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Lancet 2012; 379: 933–42

See Comment page 877

This is the fi rst in a Series of two papers about stem cells

Cardiac Arrhythmia Service, Massachusetts General Hospital Heart Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA (L M Ptaszek MD, M Mansour MD, J N Ruskin MD); Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA, USA (L M Ptaszek, Prof K R Chien MD); and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA (L M Ptaszek, K R Chien)

Correspondence to:Prof Kenneth R Chien, Cardiovascular Research Center, Massachusetts General Hospital, 185 Cambridge Street, CPZN 3250, Boston, MA 02114, USAkchien@harvard.edu

Stem Cells 1

Towards regenerative therapy for cardiac diseaseLeon M Ptaszek, Moussa Mansour, Jeremy N Ruskin, Kenneth R Chien

Development of regenerative therapeutic strategies to reverse the progression of advanced heart failure is one of the most urgent clinical needs of this century. Insights gained from clinical trials of adult stem cells, together with fundamental scientifi c advances in cardiac stem cell and regenerative biology, are beginning to yield potential new targets and strategies for regenerative therapies. Of particular importance are new scientifi c discoveries related to intrinsic cardiac regeneration, renewal factors that can trigger regeneration, and tissue-engineering technology, which are beginning to change the way investigators view the scientifi c and clinical position of cardiovascular regenerative therapy.

IntroductionHeart failure is one of the key causes of morbidity and mortality worldwide.1 The eff ects of heart failure are growing very rapidly, especially in developing countries.2 Available medical and device-based therapies can ameli-orate the eff ects of heart failure, but cannot reverse the loss of functional myocardium, which is the underlying cause of the problem.3 The only available cure for advanced heart failure is orthotopic heart transplantation, which is not a viable strategy in the general population because of a relative absence of donor hearts.4 Heart failure is thus evolving into a global epidemic for which medicine does not have an available solution. Design of an effi cacious regenerative therapeutic strategy has therefore become a holy grail of modern cardiovascular science. Recent breakthroughs in stem cell biology provide large strides toward this goal. This report discusses key opportunities and challenges in movement from academic stem cell biology towards the development of cardiovascular regenerative therapy.

Attempted cardiac regeneration with non-cardiac progenitor cellsThe severity of heart failure and the inability of available treatments to abrogate its eff ects have spurred intense interest in cardiac regeneration. Several organs, notably the liver, have clinically relevant regeneration after injury.5 Although cardiac regeneration is reported in lower verte-brates such as amphibians and zebrafi sh, equivalent regenerative capacity is not possessed by adult mammals.6,7 Cardiomyocyte proliferation is present in neonatal mice, but diminishes rapidly after birth,8 and dividing cardio-myocytes are rare in the hearts of adult human beings.9,10 Cardiomyocyte turnover rate is about 1% per year in young adults, and decreases to 0·5% per year in elderly individuals.11 Turnover increases in response to injury, and higher numbers of immature, dividing cardiomyocytes are noted at infarct borders;12 however, the clinical course of heart failure shows that this pool of dividing cells cannot independently reverse the eff ects of a large insult.13

When development of cell-based regenerative therapies for heart failure was fi rst started, the belief was that

cardiac cell populations with regenerative potential, such as dividing cardiomyocytes and cardiac progenitors, were too rare and diffi cult to isolate to be of practical use. Motivated by the magnitude of the clinical issues that might be solved with cardiac regenerative therapy, several investigators chose to assess the regenerative capacity of non-cardiac progenitor cells rather than wait for characterisation of cardiac progenitors. This approach was based on the assertion that non-cardiac progenitor cells in the mesodermal lineage have suffi cient develop-mental plasticity to diff erentiate into cardiac cells if situated in the appropriate niche.14

A myocardial infarction can lead to loss of up to a billion cardiomyocytes, or about 25% of myocardial mass. Skeletal myoblasts, bone marrow cells, and peripheral blood stem cells were among the fi rst cell types investi-gated for cardiac regeneration, because they were plenti-ful and comparatively well characterised (fi gure 1). The fi rst studies in animals involving transplantation of skeletal myoblasts into infarcted myocardium showed improve ment in cardiac function after transplantation. This fi nding added credibility to assertions about the plasticity of non-cardiac progeni tors.15 The mechanism by which these cells produced the reported benefi t was not understood, but these data led to initiation of studies in human beings involving myoblasts, including the

Search strategy and selection criteria

We searched PubMed for published articles with the terms “cardiovascular disease”, “heart failure”, “cardiac regeneration”, “cardiac progenitor”, “stem cell”, and “stem cell therapy”. Most selected publications were published in the past 5 years. Older publications were included if they were well regarded or widely referenced. We also included references listed in articles identifi ed in the initial searches, and reports from governmental organisations. The date of the last search was Nov 30, 2011. The CADUCEUS trial39 was published when this Review was in galley form. Review articles pertaining to extensively covered topics were cited to accommodate space constraints.

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Non-cardiac progenitors

Key clinical issues:

Cardiac cells and putative cardiac progenitors

Limited durability, maturation of cells in situ• Ideal progenitor identified?

Complex three-dimensional structures not recreated• Recreation of three-dimensional cardiac structures?

Arrhythmias noted after cell injection• Electromechanical dissociation of engrafted cells?

Limited cell survival• Tissue milieu supportive of cell growth?• Immune rejection?

Limited delivery of cells to target area,haematogenous dissemination of cells• Efficiency of the delivery system sufficient?

Scalability• Clinically relevant number of therapeutic cells available?

2000 2004 2005 2008 2010 In progress In progress

Bone marrow:mesenchymal,endothelialstem cells

Peripheral blood: peripheral blood stem cells and G-CSF

Skeletal muscle:myoblasts

Adipose: mesenchymal stem cells

Cardiac muscle:C-Kit positive cells,cardiospheres

Induced pluripotentstem cells:cardiac progenitors,cardiomyocytes

?

