European Heart Journal Supplements (2006) 8 (Supplement E), E43E54 doi:10.

1093/eurheartj/sul031

Stem cells for myocardial repair

Oren Caspi and Lior Gepstein*
Sohnis Family Research Laboratory for Cardiac Electrophysiology and Regenerative Medicine and the Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 2 Efron Street, PO Box 9649, 31096 Haifa, Israel

KEYWORDS
Stem cells; Cell therapy; Heart failure; Ischaemic heart disease

The adult human heart has limited regenerative capacity and therefore any signicant myocardial damage results in progressive deterioration in its function. Cell-based cardiac repair represents an exciting approach for rebuilding the injured heart. Consequentially, during the last decade, signicant efforts have been made to assess the potential role of skeletal myoblasts, foetal cardiomyocytes, bone marrow stem cells, endothelial progenitors, resident cardiac progenitor cells, and embryonic stem cells (ESC) to restore the myocardial performance. Clinical translation of these basic studies has progressed rapidly with several Phase I and II clinical trials, utilizing autologous bone marrow cells and skeletal myoblasts, being well underway. In this review, we will describe these basic and clinical efforts. A specic emphasis will be placed on the potential role of human ESC for myocardial repair. These unique pluripotent cell lines can be propagated in the undifferentiated state in culture and coaxed to differentiate into cell derivatives of all three germ layers, including cardiomyocytes. The potential applications of this unique differentiating system in the emerging eld of cardiovascular regenerative medicine will be discussed with special emphasis on the steps required to fully harness their unique potential.

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Introduction
One of the most exciting areas in basic and applied research today involves the use of stem cells. These unique cells have the capability to transform and replenish the different tissue types that make up the body, and also represent the fundamental building blocks of human development. The recent advances in the areas of stem cell biology and tissue engineering coupled with parallel achievements in molecular and cell biology have paved the way to the development of a new eld in biomedicine, regenerative medicine. This approach seeks to develop new biological solutions to replace or modify the function of diseased, absent, or malfunctioning tissue. The adult heart represents an attractive candidate for these emerging technologies because adult cardiomyocytes have limited regenerative capacity. Hence, any

* Corresponding author. Tel: 972 4 829 5303; fax: 972 4 852 4758.
E-mail address: mdlior@tx.technion.ac.il

signicant loss of heart cells is mostly irreversible and may lead to progressive loss of ventricular function and nally to the development of heart failure. Despite advances in the pharmacological, interventional, and surgical therapeutic measures, the prognosis for heart failure patients remains poor. Non-pharmacological treatments like heart transplantation for end-stage patients are of limited impact as the chronic lack of donors limits the number of patients that could benet from heart transplantations. Given these circumstances, the development of novel therapeutic strategies for the treatment of heart failure has become imperative. Although the pathophysiological mechanisms underlying the development of progressive ventricular dysfunction and clinical heart failure are multi-factorial, the initial rationale underlying the cell replacement strategy was simple, to attempt to restore myocardial performance by augmenting the number of functional cardiomyocytes within the diseased hearts.1,2 While these initial experimental strategies mainly focused on repopulating the diseased myocardial areas with a new pool of

& The European Society of Cardiology 2006. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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functional myocytes (skeletal myoblasts or foetal cardiomyocytes),3,4 it is now clear that cell therapy may lead to improvement of myocardial performance through a variety of mechanisms. Examples of such indirect mechanisms include improving the survival of host cardiomyocytes by induction of angiogenesis, augmenting a possible endogenous repair mechanism, and altering the negative remodelling process that occurs following myocardial infarction by changing the geometry and structural properties of the scar. Since the initial pioneering cell therapy experiments more than a decade ago, numerous basic studies and several early-stage clinical trials were performed using a variety of cell types with the hope of improving myocardial performance. In the current review, we will briey describe these efforts (for a more detailed discussion please refer to a number of excellent reviews).1,2,5 We will then focus on one unique cell type, the human embryonic stem cell (hESC), and describe the possible future role of these cells in cardiovascular regenerative medicine as well as the steps required to harness their potential.

and neonatal) were demonstrated to be better candidates than more mature cardiac cells due to their superior in vivo survival.13 More recently, it was demonstrated that these cells could survive and improve cardiac function for up to 6 months in a rat model of chronic myocardial infarction.7 Cardiomyocyte cell transplantation was associated with smaller infarcts,14 prevented cardiac dilatation and remodelling following infarction,15 and also improved the ventricular function in some of these studies.

