[9–11]. Together with the seminal discovery of circulating [12]and resident [13,14] endogenous cardiac stem cells, this findinghas dramatically shifted our perspective regarding the potentialfor regeneration of the damaged human heart. Although initialattempts at cell-mediated cardiac repair were performed with thegoal of directly replacing lost myocytes via transdifferentiationof exogenous stem cells, the recognition of endogenous cardiacregenerative potential has led to new approaches involving theadministration of cell populations derived from the heart itselfand/or activation of the host’s own reparative responses to pro-mote myocardial regeneration and improve cardiac function. Inthe present review, we will summarize recent progress in ourunderstanding of the heart’s capacity for repair, discuss how therecognition of endogenous regenerative potential has directedthe development of novel cell-based therapeutic approaches andexamine the mechanisms by which these approaches appear torevitalize damaged and dysfunctional myocardium.

ENDOGENOUS REPAIR CAPACITY OF THEMAMMALIAN HEART

For many decades, the mammalian heart has been considered aterminally differentiated organ without the intrinsic capacity forregeneration. Under this view, it was believed that the number ofcardiomyocytes was established shortly after birth and remainedlargely unchanged throughout the adult lifespan, with changesin heart mass attributed exclusively to hypertrophy of existingmyocytes [15]. However, accumulating evidence suggests thatcellular turnover does in fact occur in the adult mammalian heart[8]. Using the innovative approach of radiocarbon DNA dating,Bergmann et al. [9] provided evidence of cardiomyocyte renewal

in human adult hearts and estimated that ∼50 % of the cardi-omyocyte compartment is replaced throughout a typical life span.This notion of cardiomyocyte proliferation was supported by es-timates of cardiomyocyte DNA synthesis in post-mortem tissuefrom cancer patients who had been treated with the thymidineanalogue iododeoxyuridine, which is incorporated into the DNAof cycling cells and thus allows assessment of myocyte prolif-eration over time [16]. Mathematical modelling suggested thatcardiomyocytes turn over at a much higher rate (∼20 % per year)than had been proposed by Bergmann et al. [9], but neverthe-less reinforced the emerging concept that myocyte turnover andtissue regeneration occur throughout adulthood. The discrepantfindings regarding the magnitude of cardiomyocyte turnover mayhave been resolved by a recent collaborative effort involvinginvestigators from each of the previous studies in which retro-spective radiocarbon DNA dating was performed on fresh tissuesamples from healthy and failing human hearts [11]. This methodminimizes the requirement of mathematical modelling that washeavily relied upon in earlier reports and may have contributedto an underestimation of myocyte turnover [17]. Using this im-proved approach, the authors found that from 20 to 78 years ofage, the entire cardiomyocyte compartment is replaced ∼8 timesin healthy hearts, with an even greater turnover rate apparent inpatients with heart failure. This finding challenges the prevailingdogma of the heart as a post-mitotic organ and indicates that themammalian heart retains a significant degree of plasticity andcell turnover throughout adult life.

The recent discovery of CSCs (cardiac stem cells) has alsoplayed a pivotal role in promoting the modern view of the heartas an organ capable of regeneration. Although a specific hierarchyof CSCs and cardiac progenitor cells has yet to be defined, severalgroups of investigators have identified cell populations with char-acteristics of stem cells that reside in the post-natal heart [18].The most extensively studied cell population is that character-ized by expression of the tyrosine kinase receptor c-kit. Initiallyidentified by Beltrami et al. [19], Lin− /c-kit+ cells are self-renewing, clonogenic and multipotent, with the ability to differ-entiate into cardiomyocytes, smooth muscle cells and endothelialcells [14]. These CSCs are thought to reside within specializedniches primarily located in the atrium and apex, where they main-tain their ‘stemness’ via connexin- and cadherin-mediated struc-tural connections with mature cardiomyocytes, fibroblasts andother stem cells [20]. Additional cell populations characterizedby the expression of surface markers {SCA (stem cell antigen)-1[21]}, transcription factors (Islet-1 [22]) or the ability to effluxfluorescent dye (side-population cells expressing ATP-bindingcassette transporters [23]) have also been found in the heart andshown to possess characteristics of stem cells. More recently, theepicardium has been identified as a potential source of cardiacprogenitor cells that are capable of regaining their embryonicpotential to form new vascular cells and cardiomyocytes follow-ing stimulation by thymosin β-4 [24]. Although initial reportssuggested minimal overlap between these putative CSC popula-tions, subsequent studies have shown expression of some surfaceantigens in the same line of cells at different stages of matura-tion [18]. Regardless, it is clear that cells with the capacity toform cardiomyocytes exist in the post-natal mammalian heart,

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Stem-cell-mediated cardiac repair

providing further evidence of endogenous cardiac regenerativepotential.

The high rate of morbidity and mortality in patients withischaemic heart disease clearly demonstrates that the endogen-ous capacity for cardiac repair is insufficient to offset the loss ofmyocytes that typically occurs with acute or chronic myocardialischaemia. However, evidence of some degree of myocyte regen-eration after injury has provided further support for the notionthat the heart is capable of a limited amount of self-repair. Forexample, Richard Lee’s laboratory has recently used genetic fate-mapping with stable isotope labelling and multi-isotope imagingMS in mice to demonstrate that the formation of new cardiomyo-cytes occurs at a low rate during normal aging, but is increasedin areas adjacent to infarcted myocardium [25]. Interestingly, thegenesis of new myocytes primarily occurred via the division ofpre-existing cardiomyocytes that had re-entered the cell cycle. Al-though these results suggest that endogenous progenitors have aminimal role in repair after injury, other investigators haveperformed lineage-tracing experiments with transgenic mice todemonstrate that progenitor cells are mobilized and/or recruitedto the heart and form cardiomyocytes after myocardial infarctionor pressure overload [26]. To investigate the mechanisms involvedin this process, specific growth factor signalling pathways havebeen examined based on immunohistochemical evidence thatc-kit+ CSCs express the HGF (hepatocyte growth factor) re-ceptor c-Met and the IGF (insulin-like growth factor)-1 receptor(IGF-R) [27]. In vitro experiments revealed that HGF promotedCSC migration and IGF-1 enhanced cell survival and prolifera-tion, which lead the authors to administer these growth factorsto the infarcted rat heart in an attempt to stimulate a regenerat-ive response in vivo. Fluorescent labelling of CSCs in the atrio-ventricular groove enabled tracking of these cells, which migratedtowards the infarcted area and appeared to begin differentiatinginto small myocytes. Moreover, the combined HGF/IGF-1 treat-ment resulted in a significant improvement in ventricular func-tion and animal survival, supporting the idea that activation ofendogenous CSCs may be a viable approach to promote cardiacrecovery after injury.

Collectively, the documentation of cardiomyocyte turnoverthroughout adulthood, evidence that new cardiomyocytes canform from the division of pre-existing cardiomyocytes and theidentification of resident cardiac stem and progenitor cells thatcontribute to myocyte renewal have re-shaped our understandingof myocardial biology. It is now apparent that cardiac cellular ho-moeostasis throughout life is a reflection of the balance betweenmyocyte death and myocyte renewal, with profoundly negativeimplications for organ function when this balance is shifted insuch a way that cell death exceeds cell regeneration. Althoughendogenous progenitor cells and division of pre-existing cardi-omyocytes may contribute to cardiac regeneration after injury,the adult heart’s capacity for repair is clearly of insufficient mag-nitude to offset the dramatic loss of myocytes that characterizesacute or chronic ischaemic heart disease. Nevertheless, the re-cognition of natural regenerative mechanisms in the mammalianheart has stimulated interest in the development of therapeuticapproaches that exploit the endogenous potential for cardiacrepair.

