Transplantation of progenitor cells and regeneration ofdamaged myocardium: more facts or doubts? Insights fromexperimental and clinical studiesMassimo F. Piepoli

The development of postinfarction left ventriculardysfunction, particularly in patients with a large myocardialinfarction, remains a major challenge. Recent experimentalstudies, showing that bone marrow cells may repair cardiactissue, have o�ered renewed hopes for the prevention ofthis ominous occurrence. This optimism is furthersupported by encouraging results from some clinical trialsin both acute myocardial infarction and in chroniccardiomyopathy, although the degree of bene�ts, theunderlying mechanisms, and the cell types involved remainto be elucidated. This review summarizes the most relevantexperimental experiences and all clinical studies supportingbut also questioning the use of bone marrow cells in

myocardial repair.

IntroductionThe regeneration or replacement o� unctional tissuesafter heart damage has been traditionally considered a‘mission impossible’ in cardiology. Reperfusion of theischaemic myocardium was the only intervention avail-able to restore the various cellular functions a�ected bymyocardial ischaemia. Recently myocardium self repairby regeneration from autologous and undi�erentiatedprimitive cells or di�erentiated cells with proliferativeproperties has been theorized [1]. However, this repara-tive process has demonstrated limited clinical importancedue to the poor capacity of regeneration and proliferationof autologous human cardiomyocytes to prevent eitherthe scar formation that follows myocardial infarction andthe loss of heart function occurring in patients withcardiomyopathy and heart failure.

Recent hopes have risen from experiences that demon-strated the possibility of replacement and regenerationo� unctional cardiac muscle achieved either by stemcells.

The term ‘stem cell’ arose from the concept that thesecells have properties analogous to those of the stemof a plant. In plants, the stem may grow to producemore stem, that is, more o� t, or di�erent structuressuch as leaves or �owers. This elegantly illustrates thetwo key properties that de�ne stem cells. Firstly, theyhave the ability to renew themselves for long periodsthrough cell division. Secondly, under speci�c con-ditions they can di�erentiat e into a spectrumof di�erentcell types.

Stem cells have a hierarchy in terms of their ability todi�erentiate into other cell types. This ability is termedtheir di�erentiation ‘potential’. In nature, the stem cellwith the greatest ability to di�erentiate into variousdi�erent cell types is the zygote, which is termed ‘toti-potent’ as all cell types of the body arise from it. Anembryonic stem cell, which arises from subsequentdivision of a zygote, is termed ‘pluripotent’ as it is capableof di�erentiating into any cell type from any of the threegerm layers (i.e., endoderm, mesoderm, ectoderm). Adultstem cells, which are present in all adult mammals, aretermed ‘multipotent’ given their ability to di�erentiateinto di�erent tissue types. Finally, the committed pro-genitor cell is termed ‘unipotent’ as it is destined only tobecome one cell type.

Broadly, stem cells can be initially classi�ed into embryo-nic or nonembryonic with the nonembryonic categorybeing subsequently split into adult stem cells and cordblood stem cells. One of the central and recurring ques-tions of all stem cell treatment modalities is which type ofstem cells to use in which setting?

For heart regeneration di�erent approaches have beenproposed, such as the transplantation of allogenic cells(e.g. embryonic stem cells, bone marrow mesenchymalcells or skeletal myoblast) or by stimulation of autologousstem cells and/or resident progenitor cells.

Embryonic stem cellsThe most primitive of all stem cells are the embryonicstem cells that develop as the inner cell mass in the

Original article

human blastocyst at day 5 after fertilization: embryonicstem cells have vast developmental potential as they cangive rise to cells of the three embryonic germ layers.They can undergo cell proliferation and form embryo-like aggregates (termed embryoid bodies) in vitro, someof which can spontaneously contract. The beating embry-oid bodies contain a mixed population of newly di�er-entiated cell types including cardiomyocytes. Embryonicstem cells spontaneously di�erentiate into endothelialprogenitor cells (EPC), haemangioblasts, mesenchymalstem cells (MSC). Haemangioblasts further di�erentiategenerating both haematopoietic stem cells (HSC) andEPC, which give rise to both vascular blood and myocytecomponents. Under the appropriate conditions (most ofwhich remain to be determined), cardiomyocytes canform from embryoid bodies as well as from EPC andMSC (Fig. 1).

Embryonic stem cells have raised particular interest,since as the prototypical stem cell, they unequivocallyful�ll all of the criteria of stemness: clonality, self-renewal, and multipotency [2,3]. Embryonic stem cellscan di�erentiate into all cell types required in the adultand hold the potential to completely regenerate themyocardium. Ethical issues that ensued from humanembryonic stem cell derivation requiring the destructionof human embryos, technical hurdles with maintainingsurvival of transplanted cells, and the concerns ofimmunologic rejection and teratoma formation stand asobstacles in the path of embryonic stem cell-basedtherapy [4]. However, these limitations may be overcomewith advances in our understanding of the speci�cationand di�erentiation of embryonic stem cell-derived car-