Skeletal myoblasts
Experimental cell therapy approaches for cardiac repair began with the use of skeletal myoblasts derived from skeletal muscle satellite cells.3,16 Interestingly, besides their non-cardiac origin, skeletal myoblasts have almost all the properties of the ideal donor cell type. Skeletal myoblast satellite cells can be harvested from autologous sources, they can be rapidly expanded ex vivo to clinically applicable numbers of myoblasts without a risk for tumorgenicity, and they have the capabilities to withstand ischaemia better than many other cell types. Although it was originally hoped that these cells would adapt a cardiac phenotype, it is now clear that myoblasts remain committed to form only mature skeletal muscle in the heart that possess completely different electromechanical properties than those of heart cells. Moreover, given the inability of myoblasts to form electromechanical connections with host cardiomyocytes (due to lack of expression of adhesion and gap junction proteins), it is not surprising that physiological studies failed to demonstrate synchronous beating of the grafted cells within the host tissue.17 Interestingly, despite these apparent shortcomings, several studies in small and large animal models of infarction demonstrated benecial effects of grafting of these cells on ventricular performance.3,18 Given their autologous origin, the encouraging preclinical results, and despite the lack of complete understanding of the mechanisms underlying their benecial effects, skeletal myoblasts were the rst cell type to reach the clinical arena.19,20 These clinical trials utilized autologous skeletal myoblasts that were harvested by a muscle biopsy, expanded ex vivo, and later grafted into the hearts of chronic heart failure patients. The pioneering Phase I clinical trials were performed either using a direct surgical approach [during coronary artery bypass graft surgery (CABG)] or using a percutaneous endocardial catheter-delivery approach and have demonstrated both the feasibility of the procedure and the ability of the cells to engraft in the infarcted myocardium (Table 1).1925 These trials, however, have also raised a number of issues for concerns.1 Pathological studies in some of the patients revealed that engrafted cells populated only a small proportion of the infarct. Thus, methods should be developed in the future to improve the efcacy of cell engraftment and survival.2 An important safety concern was the disturbingly high incidence of ventricular arrhythmias noted in the initial stages of some of

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Cell therapy approaches for myocardial repair

During the last decade, different cell types have been proposed for cell-based cardiac repair. These include foetal cardiomyocytes,4,6,7 skeletal myoblasts,3 bone marrow-derived haematopoietic8 and mesenchymal9 stem cells, mouse10 and hESCs,10,11 and more recently resident cardiac progenitor cells.12 Although a variety of cell types have been suggested, the ideal donor cell should probably exhibit the electrophysiological, structural, and contractile properties of cardiomyocytes, and should be able to integrate structurally and functionally with host tissue. In addition, it has to retain an initial high proliferative potential that may enable improved colonization of the scar tissue. The ability to engineer the candidate cell to promote desirable characteristics such as resistance to ischaemia and apoptosis and improved contractile properties may be another advantage of such an ideal cell type. Finally, the optimal candidate cell should be of autologous origin or retain minimal immunogenicity and should be readily available in large quantities for transplantation. Unfortunately, none of the candidate cell sources exhibit all of the aforementioned properties.

Foetal cardiomyocytes
Early cell transplantation studies focused on using foetal or neonatal rat cardiomyocytes, as these cells have the inherent electrophysiological, structural, and contractile properties of cardiomyocytes and still retain some proliferative capacity.4,6,7,13 In these pioneering studies, foetal cardiomyocytes transplanted into healthy mice hearts were demonstrated to survive, align with host cells, and form cell-to-cell contacts with host myocardium.4 Interestingly, early-stage cardiomyocytes (foetal

Stem cells for myocardial repair

Table 1 Skeletal myoblasts transplantation in chronic heart failure Author/study name Herreros et al.22 Number of patients 12 Cell delivery Additional procedures CABG Cell dose 2.21 108 Endpoint assessment methodology Echocardiography, PET Control group Results Adverse effects

Directintramyocardial

None

Menasche et al.19

10

Directintramyocardial

None

8.71 108

Echocardiography

None

Pagani et al.23

Directintramyocardial

LVAD

3 108

Histological analysis

None

Siminiak et al.24

10

Directintramyocardial

CABG

1.7 107

Echocardiography

None

"LVEF "Regional contractility in transplanted segments "Viability of infarct zone (F-FDG PET) "NYHA functional class "LVEF "Regional contractility in transplanted segments Myoblasts survive and form myobers "Small vessel formation "LVEF

None reported

Four delayed episodes of sustained VT

None reported

Smits et al.20

Siminiak et al. -PONZAN Trial25 Dib et al.21

10

Percutaneous electromechanical mapping Percutaneous coronary sinus Directintramyocardial

None

1.96 108

LV angiography nuclear radiography DSE MRI

None

"LVEF, LV Wall thickening in target areas (MRI) "LVEF "NYHA functional class "LVEF "Viability of infarct zone (F-FDG PET) Long term survival of engrafted cells (up to 4 years)

Four episodes of VT (no additional episodes following administration of amiodarone prophylaxis) One episode of non-sustained VT

None

Up to 1 108

Echocardiograpy

None

None reported

30

LVAD (n 6) CABG (n 24)

130 107

PET, echocardiography, MRI

None

Three episodes of non-sustained VT

F-FDG-PET-F, F-labelled uoro-deoxyglucose positron emission tomography; VT, ventricular tachycardia; NYHA, New York Heart Association; LVAD, left ventricle assist device; DSE, dobutamine stress echo.

