Heart Failure Reviews, 8, 221–227, 2003

C© 2003 Kluwer Academic Publishers. Manufactured in The Netherlands

Myoblast-Based Cell TransplantationPhilippe MenascheDepartment of Cardiovascular Surgery, Hopital EuropeenGeorges Pompidou, Paris, France

Abstract. Cell transplantation is emerging as a new treat-

ment designed tot improve the poor outcome of patients

with cardiac failure. Its rationale is that implantation of

contractile cells into postinfarction scars can functionally

rejuvenate these areas. Primarily for practical reasons, au-

tologous skeletal myoblasts have been the first to be tested

clinically but bone marrow stromal and hematopoietic stem

cells may represent an interesting alternative in select sit-

uations because of their autologous origin and their pur-

ported plasticity. However, several key issues still need to

be addressed including (1) the optimal type of cells, (2)

the mechanism by which cell engraftment improves cardiac

function, i.e., increased contractility or limitation of remod-

eling, (3) the most effective strategies for optimizing cell

survival, and (4) the potential benefits of cell transplanta-

tion in nonischemic heart failure. In parallel to the experi-

mental studies designed to address these issues, initial clin-

ical trials are underway and should hopefully allow to know

whether the hopes raised by cellular therapy are met by clin-

ically meaningful improvements in function and outcome in

patients with severe left ventricular ischemic dysfunction.

Key Words. heart failure, cell therapy, skeletal myoblasts,

bone marrow stem cells, transplantation

Cell transplantation is an emerging technique incardiology which has been developed to improvethe outcome of patients with cardiac failure. Thealready high incidence of this condition (approxi-mately 500,000 new cases per year in the USA),is expected to further increase in the forthcomingyears because of the ageing of the population. Themortality of cardiac failure is high as it can reach60% within 1 year for patients in New York HeartAssociation functional class IV and, not unexpect-edly, these figures translate into tremendous fi-nancial costs estimated to consume 1–2% of thetotal health care budget of western countries. Arecent study [1] shows that over the last decade,the percentage of healthcare expenditure relatedto heart failure in the National Health Service(United Kingdom) has raised from 1.2 to 1.9% (ex-cluding additional costs related to secondary heartfailure admissions and long-term nursing-homecare).

Over the past years, improvements in medicaltherapy have favourably impacted on patient out-comes. However, some forms of cardiac failure re-

main refractory to an optimal medical manage-ment, thereby requiring implementation of moreagressive approaches like ventricular resynchro-nisation or cardiac surgery. In this setting, oper-ations could so far be categorized as those aimedat “reshaping” the dilated left ventricle (the Dor’sendocardial patch plasty) or radically replacingit (transplantation). The limitations of these ap-proaches (inconsistent efficacy of remodeling pro-cedures when the scar is akinetic rather thandyskinetic, organ shortage and complications ofimmunosuppression in the case of cardiac trans-plantation) justify the search for alternate ther-apeutic options. In parallel with the develop-ment of permanent implantable assist devices,cell therapy might be one of these new treatmentmodalities.

Conceptual Basis

The objective of cell therapy is to repopulatepostinfarction scar tissue with contractile cellsthat can engraft in sufficient numbers and be func-tionally integrated into the host tissue so as to“rejuvenate” these akinetic areas. Conceptually,this objective can be achieved through three dis-tinct approaches. The first consists of stimulatingresidual cardiomyocytes to reenter a mitotic cycle[2,3] but the level of this proliferation is too lowto compensate for the loss of cardiomyocytes re-sulting from a large infarct. The second strategyis based on the genetic transformation of in-scarfibroblasts into contractile cells through transfec-tion with master genes which control the skeletalmuscle differentiation programme. Although thisapproach has yielded some successful experimen-tal results [4], its clinical applicability remainsquestionable. The third approach consists of in-jecting exogenous contractile cells into the scar.From a clinical standpoint, this “transplantation”strategy is likely to be the most realistic and, con-sequently, has been extensively investigated in the

Address for correspondence: Dr Philippe Menasche, Depart-ment of Cardiovascular Surgery, Hopital Europeen GeorgesPompidou, 20, rue Leblanc, 75015 Paris, France. Tel.: (331) 5609 36 51

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222 Menasche

laboratory setting before being tested in the firsthuman trial. Of note, although most of these exper-iments have focused on ischemic, segmental car-diomyopathies, preliminary studies yet suggestthat the putative benefits of cellular transplanta-tion might extend to idiopathic [5] or doxorubicin-induced [6] globally dilated cardiomyopathies.