Attempted identificationof an ideal cardiacprogenitor cell population

Revascularisation

Improvement of deliverytechnology

Figure 1: Milestones in clinical trials of cardiac regenerative therapiesEarly clinical trials aimed to discover a candidate cardiac regenerative therapy from readily available populations of non-cardiac progenitor cells. Modest improvements in cardiac function after myocardial infarction were reported with these cells, but improved knowledge of cardiac progenitor cells and the advent of induced pluripotent stem cell technology allowed investigation of the rarer cardiac cells with regenerative potential, including putative progenitor cells; however, clinical validation is needed. G-CSF=granulocyte colony-stimulating factor.

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multicentre, randomised myo blast autologous grafting in ischaemic cardiomyopathy (MAGIC) trial;16 however, studies of skeletal myoblasts in animals, which were started after MAGIC was in progress, raised several doubts about such grafting. Skeletal myoblasts became skeletal myocytes in the heart rather than cardiomyocytes, and engrafted skeletal myocytes were not electro-mechanically coupled with native myocardium.17 The results of the MAGIC trial reinforced these issues: patients treated with autologous myoblast injections had cardiac arrhythmias and no signifi cant improvement in ventricular contractile function.16

Early studies in animals suggested that bone marrow cells might have potential as cardiac regenerative therapeutics. Bone marrow cells injected into rodent hearts seemed at fi rst to diff erentiate into cardiomyocytes in situ.18 Studies of human recipients of orthotopic heart transplants reported that cells with the recipient’s geno-type were present in the transplanted heart.19,20 Many clinical trials followed these reports. Although injection with bone marrow cells was subse quently reported to be safe, the associated benefi t was variable. In some trials, benefi t was present, but short-lived.21 In other trials, no benefi t was noted.22,23 A meta-analysis reported the aggregate long-term outcome from 29 randomised control trials studying the treatment of acute myocardial infarction with either an intracoronary injection of bone marrow cells or intravenous injection of granulocyte colony-stimulating factor (G-CSF) to mobilise bone marrow cells.24 This analysis showed a small improve-ment in left ventricular ejection fraction after injection of bone marrow cells (~3%).24 No signifi cant benefi t was associated with treatment with G-CSF.

A small improvement in left ventricular function after injection of bone marrow cells was also noted in patients enrolled in the REPAIR-AMI25 and STAR-heart26 studies, the largest randomised trials of injection of bone marrow cells in the context of acute and chronic myocardial infarction, respectively. The STAR-heart study reported a statistically signifi cant decrease in mortality after injection of bone marrow cells compared with placebo. Notably, treatment with angiotensin-converting enzyme inhibitors and angiotensin receptor blockers after myocardial infarction is associated with mortality benefi t, even in the absence of a large improvement in left ventricular systolic function.27 This fi nding raises an important point about the criteria by which regenerative therapies are judged: full reversal of cardiomyocyte loss might not be necessary to change the natural course of heart failure. Large studies will be needed to further investigate the degree of mortality benefi t, and cost eff ectiveness, of treatment with bone marrow cells.

The small and inconsistent benefi t associated with treatment with bone marrow cells was initially attributed to ineffi cient delivery, because only 10% (or less) of delivered cells remained in the heart.28 Indeed, one meta-analysis suggested there might be a relation between

bone marrow cell dose and therapeutic eff ect.24 Animal experiments started after the fi rst wave of clinical trials proposed another issue: assumptions about plasticity of the bone marrow cells were incorrect.29 The reported change in cell morphology after injection into the heart, originally interpreted as transdiff erentiation, was actually caused by fusion of injected and native cells.30 Haemopoietic stem cells were also noted to adopt a mature blood cell phenotype in the heart.31

More recent data suggest that the clinically observed benefi t associated with injection of bone marrow cells is not the result of engraftment, but rather the release of paracrine factors.32 These fi ndings raise questions about the plasticity of other non-cardiac cell types proposed as candidate regenerative therapeutics, including adipose-derived stem cells.33 Clinical trials involving such cells are in progress, including phase 1 trials of adipose-derived stem cells (NCT00426868, NCT00442806) and a phase 2 trial of mesenchymal cells (NCT00721045).

Endogenous cardiac progenitor cellsAugmentation of endogenous regenerative activity is a compelling strategy for cardiac repair. In theory, such amplifi cation can be achieved with two distinct approaches. One approach would be to stimulate expan-sion of cardiomyocytes or putative cardiac progenitor cells with a drug or paracrine factor, in the same way that erythropoietin is given to stimulate bone marrow progenitor cells to produce erythrocytes. The second approach involves propagation of cardiac cells with regenerative potential ex vivo followed by implantation of these cells directly into an injured area.

Early attempts to identify stem cells in rodent hearts made use of several techniques and yielded various phenotypically distinct cell populations, generally termed cardiac progenitor cells.34,35 Several, but not all, of these populations express the c-Kit and Sca-1 proteins. Early studies suggested that transplantation of cells express-ing c-Kit into the heart could stimulate formation of new blood vessels and myocardium.36 Suspension culture of cells obtained from human myocardial biopsy samples yields spheroid cell clusters (termed cardio spheres) that contain a mixed population of c-Kit-expressing and Sca-1-expressing cells. It was reported that cardio -spheres could stimulate cardiac regeneration after infarction.37 These results led to the initiation of seve -ral phase 1 clinical trials involving heart-derived cells: ALCADIA (NCT00981006), SCIPIO (NCT00474461), and CADUCEUS (NCT00893360). Preliminary results from the SCIPIO and CADUCEUS trials were recently published.38–40 Both studies were designed to assess the feasibility and safety of intracoronary injection of auto-logous heart-derived cells after recent infarction. The SCIPIO study38 involved c-kit-expressing cells cultured from explanted atrial tissue, whereas the CADUCEUS study39 involved cardiospheres cultured from biopsy-obtained right ventricular tissue. Neither study showed a

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signifi cant increase in adverse events associated with cardiac cell injection, although more adverse events were noted in the treatment group of the CADUCEUS study. Both studies reported reduction in myocardial scar mass following cell treatment, but only the SCIPIO trial reported an improvement in left ventricular ejection fraction. The reported improvements should be viewed with caution, since the number of patients in the treatment arm of each study was small (16 in SCIPIO and 17 in CADUCEUS), and neither study included a placebo group because of the invasive nature of the treatment. Larger studies, powered to show clinically meaningful outcomes, will be needed to demonstrate the safety and effi cacy of these treatment strategies.