diomyocytes, alongside methods to limit tumourigenesis,including genetic preprogramming or in-vitro di�eren-tiation before injection. One alternative was proposed byTakahashi and Yamanaka: [5] in mouse embryonic oradult �broblast cultures could generate pluripotent stem-cell-like cells (induced pluripotent stem (iPS) cells),showing the essential characteristics of embryonic stemcells in terms of morphology, cell-surface markers, gene-expression pro�les and telomerase activity. Furthermore,iPS cell clones could be maintained in culture for severalmonths at least and could be induced to di�erentiate intoderivatives of all three embryonic germ layers bothin vitro and in vivo. These studies open up excitingprospects. However, the present methods for generatingiPS cells require genetic integration by retroviruses orlentiviruses: [6] ongoing work focuses on in�uencingcardiomyocyte speci�cation from embryonic stem cellsby manipulating signalling to enable the therapeuticstrategy of preselecting committed embryonic stemderived cardiac progenitors for transplantation [7]. Inconclusion embryonic stem cells cannot be considereda viable option for cellular cardiomyoplasty until theseveral open issue have not been successfully solved.

Although di�erent types of cells have been studied, inclinical settings adult skeletal myoblast cells and bonemarrow stem cells have beenmore extensively evaluated,together with stimulation of stem and progenitor cellsbecause their use bypasses much of the ethical and legalissues raised by the use of embryonic stem cells.

Adult skeletal myoblast cellsThe �rst clinical studies of myocardial regeneration wereperformed using adult skeletal myoblast cells. Whentransplanted, these stem cells can successfully homeand engraft within a damaged myocardium, preventingprogressive ventricular dilatation and improving cardiacfunction [8,9]. The myoblasts can be delivered into themyocardium by either intramural implantation or arterialdelivery [10,11]. Skeletal muscle satellite cells can pro-liferate abundantly in culture; and can be easily grownfrom the patients themselves (autologous) thereby,avoiding potential immune response. Myoblasts are rela-tively ischaemia-resistant (compared with cardiomyo-cytes which become injured within 20min) and theycan withstand several hours of severe ischaemia withoutbecoming irreversibly injured [12]. In animal model ofdilated cardiomyopathy the use of skeletal myoblasts,delivered by multiple intramyocardial injections, wase�ective in restoring left ventricular function, demon-strating that the functional bene�ts of transplantedskeletal myoblast can be extended to nonischaemic car-diomyopathy [13].

Human studiesAfter an initial case report [14], few small, nonrando-mized trials investigating the safety and feasibility of

Fig. 1

Embryonic stem cells spontaneously di�erentiate into endothelialprogenitor cells, haemangioblasts, mesenchymal stem cells andembryoid bodies. Haemangioblasts further di�erentiate generating bothhaematopoietic stem cells (HSC) and EPC, which give rise to bothvascular blood and myocyte components.

myoblast transplantation in patients with ischaemia car-diomyopathy have been published which have shown theef�cacy of autologous skeletal myoblast transplantationin patients with left ventricular (LV) dysfunction [9]. Incontrast more recently a large randomized controlled trialfailed in showing any signi�cant bene�cial e�ects inglobal or regional LV function (Table 1) [15–23].

Thus, several aspects warrant further investigation. Thetransdi�erentiation to a cardiomyocyte-phenotype hasnot been unequivocally demonstrated [24]. Grafted myo-blasts may display incompatible ‘wiring’ or cell-to-cellconnections with resident cardiomyocytes and do notrespond in the same way to electrical signalling andstimuli [25]. More importantly, patients receivingskeletal myoblast transplant have experienced severeand often life-threatening arrhythmias. In a �rst clinicaltrial, four out of 10 patients with severely reduced LVEFundergoing CABG after myoblast injections into scartissue developed sustained ventricular tachycardias bothacutely and several months after the operation [15]. In thelarger MAGIC trial an increased risk of ventriculararrhythmias occurring in the transplanted patients wasrecently con�rmed [23]. In the absence of electromecha-nical coupling, the underlyingmechanisms remain uncer-tain. It has been proposed that the ability of myoblasts to�re bursts of action potentials may induce deleteriousextrasystoles, through electrotonic interaction. On thecontrary, the arrhythmias may be promoted by the med-ium used to introduce the cells, rather than by the cellsthemselves [19]. Parenthetically, the functional bene�tsof myoblast transplantation may be related to the limita-tion of adverse postinfarction remodelling and/or theparacrine e�ects of transplanted myoblasts on recipienttissue, rather than to a grafted-myoblast contribution toenhance ventricular systolic function.

On the basis of these limitations, no �rm conclusionsregarding ef�cacy can be drawn at this time.

Bone-marrow stem cellsInterest in bone marrow derived stem cells (BMC) hasbeen mainly motivated by their potential to di�erentiatein cardiomyocytes, or endothelial cells and these pro-perties are enhanced by speci�c growth factors andcytokines.

Experimental studiesThe bene�cial e�ects of BMCwere shown onmyocardialdamage in mice [26] in which implanted BMC coulddi�erentiate into myocytes and coronary vessels andthereby ameliorate the function of the injured heart.Similar �ndings of cell-mediated repair of myocardialinfarction were reproduced in mouse model [27].

