MINERVA CARDIOANGIOL 2005;53:549-64

Cardiac stem cell therapy

A
for myocardial regeneration
A clinical perspective

C
T ® DI
B. DAWN, E. K. ZUBA-SURMA, A. ABDEL-LATIF, S. TIWARI, R. BOLLI

H E
Myocardial infarction and other pathologic Institute of Molecular Cardiology
IG M
conditions of the heart result in loss of car- University of Louisville, Louisville, KY, USA
diomyocytes, scar formation, ventricular
remodeling, and eventually heart failure. Since
pharmacologic and interventional strategies
fail to regenerate dead myocardium, heart fail-
the potential utility of cardiac stem cells for
R A
ure continues to be a major health problem
worldwide. Recent studies in animal models therapeutic myocardial regeneration.
of myocardial infarction and heart failure have Key words: Cardiac stem cell - Myocardial regen-
Y V

demonstrated that various subsets of adult eration - Myocardial infarction - Cardiomyo-

primitive cells can regenerate functional car- pathies.
diomyocytes and cardiac vasculature with
P R

improvement in cardiac structure and func-

tion. Small clinical trials of cell therapy in
patients with myocardial infarction and
ischemic cardiomyopathy have recapitulated M yocardial infarction (MI) leads to loss
of cardiomyocytes and scar formation
O E

these beneficial effects in humans with infarct which over time result in progressive left
size reduction and improvement in ejection ventricular (LV) remodeling and congestive
fraction, myocardial perfusion, and wall
C IN

heart failure.1 Until recently, the heart was

motion. Several phenotypically distinct cell
populations have been utilized for cardiac
regeneration, and the relative merits of one
cell over another remain to be determined. The
recent discovery of adult cardiac stem cells has
M
viewed as a terminally differentiated organ
that lacked the ability to heal itself 2 and car-
diac regeneration with functional new car-
diomyocytes was considered impossible.

sparked intense hope for myocardial regener-
ation with cells that are from the heart itself
and are thereby inherently programmed to
reconstitute cardiac tissue. The purpose of this
review is to summarize the evidence regard-
ing the feasibility of cardiac repair in humans
via adult stem/progenitor cells, and to discuss
This publication was supported in part by the National
Institutes of Health grants R01 HL-72410 (B.D.), HL-55757, HL-
68088, HL-70897, HL-76794, and HL-78825 (R.B.) and National
AHA Scientist Development Grant 0130146N (B.D.).
Address reprint requests to: B. Dawn, MD, Division of
Cardiology, University of Louisville, Louisville, KY 40292,
USA. E-mail: buddha@louisville.edu
Thus, therapy of heart failure has focused
predominantly on improving patient symp-
toms with pharmacotherapy, restoring blood
supply via interventional strategies, and using
assist devices and cardiac transplantation in
end-stage disease.3 However, while these
therapeutic approaches can only ameliorate
the symptoms of heart failure, they fail to
reconstitute dead myocardium with func-
tional new cardiomyocytes.
Over the past decade, several studies have
reported the ability of adult stem/progenitor
cells to differentiate into cardiomyocytes as