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these trials. For example, 10 out of the rst 22 patients, reported in the literature, undergoing skeletal myoblast transplantation experienced some form of ventricular arrhythmias.19,20 Although direct causal relationship is hard to prove in these non-controlled trials given the expected high incidence of ventricular arrhythmias in this patient population, this potentially life-threaten side effect warrants further considerations. It is postulated that due to their lack of gap junctions, the engrafted myotubes are completely uncoupled to the surrounding ventricular myocytes and may therefore act as anatomical obstacles, increasing tissue inhomogeneity, slowing conduction, and increasing the likelihood for the formation of re-entrant arrhythmias. Given the fact that these Phase I clinical trials were not randomized, placebo controlled, or blinded, that they used widely different cell transplantation protocols in a relatively small number of patients, and that they were associated with other confounding factors such as concomitant LVAD implantation or revascularization, it is difcult to draw any signicant conclusions about their efcacy. Ongoing Phase II clinical trials will hopefully address these concerns and thoroughly evaluate the safety and efcacy of myoblast transplantation.

culprit coronary artery following ST elevation acute myocardial infarction (STEMI) (Table 2) and intramyocardial cell injection for chronic IHD (CIHD) (Table 3). Acute myocardial infarction Initial Phase I and II clinical studies assured the feasibility and safety of the intracoronary cell infusion in the setting of STEMI.2934 Strauer et al.29 evaluated in 10 patients the effect of BMMC infusion, 7 days following STEMI, on LV function when compared with control patients who refused to enter the study. Patients who received BMMC infusion displayed improvement in the extent of the hypokinesis/dyskinesis area but did not show any signicant change in the left ventricular ejection fraction (LVEF). The following TOPCARE-AMI study compared blood- and bone marrow-derived progenitor cells (delivered 4 days following MI) and demonstrated that both cell types had a similar but mild positive effect on cardiac function when compared to historical controls (improvement of EF from 52 to 60% over 4 months whereas in controls, EF improved from 51 to 54%).30 Wollert et al.34 (the BOOST trial) conducted a randomized study in 60 patients and reported, using MRI, a substantial improvement in global LVEF in the BMMC infusion group (50.056.7% in treated vs. 51.352% in controls) following 6 months of follow-up. More recently, however, it was reported that following 18 months, there were no signicant differences in global LVEF, although regional contractility in target areas remained signicantly improved.32 These results were recently reinforced in the rst double blinded, randomized, sham controlled, trial conducted by Janssens et al.33 Following a period of 4 months, patients treated with BMMC had an improved viability in the infarct area and regional systolic function but a similar global LVEF function when compared to sham-infused controls. Chronic IHD Similar to the skeletal myoblast trials, delivery of bone marrow stem cell to the chronically ischaemic myocardium was performed either using direct intramyocardial injection (as an adjunct to CABG)35,36 or using a percutaneous catheter-based endomyocardial injection approach.3739 The hypothesis underlying these studies was that the injected cells may improve the perfusion and survival of the ischaemic tissue. These initial trials included only a small number of patients, lacked a control group, and some also included a confounding revascularization procedure.36,37,39 More recently, Perin et al.38 reported a controlled, non-randomized, trial that included 21 patients with end-stage IHD treated with percutaneous transendocardial injection of BMMC. The treated patients showed improved functional class and myocardial performance with no signicant adverse effects. More recently, Patel et al.35 reported on the rst controlled randomized trial evaluating BMMC in IHD. BMMC were implanted in conjunction to CABG surgery in 10 patients. Cell transplantation resulted in a substantial improvement of both symptoms and myocardial performance.

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Bone marrow-derived stem cells

Studies in animal models of ischaemia and Phase I & II clinical trials suggest that delivery of haematopoietic stem cells (HSCs) and circulating endothelial progenitor cells (also originating from bone marrow stem cells) may result in improvement in the ventricular function in ischaemic heart disease (IHD) patients. Nevertheless, the initial assumption regarding the capability of bone marrow-derived HSCs to regenerate the heart by transdifferentiation into cardiomyocytes8 has been recently challenged by a number of studies demonstrating cell fusion of the progenitor cells with host cells.26 Moreover, recent studies suggest that the HSC continue to differentiate along the haematopoietic lineage27,28 and the functional improvement observed may not be related to transdifferentiation into the cardiac lineage, but rather from indirect mechanisms. The main assumption is that the infused bone marrow mononuclear cells (BMMC) stimulate neoangiogenesis and thus prevent late myocardial remodelling, thereby decreasing myocyte apoptosis, collagen disposition, and scar formation.5 Fueled in part by hopes for cardiac transdifferentiation, as well as by the considerable body of data supporting angiogenic activity,5 bone marrow studies moved remarkably quickly from small animals to clinical trials. As expected, from a rapidly developing therapeutic modality, there is a signicant heterogeneity between the conducted studies concerning the cell type used [bone marrow is a heterogeneous tissue, containing rare haematopoietic and mesenchymal stem cells (MSC) (0.01%)], the indications, and the clinical endpoints assessed in these studies. In general, the clinical trials using bone marrow-derived stem cells can be grouped into two groups: intracoronary cell infusion to the