Bone marrow contains several stem cell populationswith overlapping phenotypes, including endothelial

Cardiac regeneration Piepoli 3

Table1

Clinical

stud

iesusingskeletal

myo

blasts-based

celltherap

yin

human

s(w

ithchronicisch

aemic

heartfailu

re)

Autho

rs,(year)

Patie

nts(n)

Design

Way

andtim

eof

deliveryafterMI

Dosag

eof

delivery(cells/m

l)Fo

llow-up

(mon

ths)

Baselin

eLV

EF(%

)Ch

ange

sin

LVEF

(unit%

)Other

Major

Find

ings

Men

ache

’(20

03)[15,16

]10

treated

Non

-RCT

TEpdu

ringCA

BG;3

–228

mon

ths

8.7

1.9

108

5224

4"(þ

4)"Re

gion

alwallm

otion

Smits

(200

3)[17]

5treated

Non

-RCT

TEn;

24–1

32mon

ths

1.9

1.1

108

636

11"(þ

9)"Re

gion

alwallm

otion

Siminiak(200

4)[18]

10treated

Non

-RCT

TEpdu

ringCA

BG;4

–108

mon

ths

4–5

106

1235

.2"(þ

6.8)

"Re

gion

alwallm

otion

Chachq

ues(200

4)[19]

20treated

Non

-RCT

TEpdu

ringCA

BG;N

A3.0

0.2

108

1428

3"(þ

24)

"Re

gion

alwallm

otion

Dib

(200

5)[20]

30treated

Non

-RCT

TEpdu

ringCA

BGor

LVAD;N

A3

108

2428

"(þ

8)"Re

gion

alwallm

otionan

dviab

ility

Gavira

(200

5)[21]

12treated

Non

-RCT

TEpdu

ringCA

BG;3

–168

mon

ths

1.9

1.2

108

1236

8"(þ

20)

"Re

gion

alwallm

otionan

dviab

ility

Causmic

(200

8)[22]

12treated11

controls

RCT

TEn;

24–1

32mon

ths

3–60

107

12<40

""Re

gion

alwallm

otionan

dviab

ility

MAGIC

(200

8)[23]

97treated;

30controls

RCT

TEpdu

ringCA

BG;

>4wee

ks4–

810

86

25–2

8¼

#LV

ESVan

dLV

EDV

AMI,acutemyo

cardialinfarction;CA

BG,coron

aryartery

by-passgraftin

g;CD

133

þbo

nemarrow-derived

CD13

3þcells;C

PC,circ

ulatingbloo

d-de

rived

prog

enito

rcells;IC,

intracoron

ary;IM

,intramyo

cardial;LV

AD,leftv

entricular

assist

device

implan

tatio

n;MNC,

Bone

marrow-derived

mon

onuclear

cells;M

SC,b

onemarrow-derived

mesen

chym

alcells;N

A,n

otavailable;NC,

Bone

marrow-derived

nucleatedcells;R

CT,ran

domised

controlledtrial,RP

CT,

rand

omised

placeb

ocontrolledtrial;TEn,

tran

send

ocardial;T

Eptran

sepicardial;year,y

earof

publication.

stem/precursor cells (EPCs), haematopoietic stem cells(HSCs), mesenchymal stem cells (MSCs), and multi-potent adult progenitor cells (MAPCs) [28]. In exper-imental setting adult EPCs transdi�erentiate into activecardiomyocytes [29], although how extensively thisoccurs in clinical setting is presently unknown. Althoughthis cell-mediated myocardial repair was initially charac-terized as resulting from HSC’s ability to transdi�erenti-ate to cardiomyocytes, HSC plasticity has been dif�cultto reproduce and both its signi�cance and basis remainundetermined.

Bone marrow-derived MSC and stromal cells (thoughtto have many properties of MSC) exhibit a high degreeof plasticity allowing them to be employed as a self-renewing autologous source of progenitor cells (fromadults), with the potential for di�erentiating into cardio-myocytes and can be used in cellular cardiomyoplasty invitro and in vivo by creating three-dimensional aggre-gates upto 50% of human MSC express cardiac protein[30].

Upon treatment with speci�c agents (e.g. 5-azacytidine),MSCs can di�erentiate into synchronously beatingcardiomyocytes [31]. The injection of MSCs after theirexpansion in culture can also be used in the rescue of anabnormal mouse cardiac phenotype [32] and may provee�ective in repairing a broader array of cardiac damageincluding myocardial infarct. Furthermore, these bone-marrow derived stem/precursor cells also can prevent theprogression of cardiomyocyte apoptosis and stem cardiacremodelling [33]. However, an experimental study usingMSC subpopulation reported the appearance of micro-infarctions following intracoronary delivery to a canineheart and suggested that care must be exercised whenpuri�ed BMC subpopulation are studied [34].