Preclinical Data

The prerequisite for implanted cells to improvecardiac function is that they feature contractileproperties. Fibroblasts [7] or smooth muscle cells[8], for example, can improve postinfarct diastolicperformance but not systolic function. Contractilecells, in turn, can be categorized into naturallycontractile cells and cells whose phenotype can beoriented towards a contractile pattern. Fetal (andneonatal) cardiomyocytes as well as skeletal my-oblasts fall in the former category whereas bonemarrow-derived cells belong to the latter.

Indeed, studies of fetal and neonatal cardiomy-ocytes have yielded pivotal proof-of-concept exper-iments by showing, in rat models of myocardialinfarction, that these cells formed stable intrac-ardiac grafts, connected with host cardiomyocytesthrough gap junctions and improved left ventric-ular function [9–11]. However, in a clinical per-spective, the transplantation of fetal cells is asso-ciated with significant hurdles related to ethics,availability and immunogenicity, which accountsfor the interest generated by skeletal myoblastswhich do not raise these issues.

These myogenic precursors (known as satellitecells) normally lie in a quiescent state under thebasal membrane of skeletal muscular fibers. Incase of injury, they are rapidly mobilized, prolif-erate and fuse to regenerate the damaged fibers.In a clinical perspective, these cells feature sev-eral attractive characteristics: (i) an autologousorigin which overcomes all problems related torejection and is a key factor for large-scale clin-ical applicability, (ii) an ability to grow in largenumbers from a small biopsy, (iii) a commitmentto a well-differentiated myogenic lineage whichmakes the oncogenic risk extremely low (in ourhuman trial, none of the NOD-SCID immunode-ficient mice injected with human myoblasts hasdeveloped a tumour), and (iv) a high resistance toischemia, which is a major advantage given the hy-povascular nature of the postinfarct scar in whichthey are intended to be implanted.

Analysis of experimental data on myoblasttransplantation leads to the main following con-clusions. Morphologically, the injected myoblastsdifferentiate into typical multinucleated my-otubes which tend to repopulate areas of postin-farction fibrosis [12]. Although we and others [13]

have failed to show any true transdifferentiationof the injected cells into cardiomyocytes, engraftedmyotubes coexpress fast, skeletal muscle-type butalso slow, myosin [12], (a composite pattern whichis not observed in native in situ peripheral mus-cle) thereby suggesting some phenotypic adapta-tion to the myocardial environment, possibly as aconsequence of stretch and/or repeated electrome-chanical stimulation, as previously described af-ter dynamic cardiomyoplasty. In contrast, how-ever, to fetal cardiomyocytes, engrafted skeletalmyotubes do not establish junctions with hostcardiac cells. Indeed, N-cadherin and connexin-43 (the major proteins constitutive of fascia ad-herens and gap junctions responsible for mechan-ical and electrical coupling, respectively, in hearttissue) are expressed by cultured skeletal myo-blasts but this expression is down-regulated fol-lowing intramyocardial implantation [14]. Like-wise, our recent electrophysiological studies showthat the membrane properties of engrafted my-otubes retain the patterns typical of skeletal mus-cle and are thus quite different from those ofcardiomyocytes.

These observations functionally translate intoan improvement in left ventricular function whichhas now been demonstrated in small and largeanimal models of myocardial infarction [12,15–18]. The finding that function is only improvedin hearts where implanted cells are detectable[16] strongly suggests a mechanistic link betweenengrafted skeletal myoblasts and functional out-come, and tends to rule out the role of confound-ing factors like the angiogenic response likely trig-gered by cell injections. Of clinical relevance isour finding that the functional benefits of my-oblast transplantation are additive to those of an-giotensin converting-enzyme inhibitors [19] whichhave become standard therapy for heart failurepatients. Furthermore, our long-term follow-updata indicate that these benefits are sustainedover time, since 1-year echocardiographic values ofejection fraction were found unchanged from thosemeasured at the 2-month posttransplant timepoint [20]. This long-term improvement in func-tion could be related to the increased proportionof slow-type myosin expressed by engrafted mus-cle fibers and an attendant resistance to fatigue.Finally, we have also found a close relationship be-tween the number of injected myoblasts and themagnitude of the posttransplant functional bene-fits [21], which makes sense in view of the antic-ipated high rate of posttransplantation cell death(see below). However, opposite conclusions weredrawn from another study in which increasingdonor cell number did not increase intramyocar-dial graft size [22]. These discrepant results couldbe related to differences in experimental design(the negative study used cardiomyocytes instead