Substantial gaps remain in our knowledge about cardiac progenitor cells and the mechanisms by which they might promote regeneration. Although c-Kit-posi-tive cells are present at sites of cardiac injury, they do not have a known role in injury response.41 No consensus exists about phenotype defi nition or isolation technique of these populations, many of which have not been compared. Genetic fate mapping, a stringent scientifi c technique used to establish the derivatives of a pro-genitor cell population, suggests that c-Kit-positive cells and cardiospheres do not diff erentiate into cardio-myocytes.42,43 In mice, c-Kit-positive cells derived from the bone marrow augment cardiomyocyte renewal after infarction without direct transdiff erentiation.44 This eff ect might be attributable to the actions of paracrine factors, released by the injected cells (ie, c-kit-expressing cells or cardio spheres), on a distinct popu lation of endogenous cardiac cells.40

Genetic fate mapping and endogenous cardiac progenitor cellsThe search for cardiac cells and paracrine factors that are capable of triggering cardiac repair has been challenging. Further progress will need more precise defi nitions of the phenotypes and biological roles of relevant cell populations. Use of genetic fate mapping to study embryonic cardiogenesis has eased the defi nition of progenitor populations and helped establish their roles in forming of discrete cardiac structures.

The heart contains a complex array of structures, including muscle, valves, arteries, veins, and a conduction system (fi gure 2). Distinct cell types make up each structure. Most cells in the adult heart are derived from mesoderm and a few are derived from cardiac neural crest. Early in embryogenesis, mesodermal cells that are destined to become part of the heart segregate into two anatomically distinct groups, termed the fi rst and second heart fi elds.45 Cells in both heart fi elds express the Nkx2.5 protein.46 Genetic fate mapping studies in mice showed that progenitor cells in the second heart fi eld, which is marked by expression of the Isl-1 protein, give rise to cardiomyocytes in the right and left atria, the right ventricle, the outfl ow tract, the proximal coronary

arteries, and most of the conduction system.47 Cardiac neural crest progenitor cells, characterised by Pax3 expression, also contribute to growth of the outfl ow tract.48 Cells expressing Isl-1 can be diff erentiated into all cell lineages present in the heart.49 Knowledge of these cell populations helps investigators assess the molecular cues that establish cell fate decisions of progenitors in the second heart fi eld.50 Renewal of Isl-1-positive cells is induced by ligands of the Wnt–β-catenin pathway, suggesting that fate decisions are made at the individual-cell scale.51 Multipotent cardiac progenitors expressing ISL-1 have also been produced from human embryonic stem cells in vitro.50 Left ventricular cardiomyocytes are derived from the fi rst heart fi eld, for which a unique phenotype marker has not been identifi ed.52 Because most cases of heart failure are attributable to left ventricular failure, further characterisation of the fi rst heart fi eld is especially important.

Genetic fate mapping suggests that embryonic cardiogenesis proceeds according to a stem cell-based paradigm in which lineage-restricted progenitor cells give rise to the mosaic of cells present in the adult heart. After embryogenesis, mammalian cardiomyocytes seem to expand through symmetric division.8 An embryonic progenitor population that persists to adulthood might be involved in stimulation of cardiomyocyte division in the adult heart. This progenitor population, which is marked by expression of the Wt1 and Tbx18 proteins, resides primarily on the proepicardial surface during embryogenesis, and gives rise to fi broblasts, smooth muscle cells, and potentially cardiomyocytes, in all four heart chambers (fi gure 2).53 These Wt1-positive and Tbx18-positive cells persist in the epicardium of the adult heart and proliferate in response to myocardial injury, secreting trophic growth factors into the underlying myocardium.54,55 Activation of this cell population can also be primed with paracrine factors such as thymosin β4.56 This population might mediate the putative paracrine eff ects of cells studied in reported clinical trials, although this suggestion is unproven. Further investi gation into the mechanisms by which this epicardial population could be used to eff ect myocardial repair is warranted.

The promise of induced pluripotent stem cellsEven if the phenotypes of cardiac cells with regenerative potential can be better defi ned, their rarity might make it diffi cult to generate enough cells to produce a clinically meaningful eff ect. Embryonic stem cells are an attractive source of starting material for cell-based therapies, mainly because they are self-renewing pluri-potent cells that can be diff erentiated into tissues from all three germ layers.57 Cardiomyocytes produced from mouse embryonic stem cells in vitro are closer to the fetal phenotype than to the adult phenotype, but do show electromechanical coupling with native cardio-myocytes;58,59 however, orthotopic embryonic stem cells

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Mesoderm(Bry positive)

Right ventricle

Rightatrium

Leftatrium

Aorta

Left ventricle

Cardiogenicmesoderm

(MesP1 positive)

Cardiovascularprogenitor

(?)

Multipotentprogenitor

(Isl-1 positive,Nkx2·5 positive,

Flk1 positive)

Multipotentprogenitor

(?)

Pro-epicardialprogenitor

Epicardial progenitor(Isl-1 negative, Wt1 positive,

Tbx18 positive)

Epicardial progenitor lineage

Second heart field

First heart field

Endothelium

Endothelium

Conductionsystem

(Isl-1 negative,Hcn4 positive)

Smooth muscle Cardiac muscle

Cardiac muscle

Smooth muscle

Endothelium Smooth muscle ?