EXPERIMENTAL CELL TRANSPLANTATIONFOR CARDIAC REPAIR: EMERGENCEOF NOVEL CELL POPULATIONS

Initial attempts at administering exogenous blood- and bone-marrow-derived progenitor cells to repair damaged myocar-dium were motivated largely by the findings of Orlic et al.[28], who reported that bone marrow haemopoietic stem cellscould transdifferentiate into cardiomyocytes after transplantationinto infarcted myocardium. However, subsequent studies [29–31]raised doubts regarding the transdifferentiation capacity of bone-marrow-derived cells, and it is now generally believed that thesecells only rarely give rise to cardiomyocytes, if ever [18]. Nev-ertheless, clinical trials testing the safety and efficacy of thesecell populations have moved forward, generally showing modestimprovements in ventricular function despite the inability to dir-ectly generate a large number of new myocytes [5]. Driven by thediscovery of endogenous cardiac progenitors, more recent studieshave focused on novel cell populations that may possess super-ior cardiomyogenic potential given their apparent involvement inphysiological cardiomyocyte turnover.

CSCsFollowing the identification of stem cell populations resident inthe adult heart, attempts at isolating these cells for the purposeof experimental cell therapy soon followed based on the notionthat cardiac-derived cells may be particularly effective at regen-erating myocardium. A series of preclinical studies involvingthe isolation of c-kit+ CSCs from fragments of cardiac tissue,ex vivo expansion in culture and subsequent transplantation intodamaged myocardium have provided encouraging results. Forexample, intramyocardial injection of human c-kit+ CSCs intothe infarcted hearts of immunosuppressed rodents elicited signi-ficant improvements in cardiac function, with evidence that theexogenously delivered CSCs differentiated into cardiomyocytes,endothelial cells and vascular smooth muscle [14]. These andother [32] positive results have facilitated the translation of thisapproach to human patients with the SCIPIO (Stem Cell Infu-sion in Patients with Ischemic Cardiomyopathy) trial, a Phase Iclinical trial of autologous c-kit+ CSCs. Although only a smallnumber of patients have been studied, initial data indicate thatCSC treatment improves regional and global LV function,reduces infarct size and increases viable myocardium for up to1 year after injection [33,34].

CDCs (cardiosphere-derived cells)Soon after the discovery of resident CSCs, Messina et al. [35]described the isolation of undifferentiated cells from adult car-diac tissue specimens that would spontaneously form sphericalclusters when placed in suspension culture. These clusters weretermed ‘cardiospheres’ and were shown to consist of proliferat-ing c-kit+ cells in their core, with differentiating cells expressingcardiac and endothelial cell markers in their periphery. Build-ing on this finding, Marban’s laboratory modified the cardio-sphere isolation procedure and used cardiospheres as the basis ofcell expansion, ultimately yielding CDCs [36]. It has been pro-posed that CDCs possess greater potential for repair because

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cardiospheres recapitulate the microenvironment of the CSCniche, as evidenced by an elevated number of c-kit+ cells,up-regulation of stem-cell-related transcription factors such asNanog and Sox2, and enhanced expression of extracellular mat-rix proteins and adhesion molecules [37]. In pre-clinical modelsof acute and chronic ischaemic heart disease, administration ofCDCs improves ventricular function, reduces infarct size andincreases viable myocardium [36,38]. Interestingly, a direct com-parison of CDCs with other stem and progenitor cell populationsrevealed that CDCs exhibit superior cardiomyogenic capacity, an-giogenic potential and release of paracrine factors in vitro [39].Moreover, CDCs injected into infarcted mouse hearts yieldeda greater improvement in cardiac function, higher cell engraft-ment and superior attenuation of pathological ventricular remod-elling compared with other cell types. CDCs were even deemedsuperior to purified c-kit+ CSCs based on paracrine factor re-lease and functional benefit after transplantation, suggesting thatthe therapeutic potential of CSCs may be enhanced by cardio-sphere culture and/or administration in the context of a supportivemixed-cell milieu [39]. Preliminary results from the first clinicaltrial of CDCs have recently been published, demonstrating thatintracoronary injection of autologous CDCs is safe and elicits sig-nificant improvements in regional contractility and viable heartmass, but not LV ejection fraction, 6-months after treatment [40].

MSCs (mesenchymal stem cells)Friedenstein et al. [41] first identified MSCs as a rare popula-tion of plastic-adherent bone-marrow-derived cells capable offorming single-cell colonies. These cells have subsequently beenshown to possess multilineage potential, with the ability to differ-entiate into chondrocytes, adipocytes and osteoblasts [42]. In vitroexperiments involving co-culture with mature ventricular myo-cytes have provided evidence that MSCs can transdifferentiateinto cardiomyocytes in the appropriate microenvironment [43].For example, mouse MSCs express α-actinin, form gap junctionsand synchronously contract when co-cultured with mature ratcardiomyocytes [44]. Interestingly, separation of MSCs and car-diomyocytes with a semi-permeable membrane prevented trans-differentiation, indicating that this process requires directintercellular communication. The differentiation of MSCs isprobably regulated by multiple signalling pathways, includingthe Wnt canonical pathway and the TGF (transforming growthfactor)-β pathway, which each respond to a variety ofgrowth factors to direct gene expression [45].

The in vitro cardiomyogenic potential of MSCs, as well as theiraccessibility from a number of tissues and capacity to undergoexpansion in culture, facilitated the use of this cell population inattempts to promote cardiac repair in several pre-clinical mod-els of ischaemic heart disease. In a swine model of ischaemiccardiomyopathy, transendocardially injected MSCs were shownto engraft in the heart, differentiate into cardiomyocytes, en-dothelial cells and vascular smooth muscle cells, reduce infarctsize, and improve ejection fraction for up to 12 weeks [46]. Al-though the trilineage differentiation potential of MSCs admin-istered to the heart has been supported by other studies [47], thefrequency of engraftment and differentiation is very low com-pared with the magnitude of functional recovery that is generally

observed [48], suggesting that direct differentiation is not thepredominant mechanism by which these cells promote cardiacrepair (discussed below). Recent work by Chong et al. [49] de-scribing a population of epicardium-derived cardiac MSCs raisesthe intriguing possibility that isolation, ex vivo expansion andtherapeutic administration of MSCs native to the heart may of-fer a superior approach for cardiac regeneration, although thisremains to be tested. Regardless, the clinical application of extra-cardiac-derived MSCs for myocardial repair has progressed, withinitial evidence that MSCs derived from bone marrow [50] andadipose tissue [51] are safe and have favourable effects on cardiacstructure and function in patients with ischaemic heart disease.

MECHANISMS UNDERLYING CELL-THERAPY-MEDIATED REPAIR:STIMULATION OF ENDOGENOUSCARDIAC REGENERATION

In an effort to optimize the efficacy of cell-mediated approachesfor cardiac repair, there has been significant interest in identifyingthe physiological, cellular and molecular mechanisms underlyingsuccessful myocardial regeneration. Although initial investiga-tion centred on the ability of injected stem cells to differentiateinto cardiomyocytes, more recent studies have revealed that thefunctional benefits of cell therapy arise through several mechan-isms of action that generally involve the potentiation of endogen-ous self-repair processes.