Myoblast Transplantation 223

of myoblasts and did not include functional mea-surements). This relationship between the num-ber of injected cells and the functional outcomeshould hopefully be clarified by the dose-rangingprotocol of our forthcoming phase II trial.

The mechanism(s) by which implanted my-oblasts improve function still remain largelyelusive and are currently the subject of inten-sive experimental investigations. At least threehypotheses, which are not mutually exclusive, canbe considered.

First, the elastic properties of implanted cellscould provide a scaffold strenghtening the ven-tricular wall and subsequently limiting postin-farct scar expansion. However, experimental fe-tal cardiomyocyte transplantation fails to reverseleft ventricular dilatation while improving systolicfunction [23]; likewise, in our clinical study, end-diastolic volumes did not change postoperativelyin patients who underwent myoblast implantationan average of 6 years after their infarct. There-fore, we speculate that although early postinfarctcell transplantation may prevent ventricular di-latation, it is unlikely that it can reverse remodel-ing once it is completed.

Second, a direct contribution to systolic func-tion is indirectly marshalled from the previ-ously mentioned observation that intrinsic con-tractile properties of the cells are a prerequisitefor maximal preservation of left ventricular func-tion. This assumption tends to be supported bydata from pressure-volume loops [20] and tissuedoppler imaging [12] which provide relatively di-rect evidence that engrafted skeletal myoblastsincrease global and regional systolic function, re-spectively; likewise, in our clinical study, postop-erative echocardiographic studies show that 60%of the myoblast-transplanted infarcted segmentsdemonstrate a new-onset systolic thickening. Fur-thermore, cross-striations of engrafted skeletalmyotubes appear well preserved, both in sheep ex-periments and in the heart of one of our patientswho died late from a noncardiac cause, therebysuggesting the persisting functionality of the con-tractile apparatus which otherwise features a dis-organized pattern when it is no longer operative.However, the contribution of engrafted myoblaststo systolic function is challenged by the consistentobservation that these cells do not couple with hostcardiomyocytes through gap junctions. To furtheraddress this issue, we designed a protocol in whichrat myoblasts were transfected with the gene en-coding the green fluorescent protein before beingtransplanted into postinfarction scars. One monthlater, the left ventricular wall was explanted and,while being kept beating, examined under epifluo-rescence microscopy. Detection of fluorescent my-otubes then allowed to selectively impale themwith microelectrodes so as to record action poten-

tials. With the caveat that our study point wasrelatively early after tranplantation (1 month) andthat the observations made in this model can prob-ably not be readily extrapolated to the in vivo sit-uation, we failed to document coupling betweencardiomyocytes and engrafted myotubes, i.e.,stimulation of the cardiomyocytes did not allow torecord an action potential in a neighbouring my-otube. This observation tends to eliminate the in-volvement of potential gap junction-independentmechanisms of cell-to-cell coupling such as stretchor field effects, as we had initially hypothesized.Nevertheless, myotubes were able to contract inresponse to an action potential triggered by a de-polarizing current, thereby providing evidence forthe persisting viability of the grafted cells.

This lends some support to the third hypoth-esis that engrafted skeletal muscle cells do notincrease inotropism of the recipient heart di-rectly, i.e., by their contractions, but indirectly,i.e., through paracrine effects mediated by the re-lease of pleiotrophic factors. Among them, hepato-cyte growth factor (HGF), known to be expressedby skeletal myoblasts, is an attractive candidatewhich exerts marked cardioprotective [24,25] andantifibrotic effects [26] and whose receptor, c-Met,is widely expressed on different cell populations,including those of the ischemic myocardium [24].These data are consistent with the results of oursheep experiments showing a recolonization ofpostinfarction scars by engrafted myoblasts anda reduced collagen density in these areas [12].Thus, according to this paradigm, skeletal my-oblasts could act by releasing factors (HGF be-ing likely one among others) recruiting residentcardiac stem cells [27,28] and consequently yield-ing an endogenous regeneration from this quies-cent contractile pool. Such a paracrine mechanismwould explain the persistence of long-term func-tional benefits whereas the number of engraftedmyotubes tends to decrease over time as well asour earlier observation [29] that fetal cardiomy-ocytes and skeletal myoblasts are equally effec-tive and thus, that the presence of connexin 43-supported electro-mechanical coupling is not aprerequisite for postgrafting function to improve.