?

Cardiac muscle

Bipotentprogenitor

(Isl-1 positive,Nkx2·5 positive)

Bipotent progenitor(Isl-1 negative,

Nkx2·5 positive,Tbx5 positive)

Ventricularprogenitor

(Isl-1 positive,Nkx2·5 positive,

cTnT positive)

Endothelium

Cardiac muscle

Smooth muscle

Human(patient-specific)

skin fibroblasts

Embryonic stem cells Multipotentcardiac

progenitor(Isl-1 positive)

Patient-specificinduced pluripotent

stem cells

Mouseembryonicfibroblasts

In-vitrotransdifferentiation:+Klf4+Oct4+Sox2

In-vitroreprogramming:+KLF4+OCT4+SOX2+c-Myc

Unstableintermediate

In-vitrodifferentiation

A

B

Pulmonary artery

Figure 2: Cardiac progenitor cell populations and their contributions to cardiac structures in the adult heart(A) Cardiac structures develop from embryonic progenitor cell populations in the fi rst heart fi eld (shaded green), the second heart fi eld (shaded red), both heart fi elds (shaded purple), or epicardial progenitor lineage cells (shaded blue). Neural crest cells also contribute to the great vessels and outfl ow tract. Isl-1 expression marks the multipotent progenitors of cardiomyocytes in the second heart fi eld, and other protein expression profi les mark downstream progenitors and diff erentiated tissue types. (B) Induced pluripotent stem cell technology. In-vitro reprogramming of terminally diff erentiated fi broblasts with KLF4, OCT4, SOX2, and C-MYC leads to a pluripotent phenotype analogous to an embryonic stem cell, potentially allowing future diff erentiation. Although reliable, such reprogramming is labour-intensive and slow. An alternative technique (shown beneath the dashed line) uses Klf4, Oct4, and Sox2 to reprogramme terminally diff erentiated embryonic fi broblasts into a usable intermediary (but non-pluripotent) cell that can be diff erentiated into cardiomyocyte phenotype in less than half the time of pluripotent stem cell technique.

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are probably not suitable for clinical application, because of ethical worries, potential genetic instability, and requisite immunosuppression therapy. Takahashi and Yamanaka60 recently designed a technique for in-vitro reprogramming of terminally diff erentiated cells, such as skin fi broblasts, into pluripotent cells that closely resemble embryonic stem cells. These reprogrammed cells, termed induced pluripotent stem cells, can be derived from individual patients. Cardiac cells can then be produced from induced pluripotent stem cells in vitro. This technique makes possible autologous cell trans plantation, with a theoretical reduction in risk of immune rejection.61

Several issues with induced pluripotent stem cell technology need to be resolved before clinical use is possible. The originally reported method for production of such cells used integration of viruses to express four factors that drive reprogramming (fi gure 2). Because of the risk of malignant transformation associated with integrating viruses, an alternative method is needed,62 for which several candidates have been reported.63,64 Another issue is that of acquisition of the source cell. Induced pluripotent stem cells retain epigenetic memory of the cell type from which they were derived. Thus, cardio myocytes can more readily be produced from induced pluripotent stem cells derived from the ventricular myocyte than from those derived from fi broblast tissue.65 This feature emphasises the subtle, but important, diff erences between induced pluripotent stem cells and embryonic stem cells. Before provision of induced pluripotent stem cell-derived cardiac cells to a patient, institution of a reliable method for removal of any pluripotent cells is needed.66 Further-more, immuno suppressive therapy might also be neces-sary, because induced pluripotent stem cells can elicit an immune response when transplanted between genetically identical mice.67

Diffi culties encountered with induced pluripotent stem cells inspired the development of methods for turning fi broblasts directly into cardiomyocytes without regression to a pluripotent state. The operational assumption in this approach is that travelling a shorter developmental distance might help avoid the issues associated with pluripotent cells.68,69 Direct transdiff erentiation of mouse embryonic fi broblasts into cardiomyocytes without transit through a pluripotent intermediate has been reported (fi gure 2).69 This strategy has issues of its own; for example, cardiomyocytes produced in this way express only the atrial isoform of myosin.

Paracrine factors in cardiac regenerationParacrine factors, notably those in the renin–angiotensin system, have key roles in cardiac pathophysiological mechanisms.70 The benefi t associated with bone marrow cell therapy might be attributable to paracrine factors, but neither the identity nor the actions of these putative factors are known. Several possible activities have been proposed, including activation of putative endogenous

cardiac progenitor cells, direct stimulation of cardio-myocyte division, and modifi cation of the tissue niche (ie, increase neovascularisation and reduce scar burden).58 Use of paracrine factors to mobilise cardiac cells with regenerative potential is a compelling treat-ment strategy, especially because it is associated with a potentially smaller risk of tissue disruption than is cell injection.

Growth factors investigated as candidate cardiac therapies fall into several functional categories, such as those that promote angiogenesis (eg, vascular endo-thelial growth factor, fi broblast growth factor, and stromal cell derived factor 1), inhibit apoptosis (eg, hepatocyte growth factor and platelet-derived growth factor), mobilise progenitor cells (eg, G-CSF), and stimulate myocyte proliferation (eg, periostin and neuregulin). The potential benefi t of growth factor infusion, either in combination with cell infusion or as standalone therapy, is under investigation, and various preclinical studies of growth factor infusion after myo-cardial infarction have been undertaken.71 Paracrine factors that inhibit infl ammation might reduce scar formation and thus improve the suitability of the niche for cardiomyocyte growth; however, reduction in fi bro-sis could destabilise myocardium after infarction and increase the risk of rupture.