Vol. 53, N. 6 MINERVA CARDIOANGIOLOGICA 549

DAWN
well as cells of the vascular lineage both in
vitro and in vivo.4-11 Consistent with these
observations, in animal models of MI and
heart failure, it has been repeatedly demon-
strated that therapy with adult primitive cells
induces cardiac repair, improves LV function,
A
and halts LV remodeling.5, 6, 12-16 Collectively,
these findings have generated tremendous
enthusiasm toward formulating a safe and
C
effective strategy to achieve therapeutic car-
diac repair by cell therapy.
T ® DI
In 2003, Beltrami et al.8 isolated and char-
acterized a lin-/c-kit+ resident cardiac stem
cell (CSC) population from the adult heart
that can differentiate into all of the 3 major
functional cell types in the heart, cardiomy-
H E
ocytes, smooth muscle cells, and endothe-
lial cells, and repair infarcted myocardium
IG M
in vivo with improvement in cardiac func-
tion and anatomy. Since these cells fulfill the
criteria for adult organ-specific stem cells,17-
19 including clonogenicity and multipoten-
tiality,8, 20 it is hoped that CSCs will offer a
R A
therapeutic advantage for cardiac repair in
humans. Following a summary of the data
Y V
from the initial clinical trials with different
types of adult stem/progenitor cells, this
review will focus on the advances made in
P R
the identification and characterization of CSCs
and their potential therapeutic utility for
O E
myocardial regeneration.
C IN
Myocardial regeneration:
bench to bedside
Despite continued skepticism,21-25 cell ther-
M
apy for acute MI (AMI) and ischemic car-
diomyopathy has progressed at an unprece-
dented translational rapidity. Because of iso-
lated negative results from animal studies,24,
26 the mechanistic underpinnings of clinical
trials were questioned,26 and it was suggest-
ed that clinical trials should be deferred until
the unresolved mechanistic issues are
addressed.21, 24 Fortunately, fueled by encour-
aging results from small clinical studies that
showed improvement in LV function and per-
fusion, there has been an explosion of cell
therapy studies in humans.27 Table I 28-53 sum-
marizes studies that have employed various
550 MINERVA CARDIOANGIOLOGICA
CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION
adult stem/progenitor cell populations for
cardiac regeneration in humans. These stud-
ies may be broadly divided into several cat-
egories based on the cell types used.
Bone marrow mononuclear cells
Since bone marrow mononuclear cells
(BMMNCs) can be isolated relatively quickly
by density gradient centrifugation from total
bone marrow cells, this fraction can be pre-
pared with relative ease and speed. In a small
study by Hamano et al.,28 intramyocardial
injection of BMMNCs during coronary artery
bypass graft (CABG) surgery in patients with
ischemic heart disease improved myocardial
perfusion. In the study by Strauer et al.29
autologous BMMNCs harvested from patients
with AMI were injected following percuta-
neous coronary intervention (PCI). Compared
with patients that received standard therapy,
cell-treated patients showed improvement in
regional wall motion and contractility, small-
er infarct size, and improved myocardial per-
fusion. No significant improvement was not-
ed in LVEF. No significant adverse effect due
to cell therapy was observed.29 Several stud-
ies that used BMMNCs for cardiac repair have
since been reported, and all of these studies
30-36, 38, 40, 41 except two 37, 39 have shown
improvements in one or more parameters of
LV contractility, anatomy, and perfusion,
including global LV ejection fraction (LVEF),
regional wall motion/thickening, LV end-sys-
tolic volume (LVESV), and myocardial per-
fusion. Improvements in patient symptoms,
exercise capacity, and New York Heart
Association functional class have been report-
ed.33, 34 Increased myocardial microvessel
density at the site of BMMNC injection has
also been noted at autopsy.40 Interestingly,
BMMNC therapy has been used successfully
via the intracoronary route,29, 32, 35 as well as
transepicardial 28, 40 and transendocardial 33, 34,
36, 38 routes in patients with AMI, 29, 32, 35
ischemic heart disease,28, 34, 40, 41 and ischemic
cardiomyopathy.33, 36, 38 Despite these diverse
patient subsets and modes of delivery, no
significant adverse effect of BMMNC therapy
has been reported thus far. However, since
bone marrow-derived cells can contribute to
Dicembre 2005
CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION
tumor neovascularization,54 the potential for
angiomatous neoplasms should be examined
in longterm studies. Also, the total number of
cells injected in these studies has varied wide-
ly (Table I) and the optimal number of cells
for different modes of delivery needs to be
A
determined in dosing studies. Further, despite
documented improvement in LV function and
perfusion, the mechanistic underpinnings of
C
BMMNC therapy in humans, i.e., differentia-
tion of BMMNCs into cardiomyocytes and
T ® DI
vascular cells vs cell fusion vs favorable alter-
ations in myocardial matrix and scar due to
paracrine effects, remain largely unknown.
H E
Unfractionated bone marrow cells
Unfractionated bone marrow cells (BMCs)
IG M
have also been shown to improve LV function
and perfusion following transendocardial 42
as well as intracoronary 43 injection. In the
study by Fuchs et al.,42 electromechanical
R A
mapping (EMM) guided transendocardial
injection of BMCs improved anginal symp-
toms and myocardial perfusion. In a rela-
Y V
tively large randomized clinical trial of cell
therapy in humans, Wollert et al.43 reported
significant improvement in regional and glob-
P R
al LVEF and regional wall motion in cell-treat-
ed patients. No increase in the incidence of
O E
arrhythmia or restenosis was noted. Despite
the proven efficacy and safety, the underly-
ing mechanism of these beneficial effects
C IN
with BMC therapy remains to be elucidated.
AC133+ BMCs
M
AC133+ BMCs exhibit both hematopietic
and endothelial differentiation potential and
thereby, may represent a subpopulation suit-
able for inducing angiogenesis. In the first
study, transepicardial injection of AC133+
BMCs during CABG resulted in improved
myocardial perfusion and LVEF and smaller
LV end-diastolic volume (LVEDV).44 Two
patients experienced supraventricular tach-
yarhythmia, a relatively common occurrence
during the postperative period following
CABG. In a subsequent larger study with a
control group,45 intracoronary delivery of
AC133+ BMCs resulted in improvement in
Vol. 53, N. 6 MINERVA CARDIOANGIOLOGICA
DAWN
regional wall motion and global LVEF and
reduction in myocardial perfusion defect.
However, 1 patient experienced spontaneous
sustained ventricular tachycardia (VT) and
sustained VT could be induced in another
with poor LV function. Importantly, during
follow-up, a higher rate of in-stent restenosis
was observed in the cell-treated group.45
Circulating progenitor cells
In the TOPCARE-AMI trial,30-32 in addition
to autologous BMMNCs, the investigators
also utilized CPCs harvested from patients’
peripheral blood followed by expansion ex
vivo. Although heterogeneous in nature,
approximately 90% of these circulating prog-
enitor cells (CPCs) exhibited an endothelial
phenotype.30-32 The overall beneficial effects
noted in CPC-treated patients were similar to
those observed in BMMNC-treated patients.
In a recent randomized double-blind place-
bo-controlled study, Erbs et al.47 have report-
ed reduction in infarct size, improvement
in global LVEF, decrease in the number of
hibernating myocardial segments, and
increase in coronary flow reserve following
intracoronary CPC delivery in patients with
ischemic heart disease with chronic total
occlusion of coronary artery. These data
suggest that the benefits of late reperfusion
(the open-artery hypothesis) 55 may possibly
be enhanced by concomitant cell therapy. In
this study,47 the chronicity of the lesion was
greater than 30 days. No increase in resteno-
sis was observed during the three-month
follow-up period.47 However, in a random-
ized trial of cell therapy with granulocyte-
colony stimulating factor (G-CSF)-mobilized
peripheral blood stem cells, Kang et al.56
reported a very high rate of in-stent resteno-
sis. The trial was stopped prematurely due
to safety concerns and despite some bene-
ficial effects, data remain essentially incom-
plete without a sizable control group. 56
Importantly, Schober et al.57 has recently
reported a significant correlation between
a greater number of circulating CD34+ cells
following elective stenting and a higher rate
of restenosis in patients with coronary artery
disease.
551
DAWN CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION

TABLE I.—Clinical trials with different types of stem/progenitor cells.

Study Disease Patients treated Controls Route of delivery Number of cells (millions)

BMMNC
Hamano et al.28 IHD 5 None Transepicardial 300-2 200
(during CABG)
Strauer et al.29 AMI 10 10 Intracoronary 28±22

A
TOPCARE-AMI30-32 AMI 29 BMMNC, 11 Intracoronary 213±75 BMMNCs
30 CPC 16±12 CPCs

C
Perin et al.33 ICM 14 7 Transendocardial 25.5±6.3
(EMM-guided)
Tse et al.34 IHD 8 None Transendocardial BMMNC from 40 ml BM

T ® DI
(EMM-guided)
Fernandez-Aviles AMI 20 13 Intracoronary 78±41
et al.35
Perin et al.36 ICM 11 9 Transendocardial 30
(EMM-guided)

H E
Kuethe et al.37 AMI 5 None Intracoronary 39±23

Silva et al.38 ICM 5 None Transendocardial 30

(EMM-guided)
IG M
Kuethe et al.39 ICM 5 None Intracoronary 29±9

Yaoita et al.40 IHD 10 None Transepicardial 3 400±1 200 BMMNCs (5.2±1.6

(during off-pump CD34+ cells)
CABG)
R A
Strauer et al.41 Chronic IHD 18 18 Intracoronary 60-132
(prior MI)
Y V

BMC
Fuchs et al.42 IHD 10 None Transendocardial 78±66
(EMM-guided)
P R

Wollert et al.43 AMI 30 30 Intracoronary 2 500±900

AC133+ BMC
O E

Stamm et al.44 ICM 6 None Transepicardial 1.02-1.57

(during CABG)
C IN

Bartunek et al.45 AMI 19 16 Intracoronary 12.6±2.2

MSC
Chen et al.46 AMI 34 35 Intracoronary 48 000-60 000
M

CPC
TOPCARE-AMI30-32 AMI 29 BMMNC, 11 Intracoronary 213±75 BMMNCs
30 CPC 16±12 CPCs
Erbs et al.47 CTO 12 11 Intracoronary 69±14

Skeletal myoblasts
Menasche et al.48 ICM 10 None Transepicardial 500-1 150
(during CABG)
Smits et al.49 ICM 5 None Transendocardial 196±105
(EMM-guided)

Herreros et al.50 IHD 11 None Transepicardial 100-393

(during CABG)
Siminiak et al.51 ICM 10 None Transepicardial 0.4-50
None (during CABG)

552 MINERVA CARDIOANGIOLOGICA Dicembre 2005

CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION DAWN

ABLE I.—Clinical trials with different types of stem/progenitor cells.

Timing Follow-up duration Results Adverse events

NR 12 months Perfusion ↑ in 3 of 5 patients None

5-9 days after 3 months Infarct size ↓; regional wall motion ↑; perfusion ↑; LVE- None

A
AMI SV ↓
4.9±1.5 days 12 months Infarct size ↓; global LVEF ↑; regional wall motion ↑; None
after AMI perfusion defect ↓; LVEDV ↓; LVESV ↓

C
NR 4 months Angina ↓; NYHA class ↓; perfusion ↑; regional wall None
motion ↑; global LVEF ↑; LVESV ↓
NR 3 months Angina ↓; perfusion ↑; regional wall motion ↑; regional None
wall thickening ↑

T ® DI
13.5±5.5 days 6 months Regional wall motion ↑; global LVEF↑; regional EF↑; None
after AMI LVESV↓
NR 12 months Exercise capacity ↑; perfusion ↑; no improvement in None
LVEF