Paracrine-factor-mediated stimulationof host cardiac repair processes by bonemarrow precursorsInitial support for the notion that mechanisms other than car-diomyogenic differentiation of donor cells contributed to cell-mediated cardiac repair came from the finding that injection ofbone-marrow-derived MSCs overexpressing the survival geneAkt1 after myocardial infarction improved cardiac function inless than 72 h [52]. Because this time course of improvementwas not consistent with direct differentiation of injected cells, theauthors postulated that the beneficial effects were mediated bythe release of paracrine factors (reviewed in [53,54]). Subsequentstudies showing a similar recovery of function following adminis-tration of concentrated conditioned medium from Akt1-modifiedMSCs supported this hypothesis [55]. Further insight into theseprocesses was provided by Young-sup Yoon’s laboratory, who ex-amined the expression patterns and sources of paracrine factorsafter transplantation of human bone-marrow-derived EPCs (en-dothelial progenitor cells) into mice after myocardial infarction[56]. Cell injection elicited a significant elevation in circulatingconcentrations of various angiogenic and anti-apoptotic paracrinefactors that persisted for at least 14 days, despite the disappear-ance of the injected EPCs after 1 week. Interestingly, the majorityof these factors were of mouse origin, indicating that the sustainedup-regulation of paracrine factors could be attributed to host cellsand/or tissues. Moreover, EPC transplantation enhanced mobiliz-ation of endogenous stem cells from the host’s bone marrow and



recruitment of these cells to the ischaemic myocardium, implicat-ing the activation of endogenous repair processes in the functionalimprovements afforded by exogenous cell administration.

Peripheral injection of MSCs improves cardiacfunction via paracrine-mediated myocardialregenerationTo take advantage of the potent paracrine actions of bone mar-row MSCs, colleagues from our Department have completed aseries of studies investigating the therapeutic benefits of extra-cardiac MSC injection in a hamster heart failure model. Bilat-eral hamstring muscle injection of MSCs significantly improvedventricular function 1 month after injection, despite the factthat the injected cells were trapped in the local skeletal mus-culature. Evidence of myocardial regeneration was provided byan ∼80 % increase in myocyte nuclear density, ∼2-fold eleva-tions in the expression of the cell-cycle markers Ki-67 and pHH3(phospho-histone H3), and a reduction in mean myocyte dia-meter [57]. In addition, intramuscular MSC injection led to anincrease in circulating growth factor concentrations, mobiliza-tion of c-kit+ , CD31+ and CD133+ progenitor cells, and asubsequent increase in c-kit+ cells in the myocardium. Takentogether, these results indicate that MSCs are capable of stimulat-ing endogenous repair processes to promote cardiac regeneration,even when they are administered via extra-cardiac injection to re-mote skeletal muscle. To delineate the mechanisms involved inthis therapeutic strategy, the activity of the skeletal muscle JAK(Janus kinase)/STAT (signal transducer and activator of tran-scription) signalling pathway was investigated [58]. Incubationof cultured skeletal myocytes with MSC-conditioned medium in-duced phosphorylation of JAK1, JAK2 and STAT3, resulting ingp130-receptor-dependent production of HGF and VEGF (vascu-lar endothelial growth factor). Similar responses were observedin MSC-injected hamstring muscle in vivo, including activationof JAK/STAT3 signalling and increased HGF and VEGF pro-duction. These host-derived factors promoted myocardial regen-eration and improvements in cardiac function, a response thatwas abolished by the JAK/STAT3 inhibitor WP1066. Collect-ively, these results indicate that MSCs activate JAK/STAT3 sig-nalling following skeletal muscle injection, causing the release ofhost-tissue-derived growth factors and activation of endogenousself-repair mechanisms to promote cardiac regeneration.

Global intracoronary MSC infusion in viabledysfunctional myocardiumBuilding on these findings, we investigated the effects ofintracoronary autologous MSC injection in pigs with hibernatingmyocardium. In this model, regional ventricular dysfunctionarises from repetitive ischaemia in collateral-dependent myocar-dium, resulting from a chronic stenosis of the LAD (left anteriordescending) coronary artery. This condition is characterizedby regional apoptosis-induced myocyte loss and compensatoryhypertrophy that is stable between 3- and 5-months afterinstrumentation, allowing assessment of cell-therapy-mediatedcardiac regeneration without the confounding effects of ongoingcell death [59]. At 4 weeks after MSC injection, regional LADwall thickening was significantly improved, despite a persistent

impairment in resting and adenosine-dilated coronary blood flow[60]. Transient elevations in circulating c-kit+ and CD133+

cells were observed at 3 days after cell injection, with corres-ponding increases in c-kit+ /CD45− and CD133+ /CD45− cellsin hibernating and remote areas of the heart. Approximately 60 %of the c-kit+ /CD45− cells found in the heart were CD133− ,suggesting that a large proportion of c-kit+ cells were residentCSCs, with fewer than half of the c-kit+ cell population derivedfrom extracardiac sources, such as the bone marrow. In addition,MSC-treated animals exhibited significantly greater expressionof Ki-67 and pHH3 in the hibernating region, indicating that celltherapy stimulated myocyte proliferation. These effects resultedin an increased myocyte nuclear density and a concomitantregression in average myocyte diameter in hibernating myocar-dium, supporting myocyte regeneration following cell injection.Importantly, ex vivo analysis of cell fate following injectionof fluorescent-labelled MSCs demonstrated rare instances ofengraftment, with fluorescent staining limited to endotheliumand vascular smooth muscle, but not cardiomyocytes. These datademonstrate that intracoronary injection of autologous bone-marrow-derived MSCs improves regional contractile function inswine with hibernating myocardium via myocardial regenerationthat appears to result from stimulation of myocyte proliferationand mobilization of endogenous progenitor cells (Figure 1).

Studies supporting MSC-mediated stimulationof endogenous repairThe notion that the benefits of cell therapy are mediated by mo-bilization of endogenous progenitor cells is supported furtherby studies using genetic lineage tracing approaches to demon-strate that administration of bone-marrow-derived c-kit+ cellspromotes cardiac regeneration by augmenting the formation ofnew cardiomyocytes from endogenous progenitors after myocar-dial infarction [61]. These results are concordant with thoseof Hatzistergos et al. [62], who injected bone-marrow-derivedMSCs into the infarct area and border zone of pigs 3 days aftermyocardial infarction. MSC-treated animals exhibited reducedinfarct size and an improvement in ejection fraction comparedwith vehicle-treated controls. Although evidence of MSC en-graftment and differentiation to cardiomyocytes and blood ves-sels was reported, activation of endogenous c-kit+ CSCs andstimulation of cardiomyocyte cell-cycling were implicated as theprimary mechanisms responsible for the beneficial effects of celltherapy. Interestingly, the same degree of tissue recovery wasnot observed following injection of MSC-conditioned medium,suggesting that cell–cell interactions are necessary to permit ac-tivation of endogenous repair mechanisms. Follow-up in vitroexperiments showed that MSCs stimulated c-kit+ CSC prolif-eration and increased CSC expression of Nkx2.5 and troponinI, suggesting that MSCs may promote CSC differentiation tocardiomyocytes. Taken together, these observations reinforce theimportance of host-derived regenerative responses in mediatingthe reparative effects of MSC therapy.

CSCs and CDCs as platforms for cardiac repairActivation of endogenous regeneration as a mechanism underly-ing cell-therapy-mediated repair is not limited to cell populations

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Figure 1 Intracoronary mesenchymal stem cells elicit myocardial regeneration that arises from mobilization of endo-genous progenitor cells and stimulation of myocyte proliferationAutologous bone-marrow-derived MSCs (44 × 106) were administered via intracoronary injection to swine with chronichibernating myocardium (n = 10). Animals were studied either 2 weeks (n = 6) or 6 weeks (n = 4) later and compared withuntreated animals with hibernating myocardium (n = 7) or sham-normal animals receiving MSCs (n = 6). (A) Myocardialc-kit+ cells were increased 2 weeks after MSC treatment and remained elevated 6 weeks after injection. In addition,intracoronary MSC administration resulted in persistent elevations in (B) Ki-67+ and (C) pHH3+ myocytes, indicating thatcell therapy stimulated myocyte proliferation. (D) Untreated hibernating myocardium was characterized by a reduction inmyocyte nuclear density and compensatory myocyte hypertrophy. MSC-treated animals exhibited a progressive increasein myocyte nuclear density and a concomitant reduction in myocyte diameter, consistent with significant myocardialregeneration. This Figure was reprinted with kind permission from Wolters Kluwer Health [Gen Suzuki, Vijay Iyer, Te-ChungLee, John M. Canty, Jr, Autologous Mesenchymal Stem Cells Mobilize cKit and CD133 Bone Marrow Progenitor Cells andImprove Regional Function in Hibernating Myocardium, Circulation Research, 109 (9) 1044–1054]. Copyright (2011).