Delivery of regenerative therapyA safe, eff ective, and practical delivery system is crucial to the success of cardiac regenerative therapy. Such a platform would have to ensure reliable delivery of a suffi cient amount of the therapeutics to trigger regen-eration, have good visualisation of the target area, and provide specifi c delivery to the target area with minimal off -target delivery and little or no risk of haematogenous dissemination.72 Several delivery techniques have been reported, ranging from direct intramyocardial injection in the context of open-chest surgery to various intra-vascular, catheter-based methods (fi gure 3).

Perhaps the most feared long-term outcome of the haematogenous dissemination of progenitor cells is metastatic tumour formation. Intramyocardial injection from the epicardial approach is probably associated with a lower risk of haematogenous spread than is injection from the intravascular approach. Most areas of the heart are accessible from the epicardial surface, apart from the interventricular and interatrial septa. Administration of regenerative therapies via the epicardial approach was fi rst undertaken during open-chest surgery. The procedural morbidity of the open-chest approach can off set the decrease in risk of haematogenous dissem-ination. Use of this approach is therefore diffi cult to justify unless another cardiac surgical procedure, such as coronary artery bypass grafting, is indicated.73

Morbidity associated with the open-chest approach motivated investigators to assess less invasive, catheter-based techniques. A percutaneous transarterial approach

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has been used for both intramyocardial and intra-coronary injections. Nonetheless, a decrease in proced-ural morbidity is off set by an increase in the risk of haemato genous dissemination and an inability to directly visualise the aff ected area. Intravascular delivery of therapeutics is ineffi cient, because the injected cells or growth factors need to move through the vessel wall. Moreover, the targeted area of myocardium is not accessible via the transarterial approach if the artery serving that region is occluded.

Delivery of regenerative therapeutics via a percutaneous transvenous approach has been proposed. Intro duction of such therapy via a peripheral intravenous catheter is associated with minimal procedural morbidity, but is not favoured, because of poor delivery effi ciency and broad dissemination, especially to the lungs.74 Epicardial coronary vein delivery is an alternative to coronary arterial delivery that has the potential to be especially useful if the coronary arteries serving the aff ected territory are occluded. Epicardial venous ana tomy is variable, so access to the aff ected territory is not guaranteed. Drawbacks of this approach include

ineffi cient delivery (injected material has to cross the venous wall) and haematogenous spread to the lungs (the coronary sinus drains into the right heart). Infl ation of an angioplasty balloon in the coronary sinus and delivery of regenerative therapies under pressure has been attempted, but is not proven to be more effi cient than the transarterial approach.75

Development of videoscopic catheter technology, which allows the operator to view internal anatomy, has eased the development of minimally invasive strategies for delivery of regenerative therapy to the epicardial surf-ace of the heart. An early version of this technology, involving insertion of catheters into the pericardial space through small incisions in the subxiphoid region, has been reported.76 This percutaneous catheter-based sys -tem provides the advantages of the epicardial approach (ie, direct visualisation and a reduced risk of haemato-genous dissemination compared with intravascular injec-tion) without the morbidity of open-chest surgery.

All reported cardiac-injection techniques are associated with low retention of delivered cells. Retention is higher with intramyocardial injection than with intravascular

Open-chest epicardial approach:intramyocardial injection

Percutaneous transvenous injection

Intracoronary

Peripheralvein

Coronary sinus

Myocardium,endocardial surface

Percutaneous transarterial approach

Percutaneous epicardial approach:intramyocardial injection

Morbidity of open-chest procedure ledto investigation of less morbidpercutaneous approaches

Advantages• Direct visualisation of affected area• Low risk of trauma to epicardial vessels• Potentially reduced risk of haematogenous spread of delivered therapeutic

Disadvantage• High morbidity of sternotomy or thoracotomy

Advantages• Procedural morbidity lower than open-chest approach• Specific injection to affected territory• Potentially reduced risk of haematogenous dissemination

Disadvantage• Adaptation of existing catheter technology required

Advantage• Low morbidity associated with percutaneous venous access

Disadvantages• Low efficiency of delivery to target area• Dissemination of delivered therapeutic in venous system

Advantage• Low morbidity associated with percutaneous arterial access

Disadvantages• High risk of systemic haematogenous dissemination of delivered therapy• Intracoronary approach might be impossible due to atherosclerosis• Intramyocardial injection might allow access to areas served by occluded coronary arteries but targeting can be technically challenging

An ideal delivery system would combineadvantages of open-chest approach(specific delivery) and percutaneousapproach (low procedural morbidity)

Figure 3: Delivery systems for cardiac regenerative therapies

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injection, but is poor nonetheless. Indeed, a cell reten-tion in pigs with intramyocardial injection of 11% has been reported, which was better than the 2·6% reported with coronary arterial injection or 3·2% with coronary sinus injection.77 Hydrogels designed to solidify at the time of injection can improve cell retention and engraftment.58 Studies in animals suggest that injection of hydrogel formulations alone can improve repair after infarction.78 Catheter compatibility is a key issue with biopolymer-based hydrogels, which solidify rapidly and therefore tend to do so within the delivery catheter. This issue will need to be addressed before clinical application is practical.