Stem cells for myocardial repair

Table 2 Bone marrow-derived stem cell transplantation in acute myocardial infarction Author/study name Janssens et al.33 Number of patients Cell type Cell delivery Cell dose 3 108 (2.8 106 CD34) Endpoint assessment methodology MRI, acetate PET Control group Results Follow-up

33 (1 day following AMI)

BMMC

Intracoronary

34, sham, randomized, double blinded 93, sham, randomized

REPAIR-AMI 32

94 (36 days following AMI)

BMMC

Intracoronary

2.4 108

MRI, LV angiography

ASTAMI 32 FernandezAviles et al.31

50 (58 days following AMI) 20 (13 days following AMI)

BMMC BMMC

Intracoronary Intracoronary

2.1 108 7.8 107

MRI, echocardiography, SPECT MRI, echocardiography, LV angiography

50, sham, randomized 13, no sham, not randomized, not blinded 30, no sham, randomized, blinding not stated

No effect on LVEF "Regional systolic function "Viability of Infarct zone "LVEF More signicant benet for cell infusion following more than 5 days #LVEF "LVEF #LVESV LV wall thickening in target areas (MRI) "LVEF "Regional contractility in transplanted segments No signicant effect on LVEF "LVEF "Regional contractility in transplanted segments "Viability of infarct zone "Velocity of infarct wall "LVEF #LVESV #Infarct size No signicant difference between BMMC and CPC

4 months

4 months

6 months 11 months

Wollert et al. BOOST Trial34

30 (6 days following AMI)

BMMC

Intracoronary

2.5 109 (9.5 106 CD34)

MRI

6 months

18 months 6 months

Chen et al.43

34 (18 days following AMI)

MSC

Intracoronary

810 109

PET, echocardiography, electromechanical mapping

35, sham, randomized, blinding not stated

Schachinger et al. TOPCARE- AMI75

20 (5 days following AMI)

BMMC (n 29) CPC (n 30)

Intracoronary

BMMC-2.1 108 (5.5 106 CD34) CPC (1.6 107)

MRI, PET, echocardiography, LV angiography

No control group

1 year

AMI, acute myocardial infarction; LVESV, LV end systolic volume; CPC, circulating progenitor cells; PET, positron emission tomography; SPECT, single photon emission computed tomography. Studies presented at the American Heart Association Conference 2005, and reported in Cleland et al.32

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Table 3 Bone marrow-derived stem cell transplantation in CIHD Author/study name Perin et al.38 Number of patients 14 Cell type Indication Cell delivery Additional procedures Cell dose Endpoint assessment methodology Echocardiography, SPECT, electrome chanical mapping, LV angiography Echocardiography, SPECT Control group Results Follow-up

BMMC

End-stage IHD

Percutaneous trans endocardial electromechani cal mapping

None

2.5 107 (6 105 CD34)

7, no sham, nonrandomized, open labelled

Fuchs et al.37

10

BMMC

Patel et al.35 Stamm et al.36

10 12

BMMC CD 133

CCS III-IV with no conventional revascularization treatment option IHD and EF , 35% IHD

Percutaneous trans endocardial electromechani cal mapping

None

7.8 107 (2 106 CD34)

None

" LVEF #Total reversible defect #NYHA Class #LVEDV, LVESV "VO2 Max #CCS score " Stress-induced ischaemia (target areas) No effect on LVEF

2 months (4 months for treated patients)

3 months

Direct intracmyocardial Direct intracmyocardial

Off pump CABG CABG

1.5 106

Echocardiography, SPECT Echocardiography, SPECT

10, no sham, randomized None

Tse et al.39

BMMC

IHD

Percutaneous trans endocardial electromechani cal mapping

None

Echocardiography MRI

None

"LVEF #NYHA class "Perfusion in target areas "LVEF #LVEDV "Traget wall thickening #Hypoperfused myocardial areas No effect on LVEF

6 months 310 months

3 months

O. Caspi and L. Gepstein

LVEDV, LV end diasystolic volume; CPC-circulating progenitor cells; SPECT, single photon emission computed tomography; CCS, Canadian cardiovascular society score.

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Stem cells for myocardial repair

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Mesenchymal stem cells

MSC represent another cell type suggested for cardiac repair. This potential multipotent stem cell is derived from the non-haematopoietic, stromal, compartment of the bone marrow. MSC were suggested by some40 (but not by the majority) studies to differentiate into cardiomyocytes both in vitro and in vivo. Transplantation of MSC into the infarcted myocardium of rats and pigs resulted in improved myocardial performance.9,41 One possible advantage of MSC is their ability to be delivered either in autologous procedures or using an allogeneic strategy as some reports suggest that they may be relatively immunoprivileged.42 These apparent attractive capabilities of the cells have recently led investigators to begin a number of clinical trials.43