H E
6.3±0.4 days 12 months No improvement in regional wall None
after PCI Motion, contractility, and global LVEF
NR 6 months Exercise capacity ↑; no change in global LVEF and per- None
fusion
IG M
1.3±0.5 years 12 months No improvement in regional wall None
after AMI motion or global LVEF
NR 5-32 months Perfusion ↑; myocardial microvessel density ↑ at autopsy 1 sudden death after 27
months
R A
27±31 3 months Infarct size ↓; regional wall motion ↑; No significant increase in reste-
months after global LVEF ↑; VO2max ↑; FDG-PET uptake ↑ nosis
AMI
Y V

NR 3 months Angina ↓; perfusion ↑ None

P R

4.8±1.3 days 6 months Regional wall motion ↑; global and regional LVEF ↑ None
after PCI
O E

>10 days but 3-10 months Global LVEF ↑; perfusion ↑; LVEDV ↓ 2 patients had SVT
<3 months
after AMI
C IN

11.6±1.4 days 4 months Regional wall motion ↑; global LVEF ↑; perfusion ↑ 1 patient had sustained VT;
after PCI higher in-stent restenosis

18 days after 6 months Infarct size ↓; global LVEF ↑; regional wall motion ↑; None
M

PCI perfusion ↑; LVEDV ↓

4.9±1.5 days 12 months Infarct size ↓; global LVEF ↑; regional wall motion ↑; None
after AMI perfusion defect ↓; LVEDV ↓; LVESV ↓
10±1 days 3 months Infarct size ↓; global LVEF ↑; coronary flow reserve ↑; None
after PCI hibernating myocardial segments ↓

3-228 months 10.9 months NYHA class ↓; global LVEF ↑; regional wall thickening ↑ 4 patients had sustained VT
after AMI
24-132 6 months Global LVEF ↑; regional wall thickening ↑ 1 patient had nonsustained VT
months after
AMI
3-168 months 6.5 months Global LVEF ↑; regional wall None
after AMI motion ↑; viability in infarct area ↑
4-108 months 12 months Global LVEF ↑; regional wall 4 patients had sustained VT
after AMI motion ↑ None
(Continued)