Furthermore the mechanism of BMC-mediated augmen-tation of cardiomyocyte number and function remainscontroversial: transdi�erentiation [28], cell fusion withpreexisting cardiomyocytes, paracrine e�ects of trans-fected cells have been hypothesized [35] Cell fusionhas been demonstrated between cardiomyocytes andnoncardiomyocytes in vivo and in vitro [36] whereasthe data in support of transdi�erentiation (particularlywith HSCs) have not always been replicable. Furtherresearch is needed to clarify these issues.

Human studies in acute myocardial infarctionInspired by the exciting experimental data, several trialswere initiated to test whether cell therapy is well toler-ated and feasible in patients after acute myocardialinfarction (AMI). Some have decried the clinical trialsas being premature without a more complete understand-ing of the underlying mechanisms [37], whereas othershave pointed out that the clinical trials are justi�ed by thepotential bene�ts of cell therapy [38] All clinical studies

included patients with AMI who had undergone primaryangioplasty and stent implantation to reopen the infarct-related artery, and cells were infused intracoronary byusing the stop-�ow balloon catheter approach. In thisregard, the clinical studies di�er signi�cantly from theanimal studies, where the infarct related artery was notreperfused and cells were directly injected into themyocardium. The clinical trials using BMC are presentedin Table 2 [22,39–51]: the combined experience fromseveral hundreds of patients suggests that intracoronarydelivery of unselected BMCs (all nucleated cells ormononuclear cell fraction only) is well tolerated in theshort-term and mid-term (3–18 months). Furthermore inthe larger study, the REPAIR-AMI trial, intracoronaryinfusion of BMC was associated with a reduction in theprespeci�ed combined clinical end point of death, recur-rence of myocardial infarction, and any revascularizationprocedure at 1 year [49].

This trial has also shown that the patients with a baselineLVEF at or below the median value derived the mostbene�t. The magnitude of LV contractile recovery wasinversely related to the baseline LVEF, con�rming similarobservations in the Transplantation of Progenitor Cellsand Regeneration Enhancement in Acute MyocardialInfarction (TOPCARE-AMI) pilot trial [41]. Thus,enhanced recovery of contractile function may bebene�cial speci�cally in patients with large infarcts anddepressed LV function. Interesting a dose-related e�ect ofautologous BMC transplantation on the myocardial func-tion has been con�rmed by a recent meta-analysis [52].

Clinical surveillance, Holter monitoring, and data from anelectrophysiological study indicate that intracoronaryBMC transfer is not associated with an increased pro-pensity to ventricular (or supraventricular) arrhythmias.Direct injection of �ltered nucleated BMCs into theacutely infarcted myocardium in rats has been found toinduce intramyocardial calci�cations. No evidence forintramyocardial calci�cations (or tumour formation) hasbeen obtained in patients in the longer follow-up studyafter intracoronary delivery of Ficoll or gelatin gradient-puri�ed BMCs [47].

No bleeding complications were noted after bone marrowharvest. Intracoronary BMC infusions did not appear toin�ict additional ischaemia damage to the myocardium orto promote a systemic in�ammatory reaction, because nofurther increases in serum troponin or CRP levels wereobserved. No increased rates o� n-stent restenosis wereobserved after transfer of unselected BMCs, but only afterselected CD133 þ population were used [45]. More evi-dences are necessary to establish that these side e�ects arecausally related to a speci�c subpopulation cell transfer.

So far no trial has demonstrated a signi�cant e�ect ofunselected BMC transfer on LV end-diastolic volumes,

suggesting a limited impact on LV remodelling afterAMI. Only MSC transplantation has shown improvementin regional wall motion and global LVEF and reduction inLV end-diastolic volume compared with a randomizedcontrol group that had received an intracoronary infusionof physiological saline, although a clear description of thecellular preparation is lacking in this report [44].

Autologous and allogeneic MSCs have been used inseveral clinical trials including Crohn disease, osteogen-esis imperfecta, and graft-versus-host disease. A multi-centre, phase I, placebo-controlled, double-blind trial ofallogeneic MSCs has been completed and will provideinformation regarding the safety and ef�cacy o� ntrave-nously administered cells in patients with acute myo-cardial infarction (MI).

Stem cell selection and preparation prior to delivery toincrease the density of the cell type o� nterest at the siteof transplantation have been investigated not only toaugment the rate of success in tissue regeneration butalso to minimize the chance of cotransplanting contami-nating undi�erentiated stem cells and hence reduce therisk of developing a teratoma. Cell sorting using cell type-speci�c membrane markers, genetic methods using cell-type-speci�c promoter or selected survival by antibioticresistance have been proposed [53].

Human studies in chronic cardiomyopathyFew trials studies have investigated the application ofstem cells in chronic cardiomyopathy mainly with leftventricular dysfunction: all but one [54] of these studieswas performed in chronic ischaemic heart diseases(Table 3) [22,54–61]. The �rst trials were uncontrolled:no procedure-related complications nor serious ventricu-lar arrhythmias were observed upto 14 months but per-fusion of the cell-injected area and LVEF wereimproved. However, because of the lack of a controlgroup, the ef�cacy of this approach was uncertain. Morerecently two trials were randomized controlled have beenpublished, both performed in ischaemic cardiomyopathybut with disappointed results: in one intracoronary infu-sions of unselected mononuclear BMCs by the stop-�owballoon catheter technique had a limited e�ect on LVEF[59], whereas in the other where BMCs were infusedeither intracoronary or transendocardially no e�ect wasreported [22].