derived from the bone marrow. Although stem cells derived fromthe heart itself possess superior cardiomyogenic potential in vitro,recent studies suggest that direct differentiation following trans-plantation is not the primary mechanism by which these cellpopulations promote cardiac recovery. For example, significantimprovements in cardiac structure (increased viable heart mass)and function (increased ejection fraction and fractional short-ening) have been observed following intracoronary injection ofc-kit+ CSCs to rats 30 days after myocardial infarction [32].Interestingly, injected cells were detected in the hearts of onlyseven out of 17 CSC-treated rats 1 month after cell transplanta-tion, although all treated animals exhibited similar increases inviable myocardium and ventricular function. Immunohistochem-ical staining against c-kit revealed an ∼10-fold increase in thenumber of c-kit+ cells in both the risk area and non-infarcted re-

gions of CSC-treated rat hearts. To determine the source of thesecells, the authors administered the thymidine analogue BrdU(bromodeoxyuridine) for 2 weeks prior to killing and co-stainedmyocardial tissue sections for c-kit and BrdU to identify newlyformed (endogenous) c-kit+ CSCs. Although ∼40 % of the totalc-kit+ population was also BrdU+ in vehicle-treated animals,the percentage of double-positive cells was significantly higherin the cell-treated animals, indicating activation of endogenousCSCs following injection of exogenous c-kit+ cells [32]. Sim-ilarly, Chimenti et al. [63] injected human adult-derived CDCsinto the infarct border zone of immunodeficient mice and quan-tified the percentage of HNA (human nuclear antigen)+ (i.e.exogenous cell-derived) myocytes and capillaries 1 week later.Although cell treatment doubled the number of myocytes and ca-pillaries, only ∼12 % of cardiomyocytes and ∼10 % of capillaries



Figure 2 Proposed paradigm of cell-therapy-mediated cardiac repair via stimulation of endogenous repair processesAlthough the initial application of cell-based therapies for myocardial repair was motivated by the idea that the deliveryof exogenous stem and/or progenitor cells could promote myocyte regeneration via direct differentiation to cardiac cells,studies to date suggest that this rarely occurs, if at all. Instead, it appears that cell administration stimulates endogenouscardiac repair processes, possibly via paracrine signalling, direct cell–cell interactions, and/or transfer of microRNAs thatinfluence the transcriptional activity of host cells. Data from experimental studies in our laboratory indicate that the repairprocess involves mobilization of c-kit+ resident CSCs in both healthy (sham control) and diseased hearts. However, onlyanimals with viable dysfunctional (hibernating) myocardium exhibit increased markers of cell-cycle activation (such as Ki-67and pHH3), indicative of myocyte proliferation. Collectively, the mobilization of endogenous progenitors and stimulation ofmyocyte cell-cycle re-entry result in myocardial regeneration, characterized by an increased density of myocyte nuclei anda reduction in mean myocyte diameter.

were also HNA+ , indicating that indirect mechanisms contrib-ute to a significant portion of cell-therapy-mediated repair. Fur-ther analyses revealed that CDC treatment increased tissue levelsof exogenous cell-derived growth factors, enhanced expression ofthe pro-survival signalling kinase Akt and reduced apoptosis, sug-gesting that paracrine-mediated effects on cell survival may havecontributed to functional recovery as well.

In summary, data from our laboratory and others indicatethat direct differentiation of exogenous cells into cardiomyocytesfollowing transplantation may not be the primary mechanism un-derlying the regenerative benefits of many cell-based therapeuticapproaches. Instead, it appears that direct and paracrine-factor-mediated activation of recently recognized endogenous mechan-isms of repair, such as mobilization of bone-marrow- andcardiac-resident progenitor cells and stimulation of cardiomyo-cyte cell-cycle re-entry, are probably responsible for myocar-dial regeneration and functional improvements derived from celltherapy (Figure 2).

IMPLICATIONS FOR FUTURE RESEARCH

Our evolving understanding of the processes involved in suc-cessful cardiomyocyte regeneration after injury has importantimplications for the development of novel strategies to optim-ize cell-mediated repair of the heart. Identification of specific

molecular and cellular mechanisms promoting the formation ofnew myocytes from endogenous progenitor cells and/or the pro-liferation of pre-existing myocytes has directed experimental ap-proaches aiming to target these pathways to enhance regeneration.In addition, recognition of the important role played by the host’sown self-repair processes in the success of cell-based therapieshas encouraged researchers and clinicians to consideravenues of myocardial repair beyond direct differentiation ofexogenous cells delivered to the damaged heart.

One active area of investigation involves the development oftechniques to modify stem and progenitor cell populations ex vivoprior to administration in an attempt to improve cell survival,proliferation, engraftment and/or production of paracrine factorsafter delivery. Several approaches have shown promising results,including cell preconditioning with hypoxic culture conditions,genetic modifications, and pharmacological augmentation of re-parative capacity [64]. For example, incubation of cardiosphere-derived c-kit+ progenitor cells in a hypoxic environment for 6 hbefore delivery enhanced their therapeutic efficacy, as these cellselicited a larger reduction in infarct size and greater improvementin ventricular function than cells cultured under normoxic condi-tions [65]. This effect was abrogated by the addition of a CXCR(CXC chemokine receptor)-4 inhibitor, indicating that the bene-fits of hypoxic preconditioning occur via up-regulated activity ofCXCR-4 and/or its ligand SDF (stromal-cell-derived factor)-1.The SDF-1/CXCR-4 axis plays an important role in progenitor

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cell homing to damaged tissue and has also been shown to beinvolved in the increased mobilization of bone marrow cells tothe damaged heart after injection of MSCs genetically modifiedto overexpress IGF-1 [66]. As an alternative method to targetthe IGF system without ex vivo genomic manipulation of cellsprior to injection, combination therapy involving administrationof c-kit+ CSCs and biotinylated IGF-1 nanofibres has also beentested as a way to enhance cell-mediated cardiac regeneration.Compared with either CSCs or IGF-1 nanofibres alone, com-bination therapy elicited a greater recovery of cardiac functionand superior myocardial regeneration largely via enhanced ac-tivation of endogenous resident cardiac progenitor cells [67].Augmentation of endogenous repair was also implicated as amechanism underlying the superior reparative efficacy of c-kit+

CSCs genetically engineered to express Pim-1 kinase, a down-stream target of Akt that promotes cell survival and proliferation[68]. Thus several cell modification techniques have been provento successfully enhance the regenerative capacity of cell-basedtherapies in pre-clinical experiments. Although many of theseapproaches aim to enhance the survival and/or retention of cellsafter injection, the vast majority of exogenously deliveredcells still do not persist several weeks after delivery yet elicitpersistent functional benefit, further reinforcing the importanceof the endogenous repair system in this process.