Vol. 53, N. 6 MINERVA CARDIOANGIOLOGICA 553

DAWN
TABLE I.—Clinical trials with different types of stem/progenitor cells.
Study Disease Patients treated
Chachques et al.52 ICM 20
Siminiak et al.53 ICM 10
A
AMI: acute myocardial infarction; BMC: bone marrow cells; BMMNC: bone marrow mononuclear cells; CABG: coronary artery bypass
FDG-PET: 18fluorodeoxyglucose-positron emission tomography; ICM: ischemic cardiomyopathy; IHD: ischemic heart disease; LV: left
tion; NR: data not reported; NYHA: New York Heart Association; PCI: percutaneous coronary intervention; SVT: supraventricular
C
T ® DI
Mesenchymal stem cells
In a relatively large randomized trial, Chen
et al.46 injected bone marrow-derived mes-
enchymal stem cells (MSCs) in patients with
H E
AMI. MSCs were expanded in culture for 10
days and injected via the coronary artery 18
days (average) after PCI. Treated patients
IG M
exhibited improved regional wall motion and
global LVEF, smaller perfusion defect, and
smaller LVEDV and LVESV.46 No significant
adverse effect was noted.
R A
Skeletal myoblasts
Y V
Following the initial reports of myocardial
repair with skeletal myoblasts,58 several clin-
ical studies 48-53 have tested the effect of these
P R
cells in humans. In the first clinical study,48
transplantation of autologous skeletal
O E
myoblasts during CABG improved patient
symptoms, increased systolic wall thicken-
ing and LVEF. However, 4/10 patients expe-
C IN
rienced delayed sustained VT needing
implantable cardioverter defibrillator (ICD)
implantation.48 Increased incidence of VT fol-
lowing myoblast transplantation has been
M
reported in subsequent studies.49, 51, 53 This
arrhythmogenic potential of myoblasts is not
surprising as studies in animal models indi-
cate that these cells do not differentiate into
cardiomyocytes 58 and do not connect with
neighboring cells via gap junctions.59, 60
Intriguingly, despite these limitations,
improvement in global LVEF and/or patient
symptom was noted in all of these studies.48-
53 In coculture studies of skeletal myoblasts
and cardiomyocytes, lentiviral transduction
of connexin 43 in skeletal myoblasts has
recently been shown to decrease arrhyth-
mogenicity in vitro.61 Similar genetic modifi-
554 MINERVA CARDIOANGIOLOGICA
CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION
Controls Route of delivery Number of cells (millions)
None Transepicardial 300±20
(during CABG) maximum
None Intramyocardial 17-106
(via cardiac veins)
cation may render skeletal myoblasts safer
for clinical use in the future.
Cardiac stem cells: isolation and
characterization
Adult stem cells have been identified in
many organs, including bone marrow, skele-
tal muscle, skin, central nervous system, liv-
er, and gastrointestinal tract.17, 18 Under phys-
iologic and pathologic conditions, these res-
ident stem cells can differentiate into appro-
priate organ-specific lineages and reconsti-
tute dead or damaged tissue.17, 18 Although the
heart has traditionally been viewed as a post-
mitotic organ incapable of intrinsic repair,2
elegant work from Anversa’s laboratory has
documented that death and regeneration of
cardiac myocytes indeed play significant roles
in cardiac homeostasis.62-68 According to this
new paradigm, a small population of cycling
myocytes are continuously regenerated via
the differentiation of CSCs.66-68
Lin-/c-kit+ CSCs
Anversa et al. recently isolated and charac-
terized a resident CSC population in the adult
heart that expresses markers for primitive cells,
including c-kit, Sca-1, and MDR1, but do not
express any of the markers of hematopoietic
lineages (lin-).8, 20, 69 In vitro, CSCs are clono-
genic and are capable of self-renewal through
multiple passages.8, 69 By virtue of their multi-
potential nature, CSCs are also able to differ-
entiate in vitro into all of the 3 functionally
important types of cells in the heart, car-
diomyocytes, smooth muscle cells, and
endothelial cells identified by the expression
Dicembre 2005
CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION
ABLE I.—Clinical trials with different types of stem/progenitor cells.
Timing Follow-up duration
NR 14±5 NYHA class ?; global LVEF ?; regional wall motion ?
months
5-96 months 6 months NYHA class ?
after AMI
A
graft surgery; CPC: circulating progenitor cells; CTO: chronic total occlusion; EF: ejection fraction; EMM: electromechanical mapping;
ventricular; LVEDV: LV end-diastolic volume; LVESV: LV end-systolic volume; MSC: mesenchymal stem cells; LVEF: LV ejection frac-
tachyarrhythmia; VT: ventricular tachycardia
C
T ® DI
of cell-specific markers.8 In these studies,8, 20,
69 CSC therapy after AMI induced myocardial
repair with formation of new cardiac myocytes,
smooth muscle cells, and endothelial cells.8
The newly formed BrdU+ myocytes expressed
H E
cardiac-specific proteins, including α-sar-
comeric actin, cardiac myosin heavy chain
IG M
(MHC), α-cardiac actinin, and proteins asso-
ciated with intercellular gap junctions, N-cad-
herin and connexin 43.8 It was also demon-
strated that CSCs express CXCR4 and can effec-
tively cross the vascular barrier following intra-
R A
aortic injection, inducing cardiac repair.20
Further characterization revealed that the CSCs
Y V
express insulin-like growth factor (IGF)-1
receptor and c-Met (receptor for stem cell fac-
tor) and are able to migrate in vivo along a
P R
growth factor gradient.70 The number of CSCs
increases with the formation of numerous new
O E
myocytes in humans with aortic stenosis, a
valvular condition associated with cardiac
hypertrophy.71 Activation of CSCs in humans
C IN
with ischemic cardiomyopathy with resultant
cardiac repair has also been documented.72
Other cardiac progenitors
M
Several other investigators have since
reported the identification of distinctly dif-
ferent populations of primitive cells in the
heart that are able to differentiate into car-
diomyocytes and/or regenerate infarcted