Cardiac tissue engineeringSuccessful reconstitution of damaged cardiac tissue cannot rely solely on regeneration of cardiomyocytes because the architecture of myocardium is complex. Cardiomyocytes in the adult heart are oriented end-to-end in fi bres, which are woven into anisotropically oriented sheets whose organisation forms the basis for chamber contraction. Among these sheets are complex webs of fi broblasts, blood vessels, and conduction-system tissue. Spatial organisation of these cells during embryogenesis is thought to involve signals between cells and from the extracellular matrix that make up the tissue niche. In the adult, this niche is thought to be crucial to cardiac repair. Consistent with this notion are the reports that embryonic stem cell-derived cardio-myocytes engraft more effi ciently into native myocardium than into scar tissue.58

Perhaps the most striking example of the importance of extracellular matrix to survival of injected cells was a study79 in which a whole rat heart was decellularised and then repopulated with neonatal cardiomyocytes, resulting in a beating heart. Application of biopolymer patches, impregnated with growth factors and progenitor cells, to the epicardial surface of myocardial scar stimulates neovascularisation and decreases scar size.80 Bioactive polymer technology makes possible the creation of patches that selectively release growth factors in response to matrix metalloproteinases expressed at the site of myocardial infarction.81

One of the main drawbacks of biopolymer-based technology is the potential for adverse host reaction to biomaterials. This issue led to the development of scaff old-free tissue-engineering technology. These so-called second-generation patches are made up only of cells and the extracellular matrix that these cells secrete. One new patch contains only human cardiomyocytes, endothelial cells, and stromal cells.82 In a rat model, vessels in such patches form eff ective anastomoses with native vessels, and patch cardiomyocytes are electro-mechanically associated with adjoining myocardium. Cardiac tissue-engineering technology is a crucial complement to the study of cardiac progenitor popu-lations. Progress in cardiac tissue engineering in the

past few years has been very rapid, but these techniques are still at an early stage. Preparation of engineered tissue remains a very labour-intensive process that will need to be streamlined before large-scale clinical application is practical.

PerspectivesThe heart is made up of a complex mosaic of distinct anatomical elements that are substantially disrupted in cardiac injury. Because of this complexity, restoration of cardiac function would need recreation of the native architecture of the heart, not just regeneration of one cell type. An ideal cardiac regenerative therapy would possess a key cell and paracrine factor combination, a cardiac tissue niche optimised to enhance cell engraft-ment and diff erentiation, and a safe, minimally invasive delivery procedure that introduces the regen erative therapy specifi cally to the aff ected area with the least risk of acute and long-term side eff ects. Because of these distinct challenges, the design elements of a clinically meaningful regenerative therapy strategy for advanced heart failure will probably reside at the intersection of the discrete arenas of stem cell biology, tissue engin-eering, trans plantation, grafting, rejection biology, clinical cardio vascular medicine, and device technology. Further more, creation of inter disciplinary teams, includ-ing partnerships between academia and the private sector, will probably be needed. Physician–scientists who have world-class clinical train ing in cardiovascular medicine, together with state-of-the-art knowledge of stem cell and regenerative biology, will be key members of these teams. Regenerative therapy in cardiac disease is in an important phase: breaking down of traditional barriers between individual areas of special isation will be challenging, but necessary, if we are to move beyond stem cell biology towards the development of true cardiovascular regenerative therapy. To foster this sort of multidisciplinary collaboration, a parallel regener ative eff ort to change the existing academic culture and environment might be necessary. The fate of the next generation of leading physician–scientists in the specialty, and the fate of patients with heart failure, will probably hinge on the outcome.ContributorsLMP and KRC did the literature search, wrote the report, and drew the fi gures. MM and JNR provided guidance about the content of the text and fi gures.

Confl icts of interestWe declare that we have no confl icts of interest.

AcknowledgmentsWe apologise to those of our colleagues whose work could not be cited because of constraints on space. LMP thanks the de Gunzburg Family Foundation and the Deane Institute for Integrative Research in Atrial Fibrillation and Stroke at the Massachusetts General Hospital (Boston, MA, USA) for their support.

References1 Bui AL, Horwich TB, Fonarow GC. Epidemiology and risk profi le

of heart failure. Nat Rev Cardiol 2011; 8: 30–41.

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2 Gaziano TA. Reducing the growing burden of cardiovascular disease in the developing world. Health Aff (Millwood) 2007; 26: 13–24.

3 Whelan RS, Kaplinskiy V, Kitsis RN. Cell death in the pathogenesis of heart disease: mechanisms and signifi cance. Annu Rev Physiol 2010; 72: 19–44.

4 Stehlik J, Edwards LB, Kucheryavaya AY, et al. The Registry of the International Society for Heart and Lung Transplantation: twenty-seventh offi cial adult heart transplant report—2011. J Heart Lung Transplant 2011; 30: 1078–94.

5 Taub R. Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Biol 2004; 5: 836–47.

6 Neff AW, Dent AE, Armstrong JB. Heart development and regeneration in urodeles. Int J Dev Biol 1996; 40: 719–25.

7 Poss KD, Wilson LG, Keating MT. Heart regeneration in zebrafi sh. Science 2002; 298: 2188–90.

8 Porrello ER, Mahmoud AI, Simpson E, et al. Transient regenerative potential of the neonatal mouse heart. Science 2011; 331: 1078–80.

9 Soonpaa MH, Field LJ. Survey of studies examining mammalian cardiomyocyte DNA synthesis. Circ Res 1998; 83: 15–26.

10 Bergmann O, Zdunek S, Alkass K, Druid H, Bernard S, Frisén J. Identifi cation of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover. Exp Cell Res 2011; 317: 188–94.

11 Bergmann O, Bhardwaj RD, Bernard S, et al. Evidence for cardiomyocyte renewal in humans. Science 2009; 324: 98–102.

12 Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med 2001; 344: 1750–57.

13 Hsieh PC, Segers VF, Davis ME, et al. Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med 2007; 13: 970–74.

14 Morrison SJ. Stem cell potential: can anything make anything? Curr Biol 2001; 11: R7–9.

15 Taylor DA, Atkins BZ, Hungspreugs P, et al. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med 1998; 4: 929–33.

16 Menasché P, Alfi eri O, Janssens S, et al. The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) trial: fi rst randomized placebo-controlled study of myoblast transplantation. Circulation 2008; 117: 1189–200.

17 Reinecke H, Poppa V, Murry CE. Skeletal muscle stem cells do not transdiff erentiate into cardiomyocytes after cardiac grafting. J Mol Cell Cardiol 2002; 34: 241–49.