As an alternative to modifying cells ex vivo before autologousadministration, there is interest in allogeneic transplantation ofcell populations that may offer superior myocardial regenerativepotential. The finding that stimulation of endogenous repair pro-cesses is a primary mechanism underlying cell-therapy-mediatedcardiac regeneration facilitates this approach, since long-termengraftment and immune tolerance to administered cells is notmandatory to obtain structural and functional benefits. This ideais particularly attractive given that the majority of patients thatwould benefit from cell therapy may possess a stem cell popula-tion that is compromised by age and/or disease, thereby limitingthe quantity and quality of cells available for isolation and ulti-mately hampering the effectiveness of autologous cell transplant-ation [69]. In this context, evidence that allogeneic transplantationof several prospective cell populations, including MSCs [50] andCDCs [70], is safe and effective at improving ventricular functionin pre-clinical models of ischaemic heart disease is particularlyexciting. Further validation of allogeneic cell transfer approachesin large animals and humans will motivate future investigationaimed at identifying sources of functionally superior stem andprogenitor cells. Along these lines, accumulating evidence sup-ports the notion that CSCs isolated at an early stage of post-nataldevelopment may be particularly well suited for therapeutic use.Multiple studies demonstrate that cardiac c-kit+ cells derivedduring the first week of post-natal life exhibit superior cardiomyo-genic capacity in vitro compared with cells isolated from adulthearts [71,72], which may contribute to the transient regenerat-ive potential of mammalian hearts at this stage of life [73]. Therecent finding that human neonatal-derived CDCs elicit greatermyocardial repair than adult-derived CDCs when administeredafter infarction provides early support for the potential transla-tion of a new therapeutic paradigm in which CSCs derived fromyoung donors are utilized to promote myocardial regeneration in

older patients with ischaemic heart disease [74]. Alternatively,non-viable (i.e. lethally irradiated) stem cells could be used asin vivo ‘feeder layers’ to promote endogenous myocardial repairsince long-term cell survival is not necessary, an approach sup-ported by recent data demonstrating activation of endogenous re-generation and improvements in myocardial function followinginjection of mitotically inactivated embryonic stem cells [75].However, it must be kept in mind that potential age- or disease-related deficits in recipient repair processes may ultimately hinderthe effectiveness of cell transplantation, regardless of the sourceof administered cells, given the important role of endogenousregenerative responses in mediating repair after exogenous cellinjection.

In addition to strategies focusing on amplifying the reparativecapacity of cells prior to delivery, efforts have also been madeto identify the optimal myocardial substrate and patient popu-lation to maximize the efficacy of cell-based therapy. This maybe particularly important given the prominent role that the hostcell and tissue repair mechanisms play in mediating cell-therapy-stimulated regeneration. From this perspective, it is tempting tospeculate that targeting dysfunctional but viable myocardium,rather than infarct and border zone areas, may facilitate greaterregeneration after cell administration. This notion is supportedby data from our laboratory documenting the ability of MSCs[60], CDCs [76] and other interventions [77] to promote myo-cyte regeneration in swine with viable dysfunctional (‘hibernat-ing’) myocardium. Although there are many causes of hibernat-ing myocardium, they typically reflect the consequences of car-diomyocyte loss and compensatory cellular hypertrophy arisingfrom overload [78]. This hypertrophied cellular phenotype arisesfrom many of the pathophysiological stimuli encountered in heartfailure, including ischaemia, chronically elevated preload andneurohormonal activation, all of which increase over time dur-ing disease progression. Importantly, a large amount of chron-ically dysfunctional myocardium in patients with ischaemic car-diomyopathy is generally viable [79,80] and is thought to be amajor contributor to the progression of global ventricular dys-function. Therefore focusing cell-based therapies on regener-ating myocytes in viable dysfunctional tissue may be a moreeffective strategy than targeting fibrotic tissue or the relativelysmall border zone adjacent to the infarct, which has been the aimof many studies. Mechanistically, chronic hibernating myocar-dium may be amenable to therapeutic regeneration as a resultof chronic cellular remodelling that may facilitate myocyte pro-liferation. Originally described by Borgers and co-workers [81],hibernating myocytes are characterized by a loss of myofibrils,mini-mitochondria and a reversion to a fetal phenotype, indic-ative of cellular dedifferentiation. We have demonstrated pre-viously that this dedifferentiated cellular phenotype is presentin remote normally perfused regions of the heart, as well as indysfunctional chronically ischaemic hibernating myocardiumin swine with a chronic LAD stenosis [82], a pattern that has alsobeen observed in patients with ischaemic cardiomyopathy un-dergoing coronary revascularization [83]. Zhang et al. [84] haveshown that dedifferentiation of mature mammalian cardiomyo-cytes in vitro results in down-regulation of cell-cycle inhibitorsand re-expression of cardiac progenitor cell markers, including



c-kit, ultimately promoting cell proliferation and the formationof new myocytes. These results raise the intriguing possibilitythat myocyte dedifferentiation may be involved in facilitatingcell proliferation in hearts with hibernating myocardium. Futurestudies will be necessary to directly test this hypothesis, with theresults likely to provide insight regarding how different patho-physiological substrates respond to therapies aimed at stimulat-ing myocyte regeneration. Ultimately, this information will havesignificant implications for the clinical translation of cell therapyand the identification of patient populations that may receive thelargest benefit from these therapeutic approaches.

SUMMARY

Tremendous progress has been made in the field of cell-basedtherapeutics for cardiac repair and regeneration in the past15 years, with encouraging results from initial clinical trials sup-porting the safety of treatment approaches involving the adminis-tration of various stem and progenitor cell populations. Lookingforward, it is imperative to expand our current knowledge of themechanisms involved in successful myocardial regeneration toguide future basic and translational investigation aimed at optim-izing cell therapy for clinical use. A growing body of evidenceclearly demonstrates that a primary mechanism underlying thebeneficial effects of cell transplantation is activation of endogen-ous cardiac repair processes, the details of which remain poorlyunderstood. It is anticipated that future studies will continue toadvance our understanding of these self-repair mechanisms andlead to the discovery of new and effective methods of cell-basedmyocardial repair, ultimately resulting in an improved ability torestore cardiac function in heart disease patients and reduce thesubstantial rate of morbidity and mortality associated with thiscondition throughout the world.

FUNDING

Our own work was supported by the National Institutes ofHealth/National Heart Lung and Blood Institute [grant numbers HL-55324, HL-61610], the American Heart Association [grant number13Post14500001] and the Albert Elizabeth Rekate Fund.

REFERENCES

1 Roger, V. L., Go, A. S., Lloyd-Jones, D. M., Adams, R. J., Berry,J. D., Brown, T. M., Carnethon, M. R., Dai, S., de Simone, G.,Ford, E. S. et al. (2011) Heart disease and stroke statistics –2011 update: a report from the American Heart Association.Circulation 123, e18–e209

2 Diwan, A. and Dorn, II, G. W. (2007) Decompensation of cardiachypertrophy: cellular mechanisms and novel therapeutic targets.Physiology 22, 56–64

3 Marelli, D., Desrosiers, C., el-Alfy, M., Kao, R. L. and Chiu, R. C.(1992) Cell transplantation for myocardial repair: anexperimental approach. Cell Transplant. 1, 383–390

4 Taylor, D. A., Atkins, B. Z., Hungspreugs, P., Jones, T. R., Reedy,M. C., Hutcheson, K. A., Glower, D. D. and Kraus, W. E. (1998)Regenerating functional myocardium: improved performanceafter skeletal myoblast transplantation. Nat. Med. 4, 929–933

5 Jeevanantham, V., Butler, M., Saad, A., Abdel-Latif, A.,Zuba-Surma, E. K. and Dawn, B. (2012) Adult bone marrow celltherapy improves survival and induces long-term improvement incardiac parameters: a systematic review and meta-analysis.Circulation 126, 551–568

6 Strauer, B. E. and Steinhoff, G. (2011) 10 years of intracoronaryand intramyocardial bone marrow stem cell therapy of the heart:from the methodological origin to clinical practice. J. Am. Coll.Cardiol. 58, 1095–1104

7 Vrtovec, B., Poglajen, G., Lezaic, L., Sever, M., Domanovic, D.,Cernelc, P., Socan, A., Schrepfer, S., Torre-Amione, G., Haddad, F.and Wu, J. C. (2013) Effects of intracoronary CD34+ stem celltransplantation in nonischemic dilated cardiomyopathy patients:5-year follow-up. Circ. Res. 112, 165–173