Cardiac resident progenitor cells

The long-held belief that the adult mammalian heart has no intrinsic capacity for repair has recently brought into question when several groups have reported evidence that myocardium itself contains a resident progenitor cell population capable of giving rise to new cardiomyocytes. Initially, Beltrami et al.44 isolated and expanded c-kit positive cells from the adult rat heart. Engraftment of the cells to the acutely ischaemic myocardium resulted in cell differentiation to cardiomyocytes, smooth muscle cells, and vascular endothelium and in signicant improvement on the ventricular function. A second progenitor population was isolated on the basis of sca-1 expression from the adult mouse heart. These cells were demonstrated to express cardiac-specic markers after treatment with 5-Azacytidine in vitro and following intravenous injection they homed to the infarcted mouse myocardium.45 Similar to side population cells reported in the bone marrow, Martin et al.46 isolated Hoechst dye efuxing cells expressing the ATP-binding cassette transporter, Abcg2. These cells have the ability to express the sarcomeric protein a-actinin after co-culturing with unfractionated cardiac cells. The most recent candidate progenitor population was isolated on the basis of the transcription factor islet-1.47 Collectively, given the limited regenerative capacity of the adult heart, it is clear that the presence of the above mentioned cells within the heart does not translate to a functionally signicant cardiac differentiation following myocardial infarction. In addition, the description of four nonoverlapping, discrete, progenitor cell populations in a non-regenerating organ may stem from the immature nature of this developing area. Nonetheless, the roles of these cells in tissue maintenance, repair, and possible therapeutic applications will be an exciting area of investigation in the coming years.

Embryonic stem cells

Most of the cell types discussed above are stem cells and therefore share a number of properties.48 First, they are

capable of self renewal, meaning that they can divide and give rise to stem cell progeny with similar properties. Second, the stem cells are clonogenic, meaning that each cell can form a colony in which all the cells are derived from this single cell and have identical genetic constitution. Third, they are capable of differentiation into one or more mature cell types. The different stem cells can be categorized anatomically, functionally, or by different cell surface markers, transcription factors, and the proteins they express. One clear division of the stem cell family is between those present in adult somatic tissue known as adult stem cells and those isolated from the embryo, known as ESC. Although adult stem cells have been found to be more versatile than originally believed, they typically can differentiate to a relatively limited number of cell types. In contrast, cells in the early pre-implantation mammalian embryo have the potential to contribute to all adult tissues. At the blastocyst stage, a group of cells begins to separate from the outer cells and forms the inner cell mass (ICM). While the outer cells become the trophoectoderm, the ICM cells will ultimately give rise, through specialized progenitor cells, to all the tissues in the body and are therefore truly pluripotent. In 1981, the ICM cells, isolated from mouse blastocysts, were used to generate pluripotent stem cell lines that were termed ESC.49,50 The mouse ESC (mESC) were demonstrated to be capable of prolonged in vitro proliferation and self-renewal but also retained the ability to differentiate into derivatives of all three germ layers both in vitro and in vivo. Thus, following cultivation in suspension, the mESC tend to spontaneously create three-dimensional aggregates of differentiating tissue known as embryoid bodies (EBs).51 Among other cell types, cardiomyocyte tissue appears within this multicellular arrangement as spontaneously contracting areas that can be studied as a cluster or as dispersed cells. The availability of the mESC system have provided important insights into the mechanisms underlying earlycardiac differentiation, development of excitability, calcium handling, and electromechanical coupling52 but also demonstrated the potential role of ESC for myocardial regeneration. By using genetically-selected mESCderived cardiomyocytes, Klug et al.10 showed that these cells can form stable intracardiac grafts in the mouse heart. Later studies, utilizing the infarcted rat heart model, demonstrated that transplantation of differentiated mESC-derived cardiomyocytes can result in short- and long-term improvement of myocardial performance.53 Interestingly, whereas studies conducted by the Puceats group indicate that transplantation of undifferentiated mESC into the immunocompetent mouse or rat heart survive, integrate, and improve the myocardial function of the infarcted heart,54 other groups reported on the generation of teratomas and rejection of the cells when undifferentiated mESC were injected into non-syngeneic or immunotolerant animals. More recently, Menard et al.55 reported that cardiac committed mESC (following incubation with BMP-2), transplanted to the infarcted sheep heart, differentiated to