Clinical Data

Surgical Approach

On June 15, 2000, we initiated the first phase Ihuman trial of autologous skeletal myoblasttransplantation [30]. This hospital-driven studyrequired three criteria to be met for patient eligi-bility: (1) severe left ventricular dysfunction (ejec-tion fraction ≤0.35), (2) history of myocardial in-farct with a residual discrete, akinetic (as assessedby echocardiography with dobutamine stress) and

224 Menasche

metabolically nonviable scar (as assessed by flu-orodeoxylucose positron emission tomography),and (3) indication for coronary artery bypassgrafting in remote (i.e., different from the trans-planted area), ischemic but viable myocardium.The study, which has included 10 patients, is nowcompleted.

The protocol used throughout the study protocolinvolves three steps. First, a biopsy of the vastuslateralis was harvested from the thigh under localanesthesia. The chunk of muscle was then mincedand shipped to the Cell Cultures Laboratory whereit was grown according to customized proceduresso as to obtain a highly purified, viable and abun-dant cell yield (an average of 870 × 106 cells wereinjected, of which at least 85% are myoblasts iden-tified by a positive staining for CD56 on cytofluo-rometry). After 2–3 weeks, cells were reimplantedacross the postinfarct scar using a customized 27gauge right-angle prebent needle so as to facilitatetangential midmyocardial cell delivery.

A detailed description of the results of this trialis beyond the scope of this review which will thenlimit to the most salient observations. First, thefeasibility of the procedure was clearly demon-strated by the ability to consistently overshootthe target numbers of cells within the preset timeframe. Second, the operation, by itself, was shownto be safe, without specific procedure-related com-plications (in particular, bleeding through themultiple needle holes was never seen). Indeed, theonly adverse event that could likely be ascribedto cell transplantation is ventricular tachycardiawhich occurred in 4 patients, all during the firstthree postoperative weeks, with a very low rate oflate recurrences since so far, only one of these 4 pa-tients who received a defibrillator experienced twoshocks whereas Holter recordings of the remain-der of the cohort has failed to detect new seriousarrhythmic events.

The mechanisms of these potentially cellgrafting-related arrhythmias are still elusive. Aninflammatory origin is plausible since cell injec-tions are known to trigger an inflammatory re-sponse and the time course of the ventricularevent would be consistent with this mechanism.However, another possibility is that differencesin membrane properties between engrafted my-otubes and host cardiomyocytes set the stage formicro reentry circuits. We admit that for this phe-nomenon to occur, engrafted cells and host car-diomyocytes should be coupled, which we havenot demonstrated so far. This does not mean thatsuch a coupling may not occasionally occur in vivo,thereby providing a sound substrate for the ar-rhythmias. In this setting, the low incidence ofdelayed arrhythmic events could be due to en-vironmentally dictated changes in myoblast ac-tion potential morphology making it closer to that

of host cardiomyocytes (an adaptive phenomenonsimilar to what we have described for the relativeproportions of myosin isoforms).

Regardless of the mechanism(s), our data sug-gest that the incidence and/or severity of thesearrhythmias can be reduced by an appropriateprophylaxis by amiodarone coupled with a closemonitoring during the early postoperative period.At most, implantation of a defibrillator may be re-quired, a strategy which in any way, i.e., indepen-dently of the cell transplant, is likely to be indi-cated at one point in many of these heart failurepatients who meet the implantation criteria of theMADIT II trial [32].

The third conclusion drawn from our trial per-tains to efficacy. By virtue of its design (small sam-ple size, lack of a control group, concomitant revas-cularization), no definite conclusion can yet bedrawn. However, the finding that approximately60% of the initially akinetic cell-grafted scarredareas demonstrated a new postoperative systolicthickening is encouraging. These data now need tobe validated by the forthcoming multicenter ran-domized phase II efficacy trial.