18 Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410: 701–05.

19 Quaini F, Urbanek K, Beltrami AP, et al. Chimerism of the transplanted heart. N Engl J Med 2002; 346: 5–15.

20 Deb A, Wang S, Skelding KA, Miller D, Simper D, Caplice NM. Bone marrow-derived cardiomyocytes are present in adult human heart: a study of gender-mismatched bone marrow transplantation patients. Circulation 2003; 107: 1247–49.

21 Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 2004; 364: 141–48.

22 Lunde K, Solheim S, Aakhus S, et al. Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction. N Engl J Med 2006; 355: 1199–209.

23 Janssens S, Dubois C, Bogaert J, et al. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial. Lancet 2006; 367: 113–21.

24 Zimmet H, Porapakkham P, Porapakkham P, et al. Short- and long-term outcomes of intracoronary and endogenously mobilized bone marrow stem cells in the treatment of ST-segment elevation myocardial infarction: a meta-analysis of randomized control trials. Eur J Heart Fail 2011; 14: 91–105.

25 Assmus B, Rolf A, Erbs S, et al, and the REPAIR-AMI Investigators. Clinical outcome 2 years after intracoronary administration of bone marrow-derived progenitor cells in acute myocardial infarction. Circ Heart Fail 2010; 3: 89–96.

26 Strauer BE, Yousef M, Schannwell CM. The acute and long-term eff ects of intracoronary Stem cell Transplantation in 191 patients with chronic heARt failure: the STAR-heart study. Eur J Heart Fail 2010; 12: 721–29.

27 Solomon SD, Skali H, Anavekar NS, et al. Changes in ventricular size and function in patients treated with valsartan, captopril, or both after myocardial infarction. Circulation 2005; 111: 3411–19.

28 Lafl amme MA, Murry CE. Regenerating the heart. Nat Biotechnol 2005; 23: 845–56.

29 Murry CE, Soonpaa MH, Reinecke H, et al. Haematopoietic stem cells do not transdiff erentiate into cardiac myocytes in myocardial infarcts. Nature 2004; 428: 664–68.

30 Terada N, Hamazaki T, Oka M, et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002; 416: 542–45.

31 Balsam LB, Wagers AJ, Christensen JL, Kofi dis T, Weissman IL, Robbins RC. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 2004; 428: 668–73.

32 Mirotsou M, Jayawardena TM, Schmeckpeper J, Gnecchi M, Dzau VJ. Paracrine mechanisms of stem cell reparative and regenerative actions in the heart. J Mol Cell Cardiol 2011; 50: 280–89.

33 Bai X, Yan Y, Song YH, et al. Both cultured and freshly isolated adipose tissue-derived stem cells enhance cardiac function after acute myocardial infarction. Eur Heart J 2010; 31: 489–501.

34 Lafl amme MA, Murry CE. Heart regeneration. Nature 2011; 473: 326–35.

35 Oh H, Bradfute SB, Gallardo TD, et al. Cardiac progenitor cells from adult myocardium: homing, diff erentiation, and fusion after infarction. Proc Natl Acad Sci USA 2003; 100: 12313–18.

36 Tang XL, Rokosh G, Sanganalmath SK, et al. Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction. Circulation 2010; 121: 293–305.

37 Chimenti I, Smith RR, Li TS, et al. Relative roles of direct regeneration versus paracrine eff ects of human cardiosphere-derived cells transplanted into infarcted mice. Circ Res 2010; 106: 971–80.

38 Bolli R, Chugh AR, D’Amario D, et al. Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial. Lancet 2011; 378: 1847–57.

39 Makkar RR, Smith RR, Cheng K, et al. Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. Lancet 2012; published online Feb 14. DOI:10.1016/S0140-6736(12)60195-0.

40 Siu C-W, Tse H-T. Cardiac regeneration: messages from CADUCEUS. Lancet 2012; published online Feb 14. DOI:10.1016/S0140-6736(12)60236-0.

41 Kubo H, Jaleel N, Kumarapeli A, et al. Increased cardiac myocyte progenitors in failing human hearts. Circulation 2008; 118: 649–57.

42 Zaruba MM, Soonpaa M, Reuter S, Field LJ. Cardiomyogenic potential of C-kit(+)-expressing cells derived from neonatal and adult mouse hearts. Circulation 2010; 121: 1992–2000.

43 Andersen DC, Andersen P, Schneider M, Jensen HB, Sheikh SP. Murine “cardiospheres” are not a source of stem cells with cardiomyogenic potential. Stem Cells 2009; 27: 1571–81.

44 Loff redo FS, Steinhauser ML, Gannon J, Lee RT. Bone marrow-derived cell therapy stimulates endogenous cardiomyocyte progenitors and promotes cardiac repair. Cell Stem Cell 2011; 8: 389–98.

45 Chien KR, Domian IJ, Parker KK. Cardiogenesis and the complex biology of regenerative cardiovascular medicine. Science 2008; 322: 1494–97.

46 Wu SM, Fujiwara Y, Cibulsky SM, et al. Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart. Cell 2006; 127: 1137–50.

47 Laugwitz KL, Moretti A, Lam J, et al. Postnatal isl1+ cardioblasts enter fully diff erentiated cardiomyocyte lineages. Nature 2005; 433: 647–53.

48 Stoller JZ, Epstein JA. Cardiac neural crest. Semin Cell Dev Biol 2005; 16: 704–15.

49 Moretti A, Caron L, Nakano A, et al. Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversifi cation. Cell 2006; 127: 1151–65.

50 Bu L, Jiang X, Martin-Puig S, et al. Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages. Nature 2009; 460: 113–17.

Series

942 www.thelancet.com Vol 379 March 10, 2012

51 Qyang Y, Martin-Puig S, Chiravuri M, et al. The renewal and diff erentiation of Isl1+ cardiovascular progenitors are controlled by a Wnt/beta-catenin pathway. Cell Stem Cell 2007; 1: 165–79.