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mature cardiomyocytes and that cell transplantation resulted in a signicant improvement in cardiac function independent of whether the sheeps were immunosuppressed or not. Given the outstanding potential demonstrated by the mESC, it was not surprising that much effort was spent on the development of similar hESC lines. This quest has ended in 1999 when two groups described the generation of hESC lines.27,28 The hESC were demonstrated to fulll all the criteria dening ESC,27,28 namely: derivation from the pre- or peri- implantation embryo, prolonged undifferentiated proliferation under special conditions, and the capacity to form derivatives of all three germ layers. More recently, we and other researchers5658 were able to generate a reproducible cardiomyocyte differentiation system from the hESC. The induction of in vitro differentiation of hESC to cardiomyocytes requires their removal from the mouse embryonic broblast (MEF) feeder layer that allows their undifferentiated propagation, and cultivation in suspension where they form EBs.56 The EBs are then cultured in adherent conditions and develop spontaneously contracting area in 10% of the clusters.56 Several lines of evidence conrmed the molecular, structural, and functional cardiac phenotype of the generated myocytes.5658 The generated myocytes expressed cardiac-specic transcription factors and cardiac-specic structural genes. Immunostaining studies conrmed the presence of cardiac-specic proteins and absence of skeletal muscle markers. Electron microscopy revealed varying degrees of myobrillar organization and the presence of nascent intercalated discs, typical of an early-stage cardiac phenotype. These studies also depicted the progressive maturation from an irregular myolament distribution to a more mature sarcomeric organization in late-stage EBs.59 Interestingly, in parallel to this ultrastructural maturation process, we could also observe a reproducible temporal pattern of early cardiomyocyte cell proliferation, cell-cycle withdrawal, and progressive ultrastructural maturation.59 The hESC-CMs were also shown to display functional properties, consistent with an early-stage cardiac phenotype.56,58,60,61 These include the presence of typical extracellular electrical activity and intracellular action potential and calcium transients. In addition, the hESC-CMs displayed appropriate chronotropic responses to adrenergic and cholinergic stimuli, demonstrating the presences of functional receptors and signalling pathways. Whole cell patch-clamp recording from the hESC-CMs demonstrated the presence of cardiac-specic action potentials and ionic currents. These studies also revealed important insights to the mechanism of automaticity, excitability, and repolarization in these developing cardiomyocytes.61 More recently, we have demonstrated, using a microelectrode array mapping technique, that this differentiating system is not limited to the generation of isolated cardiomyocytes but rather a functional syncytium is generated with stable spontaneous pacemaking activity and synchronous action-potential propagation.60 Immunostaining studies demonstrated that this functional synctium results from the formation of gap junctions between the cells.

Optimal functional improvement following cell grafting would require structural, electrophysiological, and mechanical coupling of donor cells to the existing network of host cardiomyocytes. In a recent study, we tested the ability of the hESC-CMs to integrate structurally and functionally with host cardiac tissue both in vitro and in vivo.62 Initially, primary cultures were created from neonatal rat ventricular myocytes and the contracting EBs were added to the co-cultures. Interestingly, within 24 h post-grafting, we could detect synchronous contraction in the co-cultures that persisted for several weeks. Detailed electrophysiological mapping of the hybrid cultures demonstrated tight electrophysiological coupling between the two tissue types. These ndings were reinforced by immunostaining data suggesting the development of gap junctions between the human and the rat cells. To demonstrate the ability of the hESC-CMs to survive, function, and integrate also in the in vivo heart, we assessed their ability to pace the heart and to function as a biological pacemaker.62 The hESC-CMs were transplanted to the posterolateral region of the left ventricle in a swine model of slow heart rate. Following cell grafting, a new ectopic ventricular rhythm was detected in 11 out of 13 animals studied, in six of which, it was characterized by sustained and long-term activity. Pathological studies validated the presence and integration of the grafted hESC-CM at the site of transplantation. Three-dimensional electrophysiological mapping revealed that this ectopic ventricular rhythm originated from the area of cell transplantation. Recently, our ndings were reinforced in a study conducted by Xue et al.63 using a similar model of guinea pig heart block and demonstrating cell integration by optical mapping of the epicardial surface. More recent pathological studies demonstrated the ability of grafted hESC-CMs to survive and proliferate within the uninjured arrhythmic rat heart for as long as 4 weeks.64

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From cell culture to bedsidechallenges for clinical translation of hESC-CMs

Although hESC-CMs could theoretically have the potential to fulll most of the properties of the ideal donor cell (they are the only cell source that can provide ex vivo an unlimited number of human cardiomyocytes), a number of critical obstacles are needed to be overcome prior to clinical application.11 (i) Strategies need to be developed for directing and augmenting hESC differentiation into the cardiac lineage. (ii) Purication of the differentiating cardiomyocyte population should be achieved. (iii) Upscaling of the culturing techniques is needed to yield clinically relevant number of cells for transplantation. (iv) Transplantation technique should be developed to enable proper alignment of the graft tissue, high seeding rate of the transplanted cells, and minimal damage to the host tissue. (v) Strategies aimed at preventing immunological rejection of the cells should be developed. (vi) Several regulatory issues should be addressed.

Stem cells for myocardial repair

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hESC cell line characterization and development of good manufacturing practice

Worldwide it is estimated that there are 200 hESC cell lines. However, the ability of hESC to differentiate to cardiomyocytes effectively was shown in only a minority of these lines. For effective clinical translation of the enormous potential of hESC, characterization of a wide range of lines representing the diverse human genetic pool is required. Importantly, each cell line should also be evaluated for extended periods, assuring no modications occur during prolonged passaging. The essence of uniform characterization criteria is stressed in light of the recent studies reporting karyotype abnormalities in hESC.65 The hESC were derived in medium containing serum and were plated on top of MEF feeder layer. From the perspective of therapeutic applications, a xeno-free and serum-independent culturing system is essential for ensuring high reproducibility and minimizing the risk for xenozoonoses. Recently, supporting systems based on human tissues such as human foetal broblast, adult epithelial cells, or foreskin cells were recently suggested as alternatives to the MEF feeder cells.66 More recently, some of the key regulators mediating the ability of ESC to self renew have been described. These studies allowed the generation of the creation of a feeder-free cultivation system.67