Two other phase I adjunct-to-CABG cell trans-plantation studies similar to ours have been car-ried on in the United States and in Poland. So far,no data have been published in the medical litera-ture regarding outcome of patients included in UStrial. The Polish experience has been presented atthe 2002 Meeting of the European Society of Cardi-ology. The results obtained in 10 patients confirmthe feasibility and safety of the procedure, with thecaveat of ventricular arrhythmias which also oc-curred early postoperatively in two patients. How-ever, the protocol was not designed to allow conclu-sions about efficacy. Another industry-sponsoredcognitive trial has been conducted in the U.S. andentailed grafting of autologous myoblasts at thetime of left ventricular assist device implantationused as a bridge-to-transplantation, with the ob-jective of assessing the fate of grafted cells in theexplanted heart [33].

Endoventricular Approach

In a logical attempt at reducing the invasivenessof cell transplantation, percutaneous approacheshave been rapidly developed with a dramatic sup-port of the industry. Chronologically, endoventric-ular injections have first been performed owing tothe development of dedicated catheters and nav-igation systems. However, although the technicalfeasibility of this approach has been establishedclinically, there are still no experimental data sup-porting its functional efficacy in animal modelsof myocardial infarction. Only one study [34] re-ported endoventricular injections to be superior

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to the epicardial ones on the basis of a threefoldhigher intramyocardial retention of microspheresbut it is uncertain whether these data (obtainedin noninfarcted pig hearts at a single time point)pertain to the use of cells. More recently, a transve-nous approach has been developed which usesa specifically dedicated ultrasound-guided coro-nary sinus catheter with an extendable needlefor transvenous puncture and through which amicrocatheter is advanced for cell delivery [35].Our experience with this approach has shown itseffectiveness in delivering skeletal myoblasts innormal pig and sheep myocardial tissue. Techni-cally, it is attractive because of its greater sim-plicity compared with the endoventricular routebut it now remains to assess how it function-ally compares with epicardial injections in clini-cally relevant animal models of myocardial infarc-tion. In addition to isolated and unreported (exceptthrough press release) cases of percutaneous my-oblast transfer, there has been a European trialinvolving endoventricular (and, in fewer cases,transvenous) cell injections. The study has beeninterrupted because of early postprocedural se-vere arrhythmic events but should be resumed af-ter major changes in protocol, including system-atic implantation of an automatic defibrillator.

Optimization of Cell Survival

Regardless of the route of delivery, cell death re-mains a major limitation of cell transplantation asup to 90% of cells, either fetal cardiomyocytes [22]or skeletal myoblasts (unpublished personal ob-servations) die within the first hours following theinjections. Whether multiplication of cells whichhave survived can catch up this high attrition rateis yet unknown. Using a dual-marker tracking sys-tem (injection of radiolabeled male myoblasts intorecipient females and estimation of the amountof radioactivity and Y chromosome to quantifycells that have survived and cells derived from thedonor, respectively), Beauchamp et al. [36] haveidentified a distinct, behaviorally more resistantsubpopulation of skeletal myoblasts able to main-tain its proliferation potential. However, even ifsimilar proliferation events occur in heart tissue,it is doubtful that they can fully compensate forthe initially high rate of cell death, which empha-sizes the importance of optimizing cell survival toincrease the benefits of cell transplantation.

This, in turn, requires a better understanding ofthe causes of early posttransplantation cell death.Several factors are likely to be involved, includingphysical strain during injections, inflammationtriggered by needle punctures, apoptosis and is-chemia inherent in the hypovascularity of postin-farction scars. In contrast, an immune responsecan probably be ruled out in view of the very

uncommon incidence of graft-versus-host disease(primarily described after bone marrow transplan-tation and seemingly enhanced by ciclosporine[37]. Likewise, complement activation does notseem to implicated in cell damage directly butwould be rather secondary to cell death and couldthen exert its chemotactic effects to drive in-flammatory cells to the clusters of transplantednecrotic cells [38]. In contrast, the importanceof the nutritive factor is illustrated by the find-ing that transplantation of cardiomyocytes into avascularized granulation tissue doubles cell sur-vival compared with injections made into acutelynecrotic cryoinjured myocardium [22]. Likewise,genetically engineered immortalized myoblastsoverexpressing vascular endothelial growth fac-tor [39] improve functional recovery and reduceinfarct size whereas the benefits of cardiomyocytetransplantation are enhanced when cell deliveryis preceded by intramyocardial injections of fibrob-last growth factor [40]. Thus, although it remainsto determine the optimal strategy and timing ofimplementation, it looks sound that, in the future,cell transplantation will fully benefit from an addi-tional form of revascularization whether providedby coronary surgery, angioplasty or biologicalangiogenesis.