8 Leri, A., Kajstura, J. and Anversa, P. (2011) Role of cardiac stemcells in cardiac pathophysiology: a paradigm shift in humanmyocardial biology. Circ. Res. 109, 941–961

9 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 renewalin humans. Science 324, 98–102

10 Kajstura, J., Gurusamy, N., Ogorek, B., Goichberg, P.,Clavo-Rondon, C., Hosoda, T., D’Amario, D., Bardelli, S.,Beltrami, A. P., Cesselli, D. et al. (2010) Myocyte turnover in theaging human heart. Circ. Res. 107, 1374–1386

11 Kajstura, J., Rota, M., Cappetta, D., Ogorek, B., Arranto, C., Bai,Y., Ferreira-Martins, J., Signore, S., Sanada, F., Matsuda, A. et al.(2012) Cardiomyogenesis in the aging and failing human heart.Circulation 126, 1869–1881

12 Cesselli, D., Beltrami, A. P., Rigo, S., Bergamin, N., D’Aurizio, F.,Verardo, R., Piazza, S., Klaric, E., Fanin, R., Toffoletto, B. et al.(2009) Multipotent progenitor cells are present in humanperipheral blood. Circ. Res. 104, 1225–1234

13 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 myocytesdivide after myocardial infarction. N. Engl. J. Med. 344,1750–1757

14 Bearzi, C., Rota, M., Hosoda, T., Tillmanns, J., Nascimbene, A.,De Angelis, A., Yasuzawa-Amano, S., Trofimova, I., Siggins, R. W.,Lecapitaine, N. et al. (2007) Human cardiac stem cells. Proc.Natl. Acad. Sci. U.S.A. 104, 14068–14073

15 Anversa, P., Kajstura, J., Leri, A. and Bolli, R. (2006) Life anddeath of cardiac stem cells: a paradigm shift in cardiac biology.Circulation 113, 1451–1463

16 Kajstura, J., Urbanek, K., Perl, S., Hosoda, T., Zheng, H., Ogorek,B., Ferreira-Martins, J., Goichberg, P., Rondon-Clavo, C., Sanada,F. et al. (2010) Cardiomyogenesis in the adult human heart. Circ.Res. 107, 305–315

17 Raval, Z. and Losordo, D. W. (2012) On the fabric of the humanbody. Circulation 126, 1812–1814

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

19 Beltrami, A. P., Barlucchi, L., Torella, D., Baker, M., Limana, F.,Chimenti, S., Kasahara, H., Rota, M., Musso, E., Urbanek, K.et al. (2003) Adult cardiac stem cells are multipotent andsupport myocardial regeneration. Cell 114, 763–776

20 Urbanek, K., Cesselli, D., Rota, M., Nascimbene, A., De Angelis,A., Hosoda, T., Bearzi, C., Boni, A., Bolli, R., Kajstura, J. et al.(2006) Stem cell niches in the adult mouse heart. Proc. Natl.Acad. Sci. U.S.A. 103, 9226–9231

21 Oh, H., Bradfute, S. B., Gallardo, T. D., Nakamura, T., Gaussin,V., Mishina, Y., Pocius, J., Michael, L. H., Behringer, R. R., Garry,D. J. et al. (2003) Cardiac progenitor cells from adultmyocardium: homing, differentiation, and fusion after infarction.Proc. Natl. Acad. Sci. U.S.A. 100, 12313–12318

22 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+ cardioblasts enter fully differentiatedcardiomyocyte lineages. Nature 433, 647–653

23 Martin, C. M., Meeson, A. P., Robertson, S. M., Hawke, T. J.,Richardson, J. A., Bates, S., Goetsch, S. C., Gallardo, T. D. andGarry, D. J. (2004) Persistent expression of the ATP-bindingcassette transporter, Abcg2, identifies cardiac SP cells in thedeveloping and adult heart. Dev. Biol. 265, 262–275

www.clinsci.org 117


24 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 cardiomyocytes from within the activatedadult heart after injury. Nature 474, 640–644

25 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. (2012) Mammalian heart renewal by pre-existingcardiomyocytes. Nature 493, 433–436

26 Hsieh, P. C., Segers, V. F., Davis, M. E., MacGillivray, C., Gannon,J., Molkentin, J. D., Robbins, J. and Lee, R. T. (2007) Evidencefrom a genetic fate-mapping study that stem cells refresh adultmammalian cardiomyocytes after injury. Nat. Med. 13, 970–974

27 Urbanek, K., Rota, M., Cascapera, S., Bearzi, C., Nascimbene,A., De Angelis, A., Hosoda, T., Chimenti, S., Baker, M., Limana, F.et al. (2005) Cardiac stem cells possess growth factor-receptorsystems that after activation regenerate the infarctedmyocardium, improving ventricular function and long-termsurvival. Circ. Res. 97, 663–673

28 Orlic, D., Kajstura, J., Chimenti, S., Jakoniuk, I., Anderson, S. M.,Li, B., Pickel, J., McKay, R., Nadal-Ginard, B., Bodine, D. M. et al.(2001) Bone marrow cells regenerate infarcted myocardium.Nature 410, 701–705

29 Murry, C. E., Soonpaa, M. H., Reinecke, H., Nakajima, H.,Nakajima, H. O., Rubart, M., Pasumarthi, K. B., Virag, J. I.,Bartelmez, S. H., Poppa, V. et al. (2004) Haematopoietic stemcells do not transdifferentiate into cardiac myocytes inmyocardial infarcts. Nature 428, 664–668

30 Balsam, L. B., Wagers, A. J., Christensen, J. L., Kofidis, T.,Weissman, I. L. and Robbins, R. C. (2004) Haematopoietic stemcells adopt mature haematopoietic fates in ischaemicmyocardium. Nature 428, 668–673

31 Nygren, J. M., Jovinge, S., Breitbach, M., Sawen, P., Roll, W.,Hescheler, J., Taneera, J., Fleischmann, B. K. and Jacobsen,S. E. (2004) Bone marrow-derived hematopoietic cells generatecardiomyocytes at a low frequency through cell fusion, but nottransdifferentiation. Nat. Med. 10, 494–501

32 Tang, X. L., Rokosh, G., Sanganalmath, S. K., Yuan, F., Sato, H.,Mu, J., Dai, S., Li, C., Chen, N., Peng, Y. et al. (2010)Intracoronary administration of cardiac progenitor cells alleviatesleft ventricular dysfunction in rats with a 30-day-old infarction.Circulation 121, 293–305

33 Bolli, R., Chugh, A. R., D’Amario, D., Loughran, J. H., Stoddard,M. F., Ikram, S., Beache, G. M., Wagner, S. G., Leri, A., Hosoda,T. et al. (2011) Cardiac stem cells in patients with ischaemiccardiomyopathy (SCIPIO): initial results of a randomised phase 1trial. Lancet 378, 1847–1857

34 Chugh, A. R., Beache, G. M., Loughran, J. H., Mewton, N.,Elmore, J. B., Kajstura, J., Pappas, P., Tatooles, A., Stoddard,M. F. and Lima, J. A. et al. (2012) Administration of cardiac stemcells in patients with ischemic cardiomyopathy: The SCIPIO trial:surgical aspects and interim analysis of myocardial function andviability by magnetic resonance. Circulation 126, S54–64

35 Messina, E., De Angelis, L., Frati, G., Morrone, S., Chimenti, S.,Fiordaliso, F., Salio, M., Battaglia, M., Latronico, M. V., Coletta,M. et al. (2004) Isolation and expansion of adult cardiac stemcells from human and murine heart. Circ. Res. 95, 911–921

36 Smith, R. R., Barile, L., Cho, H. C., Leppo, M. K., Hare, J. M.,Messina, E., Giacomello, A., Abraham, M. R. and Marban, E.(2007) Regenerative potential of cardiosphere-derived cellsexpanded from percutaneous endomyocardial biopsy specimens.Circulation 115, 896–908