myocardium.73-79
SCA1+ CARDIAC PROGENITORS
Oh et al.73 described a Sca-1+ cardiac prog-
enitor population that also expresses CD31,
but not c-kit or other markers of hematopoietic
lineage and endothelial cells. When injected i.v.
into infarcted mice in a Cre-lox donor/recipi-
Vol. 53, N. 6 MINERVA CARDIOANGIOLOGICA
DAWN
Results Adverse events
None
1 patient had sustained VT
ent system, these cells were able to generate
cardiomyocytes both with and without cell
fusion.73 Identification of similar Sca-1+ car-
diac progenitors have been reported by other
investigators.74 Upon oxytocin stimulation,
adherent cardiac Sca-1+ cells differentiated into
a cardiomyocytic phenotype and exhibited
spontaneous contractions.74 Interestingly, 90%
of these Sca-1+ cells did not express c-kit.74
However, the clonogenicity and in vivo regen-
erative capacity of these Sca-1+ progenitors 74
remain to be demonstrated.
CARDIAC SIDE POPULATION CELLS
Side population (SP) cells have been
described in several organs, including bone
marrow, skeletal muscle, kidney, lung, and
small intestine.80, 81 Although SP cells are typ-
ically identified by their ability to extrude
Hoechst 33342 dye, the majority of SP cells
from the bone marrow and muscle are Sca-
1+.82, 83 Martin et al.75 reported the identifi-
cation of an SP cell in the heart of embryon-
ic as well as adult mice that expresses the
ATP-binding cassette transporter Abcg2, an
integral feature of the SP cell phenotype.84
Interestingly, these cardiac Abcg2-express-
ing SP cells are Sca-1high, c-kitlow, CD34low,
CD45low, and distinct from endothelial cells.75
Subsequently, Pfister et al.77 reported a cardiac
SP cell that expresses Sca-1 and is negative for
CD31. Although these SP cells 77 differentiate
into cardiomyocytes in vitro, their ability to
survive in vivo and induce myocardial repair
remains to be proven. Subsequent work from
these authors 85 has demonstrated that the
number of cardiac SP cells decreases acute-
ly following AMI and is replenished by cir-
culating bone marrow-derived primitive cells
that lose the CD45 antigen following myocar-
555
DAWN
dial homing. These interesting results 85 sug-
gest that primitive bone marrow cells may
partake in the turnover of cardiac progenitors
during pathologic conditions.
CARDIOSPHERE-FORMING PROGENITORS
A
Messina et al.76 described the isolation of
primitive cells from human and murine hearts
C
that formed cardiosphere (CS) in culture. These
Sca-1+, c-kit+, KDR/flk-1+, and CD31+ CSs
beat spontaneously in culture and exhibit self-
T ® DI
renewal capacity.76 In addition, they express
cardiac differentiation markers, including tro-
ponin I, MHC, atrial natriuretic peptide, and
endothelial marker von Willebrand factor.
H E
These CSs survive in vivo following trans-
plantation into the heart and express markers
IG M
of cardiac lineages.76 A detailed characterization
of proliferating CSs grown in vitro from human
endocardial biopsies revealed that they are
positive for c-kit, MDR1, connexin 43, and the
cardiac transcription factor Nkx2.5.79
R A
ISL-1+ CARDIAC PROGENITORS
Y V
Using a tamoxifen-inducible Cre-lox sys-
tem, Laugwitz et al.78 described a primitive
P R
cell population in the heart that expresses
the homeobox gene isl-1 but does not
extrude Hoechst 33342 and does not express
O E
c-kit. These cells acquire cardiomyocytic phe-
notype without fusion when cocultured with
C IN
neonatal cardiomyocytes in vitro and show
electrical as well as contractile properties
reminiscent of neonatal cardiomyocytes.78
However, these isl-1+ cells have only been
isolated from very young animals and
M
humans,78 and their presence and signifi-
cance in cardiac homeostasis in adult organ-
isms remain to be ascertained. Further,
whether these isl-1+ progenitors are multi-
potential and can indeed induce cardiac
repair in vivo remains to be proven.
Cardiac stem cells: therapeutic
potential
As with any other form of therapy, cell
therapy in humans should ideally be safe
556 MINERVA CARDIOANGIOLOGICA
CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION
with minimal adverse effects, and efficacious
with significant health benefits. Although no
data are available regarding the safety and
benefits of potential CSC therapy in humans,
based on results from animals studies, ther-
apy with CSCs may potentially offer several
distinct advantages over other cell types.
Regeneration of both muscle and vessel
An organ-specific stem cell should be able
to replenish not only one type of cell, but
several important components of the organ.17,
18, 86 Further, it is important to recognize that
newly formed myocytes in the middle of an
infarct scar will not survive in the absence
of adjoining vascular structures. On the oth-
er hand, regeneration of blood vessels alone
will not enhance contractility unless accom-
panied by formation of new myocytes. Thus
far, different results have been reported by
various laboratories with reports confirming
the regeneration of either muscle or vessels,
but infrequently of both. In this regard, c-
kit+ CSCs offer a greater therapeutic poten-
tial as they express markers specific for car-
diomyocytes, smooth muscle cells, and
endothelial cells in vitro,8 and are able to
generate not only new myocytes but also
new blood vessels securing blood supply to
the newly formed cells in vivo.8, 20, 69
Connection with other myocytes
Another important hurdle for cell trans-
plantation has emerged over the past few
years during clinical trials with skeletal
myoblasts - the increased incidence of arrhyth-
mias.48, 49, 51, 53 Importantly, skeletal myoblasts
do not differentiate into cardiomyocytes 58
and do not connect with the surrounding
myocardium.59, 60 These islands of electrical-
ly active yet dissimilar cells may potentially
serve as arrhythmogenic foci. In contrast, fol-
lowing CSC transplantation in vivo, the regen-
erated myocytes express on their surface not