52 Buckingham M, Meilhac S, Zaff ran S. Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet 2005; 6: 826–35.

53 Zhou B, Ma Q, Rajagopal S, et al. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 2008; 454: 109–13.

54 Smart N, Bollini S, Dubé KN, et al. De novo cardiomyocytes from within the activated adult heart after injury. Nature 2011; 474: 640–44.

55 Zhou B, Honor LB, He H, et al. Adult mouse epicardium modulates myocardial injury by secreting paracrine factors. J Clin Invest 2011; 121: 1894–904.

56 Smart N, Risebro CA, Melville AA, et al. Thymosin beta4 induces adult epicardial progenitor mobilization and neovascularization. Nature 2007; 445: 177–82.

57 Mummery C, Ward-van Oostwaard D, Doevendans P, et al. Diff erentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 2003; 107: 2733–40.

58 Lafl amme MA, Chen KY, Naumova AV, et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol 2007; 25: 1015–24.

59 van Laake LW, Passier R, Monshouwer-Kloots J, et al. Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction. Stem Cell Res (Amst) 2007; 1: 9–24.

60 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fi broblast cultures by defi ned factors. Cell 2006; 126: 663–76.

61 Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fi broblasts by defi ned factors. Cell 2007; 131: 861–72.

62 Ott MG, Schmidt M, Schwarzwaelder K, et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 2006; 12: 401–09.

63 Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 2009; 458: 771–75.

64 Warren L, Manos PD, Ahfeldt T, et al. Highly effi cient reprogramming to pluripotency and directed diff erentiation of human cells with synthetic modifi ed mRNA. Cell Stem Cell 2010; 7: 618–30.

65 Xu H, Yi BA, Wu H, et al. Highly effi cient derivation of ventricular cardiomyocytes from induced pluripotent stem cells with a distinct epigenetic signature. Cell Res 2012; 22: 142–54.

66 Passier R, van Laake LW, Mummery CL. Stem-cell-based therapy and lessons from the heart. Nature 2008; 453: 322–29.

67 Zhao T, Zhang ZN, Rong Z, Xu Y. Immunogenicity of induced pluripotent stem cells. Nature 2011; 474: 212–15.

68 Ieda M, Fu JD, Delgado-Olguin PV, et al. Direct reprogramming of fi broblasts into functional cardiomyocytes by defi ned factors. Cell 2010; 142: 375–86.

69 Efe JA, Hilcove S, Kim J, et al. Conversion of mouse fi broblasts into cardiomyocytes using a direct reprogramming strategy. Nat Cell Biol 2011; 13: 215–22.

70 Sun Y. Intracardiac renin-angiotensin system and myocardial repair/remodeling following infarction. J Mol Cell Cardiol 2010; 48: 483–89.

71 Segers VF, Lee RT. Protein therapeutics for cardiac regeneration after myocardial infarction. J Cardiovasc Transl Res 2010; 3: 469–77.

72 Gersh BJ, Simari RD, Behfar A, Terzic CM, Terzic A. Cardiac cell repair therapy: a clinical perspective. Mayo Clin Proc 2009; 84: 876–92.

73 Pompilio G, Steinhoff GL, Liebold A, et al. Direct minimally invasive intramyocardial injection of bone marrow-derived AC133+ stem cells in patients with refractory ischemia: preliminary results. Thorac Cardiovasc Surg 2008; 56: 71–76.

74 Barbash IM, Chouraqui P, Baron J, et al. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. Circulation 2003; 108: 863–68.

75 Raake P, von Degenfeld G, Hinkel R, et al. Myocardial gene transfer by selective pressure-regulated retroinfusion of coronary veins: comparison with surgical and percutaneous intramyocardial gene delivery. J Am Coll Cardiol 2004; 44: 1124–29.

76 Kimura T, Miyoshi S, Takatsuki S, et al. Safety and effi cacy of pericardial endoscopy by percutaneous subxyphoid approach in swine heart in vivo. J Thorac Cardiovasc Surg 2011; 142: 181–90.

77 Hou D, Youssef EA, Brinton TJ, et al. Radiolabeled cell distribution after intramyocardial, intracoronary, and interstitial retrograde coronary venous delivery: implications for current clinical trials. Circulation 2005; 112 (suppl): I150–56.

78 Leor J, Tuvia S, Guetta V, et al. Intracoronary injection of in situ forming alginate hydrogel reverses left ventricular remodeling after myocardial infarction in Swine. J Am Coll Cardiol 2009; 54: 1014–23.

79 Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: using nature’s platform to engineer a bioartifi cial heart. Nat Med 2008; 14: 213–21.

80 Frederick JR, Fitzpatrick JR 3rd, McCormick RC, et al. Stromal cell-derived factor-1alpha activation of tissue-engineered endothelial progenitor cell matrix enhances ventricular function after myocardial infarction by inducing neovasculogenesis. Circulation 2010; 122 (suppl): S107–17.

81 Kraehenbuehl TP, Ferreira LS, Hayward AM, et al. Human embryonic stem cell-derived microvascular grafts for cardiac tissue preservation after myocardial infarction. Biomaterials 2011; 32: 1102–09.

82 Stevens KR, Kreutziger KL, Dupras SK, et al. Physiological function and transplantation of scaff old-free and vascularized human cardiac muscle tissue. Proc Natl Acad Sci USA 2009; 106: 16568–73.

Towards regenerative therapy for cardiac diseaseIntroductionAttempted cardiac regeneration with non-cardiac progenitor cellsEndogenous cardiac progenitor cellsGenetic fate mapping and endogenous cardiac progenitor cellsThe promise of induced pluripotent stem cellsParacrine factors in cardiac regenerationDelivery of regenerative therapyCardiac tissue engineeringPerspectivesAcknowledgmentsReferences