Probably the infusion in chronic necrotic or underper-fused areas, does not favour the growth and proliferationof BMCs, determining the inef�cacy of this technique formyocardial generation.

Stem cells and progenitor cell stimulationAs implantation of BMCs initially required surgical inter-vention and the procedure was often accompanied by ahighmortality rate, with only a 40% of successful grafting,

Cardiac regeneration Piepoli 5

Table2

Clinical

stud

iesusingau

tologo

usbo

nemarrow

cells

inAMI

Stud

y(year)

Patie

nts(n

)Design

Way

andda

yof

deliveryafterAMI

Dosag

eof

delivery

Follo

w-up

(mon

ths)

Baselin

eLV

EF(%

)Ch

ange

sin

LVEF

(%)

Other

major

�nding

s

Straue

r(200

2)[39]

10MNC;

10controls

Non

-RT

IC;5

–92.8

2.2

107

357

8¼

#infarctsize;"region

alfunc

tion;¼

LVED

VTO

PCARE

-AMI(20

02)[40]

;(200

4)[41]

29MNC;

30CP

C;11

controls

NoRT

IC;5

22.1

0.8

108;1

.61.2

107

1251

9"(þ

8)"region

alwallm

otion;#LV

ESV;

¼LV

EDV

Fernan

dez-Aviles(200

4)[42]

20MNC;

11controls

NoRT

IC;1

46

7.8

4.1

107

1151

6"(þ

6)¼

LVED

V9.3

6;

CIT

CR

oN

CN

M5

]34[)4002(

ehteuK

2.3

107

1244

11¼

¼region

alwallm

otionan

dcontractility

8.481

;CI

TC

Rslortnoc

53;

CS

M43

]44[)4002(

nehC

6.0

1010

349

9"(18þ)

"region

alwallm

otion;#infarctsize,L

VEDV

Bartun

ek(200

5)[45]

19CD

133

þ;1

6controls

Non

-RT

IC;1

11

122

106

445

2"(þ

7)"region

alwallm

otion,

perfusion;"in

sten

tsten

osis

7CI

TC

Rslortnoc

05;

CN

M05

]64[)6002(I

MAT

SA

107

641

10¼

¼LV

EDV,

¼infarctsize

BOOST

(200

6)[47]

30NC;

30controls

RCT

IC;6

5.21

109

1850

10¼

"LV

EFat

12bu

tno

tat

18mon

ths

4.21

;CI

TC

PR

obecalpC

MB

carfnU

76]84[

)6002(IM

ETS

108

448

5¼

#infarctsize

36

–3

;CI

TC

PR

obecalp301

;C

MB

301]94[

)6002(IM

A-RI

AP

ER

108

447

10"(þ

5)#infarctsize;"region

alfunc

tion

8.35.0

;CI

TC

PR

obecalp01

;C

MB

01]05[

)6002(IM

ATS-T

CT10

86

539

"(þ

5)"region

alwallp

erfusion

557

;CI

TC

Rslortnoc

22;

CM

B44

]15[)6002(

nizuleM

106

342

2"(þ

5)85

6A

N6

–2

;CI

TC

PR

slortnoc93

;C

MB

93]22[

)8002(LL

EC

NIF10

"(þ

7)After

thrombo

lytic

therap

y

Iseq

ualto‘¼

’,un

chan

ged.

Referto

Table1.

the development of noninvasive method became impera-tive. One such approach employed cytokine treatment,stem cell factor (SCF) and/or granulocyte colony-stimu-lating factor (GCSF) to mobilize endogenous BMCs anddirect their integration or homing to the infarcted heartpromoting repair.

Experimental studiesThe �rst suggestion that cytokine-induced stem cellmobilization may be used to enhance cardiac repair camefrom studies to increase EPC levels for neovasculariza-tion in hind limb ischaemia. Mice injected with SCF andGCSF exhibited a substantial increase in the number ofcirculating stem cells (from 29 in nontreated controls to7200 in cytokine-treated mice). This approach demon-strated to stimulate myogenesis and angiogenesis in theinfarcted area and to improve cardiac function after AMI[62,63]. It has been postulated that GCSF may accelerateinfarct healing by enhancing macrophage in�ltration andmatrix metalloproteinase activation, and suppress cardi-omyocyte apoptosis by activating the cytoprotectiveSTAT3 transcription factor, suggesting that stem cell–independent mechanisms may contribute to the e�ectsof GCSF after AMI.