In the setting of interventions targeted at re-ducing cell death, pretreatment of cells, eithercardiomyocytes [22] or skeletal myoblasts [41]with heat shock prior to implantation has alsobeen shown to markedly increase survival of in-tramyocardial grafts. The ability of heat shock tomarkedly reduce the number of TUNEL-positivetransplanted cardiomyocytes [22] suggests thatone of its protective mechanisms could involve lim-itation of apoptosis.

The timing of injection is also likely to affectcell survival. There is probably an optimal timewindow for cell transplantation, as suggested bythe observation that too early postinfarct injec-tions may fail because of a high rate of cell deathdue to the infarct-induced inflammatory reactionwhereas late injections may loose part of their ef-ficacy because of their inability to reverse a com-pleted remodeling process [42]. Mechanical factorsare also probably involved in cell death and needto be handled by improvements in cell deliverydevices.

Concluding Comments

Among the key questions which remain to be set-tled, at least four can be identified.

First, clarification of the mechanism(s) wherebymyoblast transplantation improves function re-mains mandatory. In line with this issue, it is im-portant to assess whether this improvement can

226 Menasche

be enhanced by the selective use of a myoblast sub-population [41] or the genetic engineering of cells-to-be-grafted so as to make them overexpressingproteins involved in angiogenesis [37] or cell-to-cell coupling like connexin 43 [42].

A second important issue is to assess how skele-tal myoblasts compare with their major “competi-tor”, i.e., bone marrow-derived stem cells. Whetherthe latter share with myoblasts the possibility ofbeing usable as autografts, they theoretically havethe additional advantage of a plasticity allowingthem to transdifferentiate into cardiac and/or en-dothelial cells under the influence of appropriateenvironmental cues. So far, data obtained with ei-ther mononuclear cells cells [43] or selected sub-populations of progenitors [44,45] suggest that thetransdifferentiation potential of bone marrow istightly dependent on the presence of still living tis-sue able to provide the appropriate signals, whichimplies cell delivery at the early stage of infarctionand in the ischemic border zones as physical cell-to-cell contact seems to be a key factor of pheno-typic conversion [46]. Conversely, at a later stage,i.e., when the predominant anatomic substrate isa fibrous scar, the benefits of bone marrow cellsare less obvious. Indeed, if these cells incur pheno-typic changes in response to environmental cues, itis even sound to postulate that their engraftmentinto a fibrous tissue may rather convert them intofibroblasts [47]. It is also noteworthy that stud-ies with bone marrow cells use injections of cul-ture medium as controls. This does not allow head-to-head comparisons between different cell typesand, in this perspective, it now seems legitimateto consider that skeletal myoblasts should repre-sent the benchmark against which bone marrow-derived stem cells should be tested.

A third practical issue pertains to the mode ofdelivery. Regardless of the cell-survival strategiespreviously mentioned, it is important to designmore elaborate injection devices and to clarify theefficacy of percutaneous approaches. Seeding ofmyoblasts onto biodegradable scaffolds is anotherarea which may have interesting implications forthe repair of wall defects and congenital malfor-mations in adult and pediatric cardiac surgery, re-spectively.

Finally, it remains important to assess to whatextent myoblast transplantation could benefit pa-tients with nonischemic cardiomyopathies. Here,the encouraging results yielded by doxorubicin-induced [6] or genetic [5] models of global car-diomyopathies need to be confirmed.

In conclusion, myoblast transfer has nowreached a stage where large randomized trialssuch as the one we are initiating (300 patients)are warranted to thoroughly assess the efficacyof the procedure. These studies should match themethodologic guidelines of drug trials as this is

the prerequisite for truly determining the poten-tial benefits of cell transplantation and the extentto which these benefits translate into clinicallymeaningful improvements of patient function andoutcome.

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