Interestingely, initial studies suggest that the pathways found to play a role in the aforementioned developmental models may also govern the in vitro differentiation of the hESC to cardiomyocytes. Lev et al.,68 from our laboratory, examined the temporal gene expression pattern of signalling proteins and transcription factors governing early hESC cardiogenic differentiation and noted a similar cascade to that noted in vivo with an early expression of cardiac promoting factors (BMP-2, WNT11), followed by the expression of cardiac-specic transcription factors (Nkx2-5, GATA4, and Mef2C), and later the expression of cardiac-specic structural genes. Similarly, the well known cardiogenic inductive role of the primitive visceral endoderm in the developing embryo was also demonstrated to play a role in the cardiomyocytes differentiation of hESC line in an elegant study conducted by Mummery et al.58 Co-culturing of a hESC line (hES2), which does not regularly differentiate spontaneously to cardiomyocytes, with END-2 cells (a visceral endoderm-like cell line) provided the missing trigger for cardiac differentiation.

Selection and purication strategies

Although the development of directed differentiation systems is essential for increasing the cardiomyocyte yield from the hESC, it is unlikely that the degree of purity that will be achieved would be sufcient for clinical purposes. Because the contracting areas derived from hESC are comprised of a mixed population of cells, selection strategies are crucial not only for increasing cardiomyocyte numbers but also for preventing the presence of other cell derivatives as well as ensuring the absence of pluripotent stem cells carrying the risk of teratomas. An elegant selection scheme to generate pure cardiomyocyte populations was suggested by Klug et al.10 in the mESC model. Undifferentiated mESC were transfected with a fusion gene encoding antibiotic resistance under the regulation of the cardiac-specic a-myosin heavy chain promoter. Differentiation was then induced and puried population of cardiomyocytes were selected based on their resistance to Neomycin. A slightly different approach was reported by Muller et al.70 using green uorescent protein driven by the cytomegalovirus enhancer and a ventricle-specic promoter (MLC-2V). In conjunction with the use of percoll gradient centrifugation and subsequent FACS, this strategy yielded a relatively pure (97%) population of ventricular cells.

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Cardiomyocyte enrichment, purication, and upscaling strategies

Although cardiomyocytes can be reproducibly generated from the hESC using the EB differentiating system, these cells typically account for only a minority of the total population within the differentiating EBs. Consequentially, developing of strategies to augment cardiomyocyte differentiation and to select the generated cardiomyocytes may be crucial for the ultimate success of these cells in myocardial repair.

Directed differntiation
The currently hESC cardiac differentiation system is essentially spontaneous and thereby characterized by relatively low efcacy. Possible strategies for increasing cardiomyocyte yield may include the use of different growth factors, overexpression of cardiac transcription factors, co-culturing with feeder layers, and mechanical factors.68 The development of a more rational directed differentiation system is hampered, however, by the relative lack of data regarding the inductive cues that lead to commitment and terminal differentiation of human cardiomyocytes. Consequentially, strategies for directed differentiation should follow the elegant developmental studies conducted in a number of model organisms, most notably the chick, amphibians, zebrash, and the mouse.69

Scaling up
The LV of the human heart contains 5.8 109 cardiomyocytes. Given the fact that 25% of the cells are lost during a typical MI leading to heart failure, cell transplantation strategies aiming to completely regenerate the myocardium would require several hundred millions of cells. Generation of this number of cells can be achieved by increasing the number of hESC used for cardiomyocytes differentiation, by using directed differentiation systems and enrichment strategies, by increasing the ability of the cells to proliferate after cardiomyocyte induction,