Human studiesAnimal studies rapidly led to initiation of clinical trials toassess the ability of G-CSF to mobilize stem/progenitorcells in patients with coronary artery disease [64] andAMI [65]. G-CSF-mobilized blood from patients con-tained 5-fold to 100-fold higher levels of HSCs, MSCs,and EPCs, compared with nonmobilized blood; however,the ability of these cells to improve cardiac remodellingand function after AMI has been disappointing [66].Some initial concerns regarding the safety of thisapproach few days after AMI was raised in clinical set-tings (Table 4) [67–73]. In a �rst clinical investigation[67], no deaths or substantial arrhythmias, aggravation ofheart failure, or angina occurred during GCSF adminis-tration and a 6-month follow-up period, but infusions ofGCSF mobilized peripheral blood-derived leukocytesand induced a 65% increase in serum creatine kinase-MB levels, indicative of mild myocardial damage. Moreseriously, 7 out of ten treated patients developed in-stentrestenosis at 6 months, which prompted a prematuretermination of the study. GCSF has the potential toactivate neutrophils, by stimulating adhesion to endo-thelial cells thereby in�uencing their recruitment at siteso� n�ammation and tissue injury. It was hypothesizedthat these systemic e�ects of G-CSF may have contrib-uted to excess neointima proliferation and restenosis.

Less worrisome results came from the FIRSTLINE-AMIstudy [68], and the REVIVAL [69] trial where GCSFtreatment after stent implantation was not associatedwith an enhanced rate o� n-stent restenosis, or otherserious adverse events: but only the FIRSTLINE-AMITa

ble3

Clinical

Stud

iesUsing

stem

cells

inch

roniccardiomyo

pathy

(stneita

P)raey(

ydutS

n)Design

Way

ofde

livery

Dosag

eof

delivery

Follo

w-up

(mon

ths)

Baselin

eLV

EF(%

)Ch

ange

sin

LVEF

(%)

Other

major

�nding

s

Stam

m(200

3)[54];(20

04)[56]

12au

tologo

usCD

133

þNon

RCT

IMdu

ringCA

BG1.5

106

1239

9"(þ

9%)

"pe

rfusion;

#LV

EDV

Perin

(200

3)[57]

14MNC;

7controls

Non

RCT

Ten

3.0

0.4

107

430

6"(þ

5%)

"Re

gion

alwallm

otion;

#LV

ESV

9CI

TC

Rno

NC

NM

81]85[

)5002(T

CAI

107

353

8"(þ

7%)

"FD

Gup

take

intheinfarctedregion

TOPC

ARE

-CHD

Asm

uss(200

6)[59]

51MNC;

35CP

C;16

controls

RCT

IC17

080

106;2

312

106

340

11"(þ

3on

lyin

MNC)

"Globa

land

region

alLV

func

tion

ABC

D(200

6)[54]

24MNC;

20controls

Non

RTIC

1.6

106CD

34þ/m

l.12

207

"(þ

5%)

NoCA

DCH

F;#LV

ESV

8neT

TC

Rno

NC

NM

01]06[

)7002(IM

MB

AT1

107

1235

4"(þ

7%)

4.9neT

TC

Rno

NC

NM

51]16[

)7002(seree

B1.4

107

323

8"(þ

4%)

"Re

gion

alwallm

otion

6A

NCI

ron

ETT

CR

)slortnocro

CIro

nET(

CN

M36

]22[)8002(

gnA

¼¼

Inwallm

otionor

scar

Iseq

ualto‘¼

’,un

chan

ged.

Referto

Table1.

study showed some positive �ndings, where thebene�cial e�ects of GCSF were magni�ed by an unex-pected decrease in LVEF in the control group. A slightlydi�erent and complementary approach has been pro-posed to amplify stem cell mobilization, with positiveresults and no complication: after being harvested bydaily G-CSF injection for 3–5 days BMC were isolatedby apheresis and delivered via an over-the-wire ballooncatheter.

Open issues and future developmentsWhat is the mechanism of action?Large debates have been promoted by the lack of anyclear information on the exact mechanisms responsiblefor the observed bene�t of this regenerative therapy. Itseems that the weight of the evidence implicates mech-anisms other than cellular dedi�erentiation and di�usion.Di�erent cell types, derived from tissues as varied as cordblood, adipose tissue and peripheral blood, behave sim-ilarly to bone-marrow cells after being injected directlyinto the heart or travelling to the ischaemic site afterintravenous injection, and therefore any reportedimprovements in cardiac function are likely to bemediated predominantly by paracrine e�ects [74].

What is the best stem cell delivery route?Multiple method of delivery for stem cells have beeninvestigated but the optimal route is still obscure. Theoptimal delivery route for autologous cell transplantationnot only varies according to the administered cell typebut will be in�uenced in the future by our ability toenhance the migratory capacity of stem cells.