37 Li, T. S., Cheng, K., Lee, S. T., Matsushita, S., Davis, D.,Malliaras, K., Zhang, Y., Matsushita, N., Smith, R. R. andMarban, E. (2010) Cardiospheres recapitulate a niche-likemicroenvironment rich in stemness and cell-matrix interactions,rationalizing their enhanced functional potency for myocardialrepair. Stem Cells 28, 2088–2098

38 Johnston, P. V., Sasano, T., Mills, K., Evers, R., Lee, S. T., Smith,R. R., Lardo, A. C., Lai, S., Steenbergen, C., Gerstenblith, G.et al. (2009) Engraftment, differentiation, and functional benefitsof autologous cardiosphere-derived cells in porcine ischemiccardiomyopathy. Circulation 120, 1075–1083

39 Li, T. S., Cheng, K., Malliaras, K., Smith, R. R., Zhang, Y., Sun,B., Matsushita, N., Blusztajn, A., Terrovitis, J., Kusuoka, H. et al.(2012) Direct comparison of different stem cell types andsubpopulations reveals superior paracrine potency andmyocardial repair efficacy with cardiosphere-derived cells. J. Am.Coll. Cardiol. 59, 942–953

40 Makkar, R. R., Smith, R. R., Cheng, K., Malliaras, K., Thomson,L. E., Berman, D., Czer, L. S., Marban, L., Mendizabal, A.,Johnston, P. V. et al. (2012) Intracoronary cardiosphere-derivedcells for heart regeneration after myocardial infarction(CADUCEUS): a prospective, randomised phase 1 trial. Lancet379, 895–904

41 Friedenstein, A. J., Chailakhjan, R. K. and Lalykina, K. S. (1970)The development of fibroblast colonies in monolayer cultures ofguinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 3,393–403

42 Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K.,Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W.,Craig, S. and Marshak, D. R. (1999) Multilineage potential ofadult human mesenchymal stem cells. Science 284, 143–147

43 Xu, W., Zhang, X., Qian, H., Zhu, W., Sun, X., Hu, J., Zhou, H. andChen, Y. (2004) Mesenchymal stem cells from adult human bonemarrow differentiate into a cardiomyocyte phenotype in vitro. Exp.Biol. Med. 229, 623–631

44 Xu, M., Wani, M., Dai, Y. S., Wang, J., Yan, M., Ayub, A. andAshraf, M. (2004) Differentiation of bone marrow stromal cellsinto the cardiac phenotype requires intercellular communicationwith myocytes. Circulation 110, 2658–2665

45 Augello, A. and De Bari, C. (2010) The regulation ofdifferentiation in mesenchymal stem cells. Hum. Gene Ther. 21,1226–1238

46 Quevedo, H. C., Hatzistergos, K. E., Oskouei, B. N., Feigenbaum,G. S., Rodriguez, J. E., Valdes, D., Pattany, P. M., Zambrano, J. P.,Hu, Q., McNiece, I. et al. (2009) Allogeneic mesenchymal stemcells restore cardiac function in chronic ischemic cardiomyopathyvia trilineage differentiating capacity. Proc. Natl. Acad. Sci. U.S.A.106, 14022–14027

47 Makkar, R. R., Price, M. J., Lill, M., Frantzen, M., Takizawa, K.,Kleisli, T., Zheng, J., Kar, S., McClelan, R., Miyamota, T. et al.(2005) Intramyocardial injection of allogenic bone marrow-derivedmesenchymal stem cells without immunosuppression preservescardiac function in a porcine model of myocardial infarction. J.Cardiovasc. Pharmacol. Ther. 10, 225–233

48 Williams, A. R. and Hare, J. M. (2011) Mesenchymal stem cells:biology, pathophysiology, translational findings, and therapeuticimplications for cardiac disease. Circ. Res. 109, 923–940

49 Chong, J. J., Chandrakanthan, V., Xaymardan, M., Asli, N. S., Li,J., Ahmed, I., Heffernan, C., Menon, M. K., Scarlett, C. J.,Rashidianfar, A. et al. (2011) Adult cardiac-resident MSC-likestem cells with a proepicardial origin. Cell Stem Cell 9, 527–540

50 Hare, J. M., Traverse, J. H., Henry, T. D., Dib, N., Strumpf, R. K.,Schulman, S. P., Gerstenblith, G., DeMaria, A. N., Denktas, A. E.,Gammon, R. S. et al. (2009) A randomized, double-blind,placebo-controlled, dose-escalation study of intravenous adulthuman mesenchymal stem cells (prochymal) after acutemyocardial infarction. J. Am. Coll. Cardiol. 54, 2277–2286

51 Houtgraaf, J. H., den Dekker, W. K., van Dalen, B. M.,Springeling, T., de Jong, R., van Geuns, R. J., Geleijnse, M. L.,Fernandez-Aviles, F., Zijlsta, F., Serruys, P. W. and Duckers, H. J.(2012) First experience in humans using adipose tissue-derivedregenerative cells in the treatment of patients with ST-segmentelevation myocardial infarction. J. Am. Coll. Cardiol. 59, 539–540

52 Gnecchi, M., He, H., Noiseux, N., Liang, O. D., Zhang, L.,Morello, F., Mu, H., Melo, L. G., Pratt, R. E., Ingwall, J. S. andDzau, V. J. (2006) Evidence supporting paracrine hypothesis forAkt-modified mesenchymal stem cell-mediated cardiac protectionand functional improvement. FASEB J. 20, 661–669

53 Gnecchi, M., Zhang, Z., Ni, A. and Dzau, V. J. (2008) Paracrinemechanisms in adult stem cell signaling and therapy. Circ. Res.103, 1204–1219

54 Mirotsou, M., Jayawardena, T. M., Schmeckpeper, J., Gnecchi, M.and Dzau, V. J. (2011) Paracrine mechanisms of stem cellreparative and regenerative actions in the heart. J. Mol. Cell.Cardiol. 50, 280–289

55 Gnecchi, M., He, H., Liang, O. D., Melo, L. G., Morello, F., Mu, H.,Noiseux, N., Zhang, L., Pratt, R. E., Ingwall, J. S. and Dzau, V. J.(2005) Paracrine action accounts for marked protection ofischemic heart by Akt-modified mesenchymal stem cells. Nat.Med. 11, 367–368

56 Cho, H. J., Lee, N., Lee, J. Y., Choi, Y. J., Ii, M., Wecker, A., Jeong,J. O., Curry, C., Qin, G. and Yoon, Y. S. (2007) Role of hosttissues for sustained humoral effects after endothelialprogenitor cell transplantation into the ischemic heart. J. Exp.Med. 204, 3257–3269



57 Shabbir, A., Ziza, D., Suzuki, G. and Lee, T. (2009) Heart failuretherapy mediated by the trophic activities of bone marrowmesenchymal stem cells: a noninvasive therapeutic regimen.Am. J. Physiol. Heart Circ. Physiol. 296, H1888–H1897

58 Shabbir, A., Zisa, D., Lin, H., Mastri, M., Roloff, G., Suzuki, G.and Lee, T. (2010) Activation of host tissue trophic factorsthrough JAK-STAT3 signaling: a mechanism of mesenchymalstem cell-mediated cardiac repair. Am. J. Physiol. Heart Circ.Physiol. 299, H1428–H1438

59 Lim, H., Fallavollita, J. A., Hard, R., Kerr, C. W. and Canty, Jr, J. M.(1999) Profound apoptosis-mediated regional myocyte loss andcompensatory hypertrophy in pigs with hibernating myocardium.Circulation 100, 2380–2386

60 Suzuki, G., Iyer, V., Lee, T. C. and Canty, Jr, J. M. (2011)Autologous mesenchymal stem cells mobilize cKit+ andCD133+ bone marrow progenitor cells and improve regionalfunction in hibernating myocardium. Circ. Res. 109,1044–1054