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3. Taylor DA, Atkins BZ, Hungspreugs P Jones TR, Reedy MC, Hutcheson KA, , Glower DD, Kraus WE. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med 1998;4:929933. 4. Soonpaa MH, Koh GY, Klug MG, Field LJ. Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 1994;264:98101. 5. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientic foundations of cardiac repair. J Clin Invest 2005;115:572583. 6. Leor J, Patterson M, Quinones MJ, Kedes LH, Kloner RA. Transplantation of fetal myocardial tissue into the infarcted myocardium of rat. A potential method for repair of infarcted myocardium? Circulation 1996;94:332336. 7. Muller-Ehmsen J, Peterson KL, Kedes L, Whittaker P, Dow JS, Long TI, Laird PW, Kloner RA. Rebuilding a damaged heart: long-term survival of transplanted neonatal rat cardiomyocytes after myocardial infarction and effect on cardiac function. Circulation 2002;105:17201726. 8. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701705. 9. Mangi AA, Noiseux N, Kong D, He H, Rezvani M, Ingwall JS, Dzau VJ. Mesenchymal stem cells modied with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med 2003;9:11951201. 10. Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J Clin Invest 1996;98:216224. 11. Gepstein L. Derivation and potential applications of human embryonic stem cells. Circ Res 2002;91:866876. 12. Leri A, Kajstura J, Anversa P. Cardiac stem cells and mechanisms of myocardial regeneration. Physiol Rev 2005;85:13731416. 13. Reinecke H, Zhang M, Bartosek T, Murry CE. Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts. Circulation 1999;100:193202. 14. Li RK, Mickle DA, Weisel RD, Mohabeer MK, Zhang J, Rao V, Li G, Merante F, Jia ZQ. Natural history of fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue. Circulation 1997;96:186187. 15. Etzion S, Battler A, Barbash IM, Cagnano E, Zarin P, Granot Y, Kedes LH, Kloner RA, Leor J. Inuence of embryonic cardiomyocyte transplantation on the progression of heart failure in a rat model of extensive myocardial infarction. J Mol Cell Cardiol 2001;33:13211330. 16. Reinecke H, MacDonald GH, Hauschka SD, Murry CE. Electromechanical coupling between skeletal and cardiac muscle. Implications for infarct repair. J Cell Biol 2000;149:731740. 17. Rubart M, Soonpaa MH, Nakajima H, Field LJ. Spontaneous and evoked intracellular calcium transients in donor-derived myocytes following intracardiac myoblast transplantation. J Clin Invest 2004;114:775783. 18. Scorsin M, Hagege A, Vilquin JT, Fiszman M, Marotte F, Samuel JL, Rappaport L, Schwartz K, Menasche P. Comparison of the effects of fetal cardiomyocyte and skeletal myoblast transplantation on postinfarction left ventricular function. J Thorac Cardiovasc Surg 2000;119:11691175. 19. Menasche P, Hagege AA, Vilquin JT, Desnos M, Abergel E, Pouzet B, Bel A, Sarateanu S, Scorsin M, Schwartz K, Bruneval P, Benbunan M, Marolleau JP, Duboc D. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol 2003;41:10781083. 20. Smits PC, van Geuns RJ, Poldermans D, Bountioukos M, Onderwater EE, Lee CH, Maat AP, Serruys PW. Catheter-based intramyocardial injection of autologous skeletal myoblasts as a primary treatment of ischaemic heart failure: clinical experience with six-month follow-up. J Am Coll Cardiol 2003;42:20632069. 21. Dib N, Michler RE, Pagani FD, Wright S, Kereiakes DJ, Lengerich R, Binkley P, Buchele D, Anand I, Swingen C, Di Carli MF, Thomas JD, Jaber WA, Opie SR, Campbell A, McCarthy P, Yeager M, Dilsizian V, Grifth BP, Korn R, Kreuger SK, Ghazoul M, MacLellan WR, Fonarow G, Eisen HJ, Dinsmore J, Diethrich E. Safety and feasibility of autologous myoblast transplantation in patients with ischaemic cardiomyopathy: four-year follow-up. Circulation 2005;112: 17481755. 22. Herreros J, Prosper F, Perez A, Gavira JJ, Garcia-Velloso MJ, Barba J, Sanchez PL, Canizo C, Rabago G, Marti-Climent JM, Hernandez M, Lopez-Holgado N, Gonzalez-Santos JM, Martin-Luengo C, Alegria E.

and by upscaling the entire process. For example, Zandstra et al.,71 using the mESC system, combined the aforementioned cardiomyocyte selection strategies with the bioreactor technology to generate more than 109 vital cardiomyocytes in a single 2 L bioreactor run.

Achieving immune tolerance

A major obstacle for the utilization of hESC derivatives in regeneration of different tissue types is the prevention of their immune rejection.72 Although initial in vitro and in vivo characterization of the immunogenicity of the hESC demonstrated that they display only minimal immunogenicity,73,74 they are still expected to be rejected. Several strategies aimed at achieving immunological tolerance are being explored. These strategies include establishing of banks of major histocompatibility complex antigen-typed hESC lines, genetically altering the hESC to suppress the immune response (for example, by knocking-out the major histocompatibiliy complexes), the concept of haematopoietic chimerism, and possibly also by nuclear transfer techniques (therapeutic cloning).72

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Summary and future potential research directions

The derivation of the hESC lines and the resulting cardiomyocytes differentiation system may bring a unique value to several basic and applied research elds. Research based on the cells may help to elucidate the mechanisms involved in early human cardiac lineage commitment, differentiation, and maturation. Moreover, this research may promote the discovery of novel growth and transcriptional factors using gene trapping techniques, functional genomics, and proteomics as well as providing a novel in vitro model for drug development and testing. Finally, the ability to generate, in vitro for the rst time, human cardiac tissue provides an exciting and promising cell source for the emerging discipline of regenerative medicine and myocardial repair. Yet, despite the enormous potential of these technologies, several hurdles need to be overcome before this strategy can become a clinical reality.

Acknowledgements
This study was partially supported by the Israel Science Foundation (grant no. 1078/04), by the American Cell Therapy Research Foundation and by the Grand Family research grant.
Conict of interest: none declared.

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