Systemic delivery of stem cells can be an easy method forthe regeneration of the injured heart but its e�ectivenessis dependent on successful homing and retention of cellsbefore the secretion of paracrine factors or transdi�er-entiation, or both. Alternatively, for a direct delivery tothe heart, twoways have been experimented: either by anintracoronary arterial route or by injection into the ven-tricular wall via percutaneous endocardial, percutaneoustranscoronary venous, or surgical epicardial approaches.Intracoronary delivery enables the application of a maxi-mum dose of cells homogeneously to the site o� njuryalthough this mode is less ef�cient for delivery to non-perfused regions of the infarct related artery. Homing ofintraarterially applied progenitor cells requires theirextravasation and migration to the surrounding ischaemiatissue. Although BMCs and haematopoietic stem cellscan extravasate [75], this has not been shown for all celltypes and larger, less motile cells, such as skeletal myo-blasts may even obstruct the microcirculation, leading toembolic MI [76]. Direct injection is the preferred deliv-ery method for chronic heart failure patients with con-siderable scar tissue. Cell homing signals such as SDF-1and VEGF are expressed at low levels at late stages of

Cardiac regeneration Piepoli 7

Table4

Clinical

stud

iesusingstem

andprog

enito

rcellmob

ilizatio

n

(stneita

P)raey(

ydutS

nngise

D)

Way

andda

yof

deliveryafterAMI

Dosag

eof

delivery

Follo

w-up

(mon

ths)

Baselin

eLV

EF(%

)Ch

ange

sin

LVEF

(%)

Other

major

�nding

s

MAGIC

(200

4)[67]

10GCS

Fan

dCP

C;10

G-CSF;

7controls

RCT

SI;

>48

h;(2

to27

0da

ys)

10mg/kg

for4da

ys6

488

"(þ

6)"insten

tsten

osis

FIRS

TLINE_AMI(20

05)[68]

25GCS

F;25

controls

RCT

SI;8

530

min

10mg/kg

for6da

ys4

484

"(þ

8)Preven

tionof

LVremod

ellin

gRE

VIVA

L-2(200

6)[69]

56GCS

F;58

placeb

oRP

CT

SI;5

days

10mg/kg

for5da

ys6

518

¼Noe�

ecton

infarctsize

G-CSF-STEMI(20

06)[70]

22G-CSF;2

2placeb

oRP

CT

SI;6

h-7

days

10mg/kg

for5da

ys3

4112

¼Neu

tral

e�ect

STEM

MI(20

07)[71]

39G-CSF;3

9placeb

oRP

CT

SI;1

2h

10mg/kg

for6da

ys6

NA

NA

Associatio

nbe

twee

ncirculating

MSC

andLV

EFZh

an-Qua

n(200

7)[72]

35treated;

35controls

Non

-RT

SIan

dIC;6

days

GCS

F:30

0–60

0mgfor

5da

ysan

dCP

C:7.5

107

650

8"(þ

7)"region

alwallm

otion;

#LV

ESV,

#LV

EDV

MAGIC

CELL-3-DES

(200

6)[73]

25treated;

25controls

RCT

IC;4

days

GCS

F:10

mg/kg

for

3da

ysan

d;CP

C:1.5

108

652

9"(þ

5)"region

alwallp

erfusion

;¼

LVED

V,¼

LVESV

SI,sub

cutane

ousinjections;‘

¼’,un

chan

ged;

forothe

rab

breviatio

ns,refer

toTable1.

disease, limiting any homing potential following intracor-onary application [77].

Injection into the injured myocardium is particularlysuited for large cells such as myoblasts and mesenchymalstem cells and is not limited by cell uptake from thecirculation or embolic risk. However, injection of pro-genitor cells into necrotic tissue, which lacks both blood�ow to provide oxygen and nutrients and healthy sur-rounding cardiomyocytes to provide paracrine support,reduces graft survival and di�erentiation.

Is there any role for the cardiac resident stem cells?Encouraging new perspectives have been harvested bythe possibility to promote cardiac resident stem cells.The �rst indication that the adult heart harboured apopulation of stem cells possessing regenerative proper-ties came from a study of AMI patients showing anelevated number o� mmature cardiomyocytes, capableof mitotic division, in the infarct border zone [78].Whether these progenitors were of endogenous or circu-lating origin was uncertain, but the identi�cation of aresident stem cell population within the heart, whichsupports myocardial regeneration, has exposed newopportunities for cardiac repair [79].

A word of cautious has been raised, however. Over thepast few years, cell populations expressing stem-cellmarker proteins such as Kit, stem-cell antigen 1(SCA1) and multidrug resistance protein 1 (MDR1) havebeen identi�ed in the human or mouse heart, or both,albeit in minuscule quantities [80]. Although the initialevidence for an adult stem cell or progenitor-cell popu-lation in the adult heart, which could potentially beharnessed for cardiac repair, was initially welcomed withenthusiasm, scepticism has since grown. These Kit-expressing cells in tissues of solid organs, including theheart, are thought to have left the bone marrow inminuscule quantities to scavenge pathogenic moleculesin peripheral tissues as part of a mechanism to promote alocal innate immune response. They are not then actualheart cells but bone-marrow cells out of place. Inaddition, many of the cells expressing Kit that weredetected in biopsied samples of adult human heart wererecently reported to coexpress markers of mast cells (cellsof the immune system) and to lack expression of cardiactranscription factors NKX2-5 and islet 1 (Isl1), crucialmarkers of the cardiac progenitor cell state in fetal hearts[81]. These cells are, therefore, not cardiac progenitorcells at all.