only N-cadherin, which is necessary for
anatomic connection with neighboring
myocytes, but also connexin 43, which con-
nects them to other myocytes electrically.8, 20
These favorable features predict less arrhyth-
mic events with CSC transplantation.
Dicembre 2005
CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION
Clonogenicity
Since the beneficial effects of cell therapy
are dose dependent,87 the number of trans-
planted cells is important for cardiac repair in
humans. Although the use of unselected bone
A
marrow cells and BMMNCs has resulted in
improvement in LV function and myocardial
perfusion in clinical trials, perhaps the ben-
C
efits would be much greater if the appropri-
ate cells, isolated in small numbers, could be
expanded in vitro prior to cell therapy and
T ® DI
administered in sufficient numbers. Although
self-renewal is a cardinal feature of a stem
cell,17, 19 many of the primitive cell popula-
tions are difficult to expand in vitro and
H E
undergo a phenotypic shift during this
process. In contrast, CSCs 8, 20, 69 are clono-
genic and retain their phenotype during
IG M
expansion in vitro. These CSCs may be har-
vested from patients with ischemic heart dis-
ease and expanded in vitro to obtain a large
number of cells for future therapeutic use.
R A
Efficacy with transcoronary delivery
Y V
In the setting of an AMI, the most conve-
nient mode of delivery is via the coronary
P R
artery during primary PCI or as an elective
procedure a few days later. However, deliv-
ery of MSCs via the intracoronary route has
O E
been reported to cause microinfarctions in
dogs, possibly because of the relatively large
cell size.88 This problem may potentially be
C IN
encountered during intracoronary delivery
of any large cell. In our experience, delivery
of culture expanded CSCs via the intracoro-
nary route in rats and pigs was not associat-
M
ed with microinfarctions (unpublished obser-
vation). Importantly, following the intra-
coronary delivery, CSCs were able to cross the
vessel wall relatively quickly to home into
the injured myocardium and regenerate new
cardiomyocytes and vasculature.20 It was also
shown that CSCs express CXCR 4,20 the recep-
tor for stromal cell-derived factor-1, that plays
a critical role in stem cell homing.89
Potential therapeutic uses
Based on the evidence discussed above, it
is apparent that CSCs are eminently poised to
Vol. 53, N. 6 MINERVA CARDIOANGIOLOGICA
DAWN
be tested in humans. CSCs may potentially be
harvested during an open-heart procedure
(e.g., CABG, LV assist device implantation,
valve repair/replacement) or catheter-based
myocardial biopsy. Because of their clono-
genicity,8, 69 CSCs can then be grown in vit-
ro to generate sufficient number of multipo-
tent cells. Because of the reported success
with cardiac repair following intramyocar-
dial injection of CSCs,8 these cells can be
injected directly into the myocardium dur-
ing CABG or via the intraventricular approach
guided by an EMM system. These stategies
may be utilized for cardiac repair in patients
with chronic ischemic as well as nonischemic
cardiomyopathy. However, following AMI,
a transcoronary route of delivery during PCI
may represent the most convenient mode of
delivery. Although AMI and ischemic car-
diomyopathy would constitute the most com-
mon clinical conditions for therapeutic use of
CSCs, potentially, CSCs may also be used to
reverse the progressive deterioration of ven-
tricular function in nonischemic cardiomy-
opathies. Importantly, in all of these scenar-
ios, stringent criteria must be formulated to
assess the benefits of CSC therapy by rigor-
ous measurements of LV function by echocar-
diography, left ventriculography, and cardiac
magnetic resonance imaging studies if pos-
sible. Myocardial perfusion should be quan-
tified by radionuclide perfusion imaging.
Finally, any impact on survival should be
assessed during longterm follow-up.
Safety issues
Major adverse effects of cell therapy iden-
tified in recent studies in humans include
potentially fatal ventricular arrhythmias and
higher incidence of restenosis (Table I). In
light of this knowledge, the safety of CSC
therapy in humans needs to be closely mon-
itored. However, sustained VT has been not-
ed primarily following transplantation of
skeletal myoblasts,48, 49, 51, 53 which are unable
to connect electrically to the neighboring
myocytes.59, 60 In contrast, in animal models,
CSC-derived cardiomyocytes have been
shown to express N-cadherin and connexin
43,8, 20 which are necessary for establishing
557
DAWN
mechanical and electrical connection, respec-
tively. A higher rate of in-stent restenosis has
been reported with intracoronary injection
of AC133+ progenitors 45 and G-CSF-mobi-
lized peripheral blood cells.56 Restenosis is
multifactorial in etiology and involves smooth
A
muscle cell activation and neointimal thick-
ening.90 Several growth factors also play crit-
ical roles in restenosis,90 and although coro-
C
nary stenting eliminates elastic recoil and
remodeling, it accentuates neointimal hyper-
T ® DI
plasia. Since CSCs have been shown to
express growth factor systems 70 and are able
to differentiate into smooth muscle cells as
well as endothelial cells,8, 20 the rate of
H E
restenosis following intracoronary injection of
CSCs following PCI will need to be moni-
tored carefully.
IG M
Cell therapy: optimizing the variables
R A
Despite the initial encouraging results from
clinical trials, many questions remain unan-
swered regarding the optimal cell therapy
Y V
strategy for myocardial repair. As summa-
rized in Table I, diverse types of cells, num-
P R
bers, and routes have thus far been employed
in patients with AMI, ischemic cardiomy-
opathy, and chronic ischemic heart disease.
O E
However, several major issues regarding the
cell type, route, and timing of therapy remain
to be addressed.
C IN
The ideal cell
Based on current evidence, the most