61 Loffredo, F. S., Steinhauser, M. L., Gannon, J. and Lee, R. T.(2011) Bone marrow-derived cell therapy stimulates endogenouscardiomyocyte progenitors and promotes cardiac repair. CellStem Cell 8, 389–398

62 Hatzistergos, K. E., Quevedo, H., Oskouei, B. N., Hu, Q.,Feigenbaum, G. S., Margitich, I. S., Mazhari, R., Boyle, A. J.,Zambrano, J. P., Rodriguez, J. E. et al. (2010) Bone marrowmesenchymal stem cells stimulate cardiac stem cell proliferationand differentiation. Circ. Res. 107, 913–922

63 Chimenti, I., Smith, R. R., Li, T. S., Gerstenblith, G., Messina, E.,Giacomello, A. and Marban, E. (2010) Relative roles of directregeneration versus paracrine effects of humancardiosphere-derived cells transplanted into infarcted mice. Circ.Res. 106, 971–980

64 Mohsin, S., Siddiqi, S., Collins, B. and Sussman, M. A. (2011)Empowering adult stem cells for myocardial regeneration. Circ.Res. 109, 1415–1428

65 Tang, Y. L., Zhu, W., Cheng, M., Chen, L., Zhang, J., Sun, T.,Kishore, R., Phillips, M. I., Losordo, D. W. and Qin, G. (2009)Hypoxic preconditioning enhances the benefit of cardiacprogenitor cell therapy for treatment of myocardialinfarction by inducing CXCR4 expression. Circ. Res. 104,1209–1216

66 Haider, H., Jiang, S., Idris, N. M. and Ashraf, M. (2008)IGF-1-overexpressing mesenchymal stem cells accelerate bonemarrow stem cell mobilization via paracrine activation ofSDF-1α/CXCR4 signaling to promote myocardial repair. Circ. Res.103, 1300–1308

67 Padin-Iruegas, M. E., Misao, Y., Davis, M. E., Segers, V. F.,Esposito, G., Tokunou, T., Urbanek, K., Hosoda, T., Rota, M.,Anversa, P. et al. (2009) Cardiac progenitor cells and biotinylatedinsulin-like growth factor-1 nanofibers improve endogenous andexogenous myocardial regeneration after infarction. Circulation120, 876–887

68 Mohsin, S., Khan, M., Toko, H., Bailey, B., Cottage, C. T.,Wallach, K., Nag, D., Lee, A., Siddiqi, S., Lan, F. et al. (2012)Human cardiac progenitor cells engineered with pim-I kinaseenhance myocardial repair. J. Am. Coll. Cardiol. 60, 1278–1287

69 Dimmeler, S. and Leri, A. (2008) Aging and disease as modifiersof efficacy of cell therapy. Circ. Res. 102, 1319–1330

70 Malliaras, K., Li, T. S., Luthringer, D., Terrovitis, J., Cheng, K.,Chakravarty, T., Galang, G., Zhang, Y., Schoenhoff, F., Van Eyk, J.et al. (2012) Safety and efficacy of allogeneic cell therapy ininfarcted rats transplanted with mismatchedcardiosphere-derived cells. Circulation 125, 100–112

71 Zaruba, M. M., Soonpaa, M., Reuter, S. and Field, L. J. (2010)Cardiomyogenic potential of C-kit+ -expressing cells derived fromneonatal and adult mouse hearts. Circulation 121, 1992–2000

72 Jesty, S. A., Steffey, M. A., Lee, F. K., Breitbach, M., Hesse, M.,Reining, S., Lee, J. C., Doran, R. M., Nikitin, A. Y., Fleischmann,B. K. and Kotlikoff, M. I. (2012) c-kit+ precursors supportpostinfarction myogenesis in the neonatal, but not adult, heart.Proc. Natl. Acad. Sci. U.S.A. 109, 13380–13385

73 Porrello, E. R., Mahmoud, A. I., Simpson, E., Hill, J. A.,Richardson, J. A., Olson, E. N. and Sadek, H. A. (2011) Transientregenerative potential of the neonatal mouse heart. Science331, 1078–1080

74 Simpson, D. L., Mishra, R., Sharma, S., Goh, S. K., Deshmukh,S. and Kaushal, S. (2012) A strong regenerative ability of cardiacstem cells derived from neonatal hearts. Circulation 126,S46–S53

75 Burt, R. K., Chen, Y. H., Verda, L., Lucena, C., Navale, S.,Johnson, J., Han, X., Lomasney, J., Baker, J. M., Ngai, K. L. et al.(2012) Mitotically inactivated embryonic stem cells can beutilized as an in vivo feeder layer to nurse damaged myocardiumfollowing acute myocardial infarction: a pre-clinical study. Circ.Res. 111, 1286–1286

76 Suzuki, G., Leiker, M., Cimato, T. and Canty, Jr, J. M. (2011)Intracoronary infusion of cardiosphere-derived cells (icCDCs)improves cardiac function by stimulating myocyte proliferation innon-infarcted hibernating myocardium with no effect in normalmyocardium. Circulation 124, A8851

77 Suzuki, G., Iyer, V., Cimato, T. and Canty, Jr, J. M. (2009)Pravastatin improves function in hibernating myocardium bymobilizing CD133+ and cKit+ hematopoietic progenitor cellsand promoting myocytes to reenter the growth phase of thecardiac cell cycle. Circ. Res. 104, 255–264

78 Canty Jr, J. M. (2012) Coronary blood flow and myocardialischemia. In Braunwald’s Heart Disease (Bonow, R. O., Mann,D. L., Zipes, D. P. and Libby, P., eds), pp. 1049–1075, Elsevier,Philadelphia

79 Sawada, S., Elsner, G., Segar, D. S., O’Shaughnessy, M., Khouri,S., Foltz, J., Bourdillon, P. D., Bates, J. R., Fineberg, N., Ryan, T.et al. (1997) Evaluation of patterns of perfusion and metabolismin dobutamine-responsive myocardium. J. Am. Coll. Cardiol. 29,55–61

80 Melon, P. G., de Landsheere, C. M., Degueldre, C., Peters, J. L.,Kulbertus, H. E. and Pierard, L. A. (1997) Relation betweencontractile reserve and positron emission tomographic patternsof perfusion and glucose utilization in chronic ischemic leftventricular dysfunction: implications for identification ofmyocardial viability. J. Am. Coll. Cardiol. 30, 1651–1659

81 Dispersyn, G. D., Ramaekers, F. C. S. and Borgers, M. (2001)Clinical pathophysiology of hibernating myocardium. Coron.Artery Dis. 12, 381–385

82 Thomas, S. A., Fallavollita, J. A., Borgers, M. and Canty, Jr, J. M.(2002) Dissociation of regional adaptations to ischemia andglobal myolysis in an accelerated swine model of chronichibernating myocardium. Circ. Res. 91, 970–977

83 Gunning, M. G., Kaprielian, R. R., Pepper, J., Pennell, D. J.,Sheppard, M. N., Severs, N. J., Fox, K. M. and Underwood, S. R.(2002) The histology of viable and hibernating myocardium inrelation to imaging characteristics. J. Am. Coll. Cardiol. 39,428–435

84 Zhang, Y., Li, T. S., Lee, S. T., Wawrowsky, K. A., Cheng, K.,Galang, G., Malliaras, K., Abraham, M. R., Wang, C. and Marban,E. (2010) Dedifferentiation and proliferation of mammaliancardiomyocytes. PLoS ONE 5, e12559

Received 30 November 2012/31 January 2013; accepted 15 February 2013

Published on the Internet 12 April 2013, doi: 10.1042/CS20120641

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stem cell stimulation of endogenous myocyte regeneration

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