Two approaches are currently under investigation tofurther investigate this issue. The �rst is to explantcardiac stem cells from the heart, induce their prolifer-ation and di�erentiation ex vivoand to engraft them backinto the damaged heart [82]; and the second, which todate has received less attention, is to stimulate cardiac

progenitor cells in situ to proliferate, migrate, and di�er-entiate in the infarcted heart without ex-vivo manipula-tion.

Among the �rst approach, a clinically applicable methodfor the isolation and expansion of adult human cardiacstem cells from percutaneous endomyocardial biopsyspecimens has been recently proposed. In culture, humancardiac stem cells self-organize into spherical clusterscalled cardiospheres. Human and porcine cardiospherescan di�erentiate into cardiac myocytes in vitro. Forin-vivo experiments, infarcts in mice with severe com-bined immunode�ciency were created: direct injection ofhuman cardiospheres into the infarct border zone led tomyocardial regeneration by histology and to functionalimprovement [83].

For the second approach, a major therapeutic goal is theidenti�cation o� actors that stimulate cardiac stem cellsto form replacement cardiomyocytes and vascular pro-genitors for regeneration of the injured heart [84].

Other open issues?The di�erent results in di�erent clinical trials (e.g.STEMI [47] vs. REPAIR-AMI [48]) may be the resultof subtle di�erences in cell handling and preparation, asdi�erences in BMC subpopulation pro�les could resultfrom di�erent technique. Other discrepancies in thesetrials were evident: methods, endpoints, imaging toolsevaluating cardiac function (MRI, echocardiography orangiography), patient selection, time from onset of AMIto percutaneous intervention, time from intervention tocell delivery: all these aspects may interfere with theobserved responses.

The intellectual property associated with cell-basedtherapies is distinctly di�erent from that associated withstandard pharmaceutical development. This can contrib-ute to the lack of major commercial investment intoclinical development programmes. This has left phys-ician researchers as the driving force for development,which encumbers them with greater responsibilities andinvestigative challenges. Individual researchers aretraditionally competitive and tend to conduct multiplesmall studies rather than larger more informative ones.Moreover, the lack o� unding for infrastructure andorganization for multicentre trials could be a majorbarrier.

ConclusionIt comes immediately evident several weaknesses ofwhat is known and what we are expecting from futureinvestigations. Still few randomized controlled studieshave been published, with di�erent cell types and prep-arations, each in a small number of patients (few hun-dreds of patients) with di�erent disease states, with shortfollow-up (from three till 18 months, but 6 months on

averaged). So although the issue on safety is generallyconsidered ruled out, with such few numbers we stillneed a ward of caution, especially if we wish to bereassured about the risk of tumour formation whichmay take several years before it can be excluded.

Open concerns are evident also in terms of ef�cacy andclinical meaning. Preliminary results of human clinicaltrials have shown a modest improvement in the cardiacfunction of patients with acute myocardial ischaemia andinfarct [85]. When transplantation was applied to patientswith chronic myocardial disease or damage secondary tomyocardial infarction the results were less de�nitive.However, no data are still available on crucial endpointssuch as increased survival or reduced hospitalization frompatients treated with cardiac regeneration. Few studieshave demonstrated reduction in LV volume suggestingthe lack of evident antiremodelling e�ect this interven-tion. Also the not ominously presented increases inLVEF are mostly in the range of the variability of themethodology used to assess it (imaging technique) raisingsome concerns regarding the real importance and clinicalimpact.

On the other side because the paucity of successfultechniques to e�ectively treat heart failure, there maybe mounting pressure to expedite the clinical applicationof cells transplant even before the mechanisms (as well asthe long-term e�ects) are fully understood.

We should proceed in a manner that maximizes both theinformation gained and the safety of patients. Patientsshould be treated with cells only as part of randomized,controlled trials and only after they understand thatneither the ef�cacy nor the long-term risks of thisapproach are established. Future trials should be poweredto examine clinical end points and patients should befollowed over the long term and for both bene�cial andadverse e�ects. The enrolment of patients with a poorprognosis (i.e., large infarcts, poor left ventricular func-tion) makes sense. They have the greatest need fortherapeutic approaches and thus have the most favour-able risk–bene�t ratio. Demonstration o� ncrementalbene�t, as compared with conventional therapy, is easierin these populations, and subgroup analyses suggest thatthey are the most likely to bene�t. However, we mustkeep in mind that the patients with too extensive myo-cardial damage and more advanced tissue degenerationcould not be the more ideal target for this therapybecause it could be less easy to get a successful homingof progenitor cells or a favourable development intofunctional tissues.

The enrolment of patients with heart failure who use leftventricular assist devices as a bridge to transplantationwould also provide a unique opportunity to examinecellular and molecular mechanisms through analyses of

cardiac tissue acquired both before cell infusion (atimplantation) and after (at transplantation). Simul-taneously, we must continue to support basic and transla-tional research that can help guide clinical investigation.

In conclusion, several issues remain to be addressed infuture studies. Only in large multicentre setting, withrigorous methodological criteria we will learn more aboutthe hopes raised but not jet con�rmed.

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Articolo pubblicato su: Journal Of Cardiovascular Medicine - 2009