M
appropriate cell for cardiac regeneration
should have several important properties.
First, it should be able to differentiate into
both cardiomyocytes and cells of the vascu-
lar lineage, so that not only new contractile
units are formed, but also blood supply to
these newly formed myocytes is secured.
Second, the newly formed myocytes should
couple mechanically as well as electrically
with native myocytes (contracting in syn-
chrony with adjacent cells). Mechanical cou-
pling will preclude ineffectual contraction,
and electrical coupling may prevent the
occurrence of arrhythmias and improve func-
558 MINERVA CARDIOANGIOLOGICA
CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION
tion. Third, the ideal cell should be able to
escape from the vasculature and home into
the infarcted or noninfarcted myocardium.
Expression of CXCR4 and other adhesion
molecules will identify cells with this capa-
bility. This will greatly improve efficacy of
cell delivery via the intracoronary route, and
improve retention of cells following intramy-
ocardial delivery. Fourth, this cell should be
easy to harvest in a timely fashion, and/or
be suitable for longterm storage with or with-
out expansion following harvest at an oppor-
tune time beforehand. Fifth, the ideal cell
should be able to self-renew in vitro without
a change in phenotype or loss of the multi-
potential nature. Finally, the adverse effects
of therapy should be minimal with this cell.
Cell transplantation should not cause the
development of teratomas or other neoplas-
tic growth, and intracoronary administration
should not cause hyperplasia involving the
arterial wall causing restenosis or a de novo
atheromatous lesion. Although numerous cell
types have been utilized in animal models,
each with somewhat distinct phenotype and
properties, no cell has been definitively
shown to fulfill all of these criteria. Although
CSCs fulfill several of these criteria, the safe-
ty profile has not been tested in the clinical
setting.
The most efficacious route
Based on patient demographics and pathol-
ogy, several different approaches can be uti-
lized for cell delivery. For chronic ischemic
cardiomyopathy, transepicardial injection dur-
ing CABG offers a very effective route, which
has been employed in several clinical studies.
Transendocardial delivery has also been suc-
cessfully employed in a number of studies.
With this method, the use of an EMM system
enables the identification of the scar area and
enables precise cell injection. The use of a
transendocardial route may potentially be
limited by the availability of this system.
Alternatively, in patients with significant coro-
nary artery disease, cells may be injected in
the coronary artery following PCI or as an
elective procedure. Interestingly, intracoro-
nary delivery of CPCs in patients with revas-
Dicembre 2005
CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION
cularized chronic (>30 days) total occlusion
47 and BMMNCs in patients with old myocar-
dial infarction 41 has been associated with
improvement in LV function. These results
suggest that intracoronary route of cell deliv-
ery may be utilized even when myocardial
A
inflammation is subacute or minimal.
In the setting of an AMI, cell delivery imme-
diately following PCI is likely to be most cost-
C
effective. However, the ready availability of
cells is a significant issue in this regard. Another
T ® DI
important issue for the intracoronary delivery
relates to the ability of the cell to cross the
vessel barrier. Primitive cells that express
CXCR4 (for example, CSCs20) and other adhe-
H E
sion molecules may be better suited for this
mode of delivery. Hofmann et al.91 reported
greater myocardial retention of CD34+ BMCs
IG M
compared with unselected BMCs following
intracoronary delivery 5-10 days after PCI fol-
lowing AMI. However, large cells may not be
suitable for intracoronary administration and
R A
intracoronary delivery of MSCs in dogs has
been reported to cause microinfarction.88
Further, injection of circulating AC133+ prog-
Y V
enitors 45 and G-CSF-mobilized progenitors 56
has been associated with a higher rate of
P R
restenosis. Thus, the method of delivery needs
to be tailored according to the cell injected.
O E
The optimal time
For nonacute conditions, including
C IN
ischemic heart disease and cardiomyopathy,
the time of delivery may be synchronized
with a planned procedure. However, since
the inflammatory reaction persists for a pro-
M
longed period of time after AMI,92 timing of
cell delivery may prove critical in the setting
of an AMI. On one hand, the increased
expression of adhesion molecules and
chemoattractants in the infarcted myocardium
93 as well as the viable myocardium may
improve cell retention.94 On the other hand,
delivery of cells into an inflammatory milieu
may cause excessive cell death. Thus far, cell
delivery after AMI in humans has been ben-
eficial at various time points (Table I). Future
studies will be necessary to identify the most
appropriate time for cell delivery in general
and CSC delivery following AMI.
Vol. 53, N. 6 MINERVA CARDIOANGIOLOGICA
DAWN
Perspectives and future directions
AMI and ischemic cardiomyopathy con-
tinue to be major health problems with
increasing incidence, and the benefits of phar-
macologic therapy appears to be limited to
symptomatic improvement with only mod-
est effects on disease progression. Because of
the compelling need to formulate a better
strategy for treating millions of patients suf-
fering from these debilitating conditions
worldwide, and because of the unprece-
dented promise of stem cell therapy, clini-
cal application of cell therapy has progressed
at a rapid pace. Thus far, data from these rel-
atively small trials have generally been pos-
itive in terms of improvements in LV function,
myocardial perfusion, and even decrease in
infarct size.
It should be noted that many of these ear-
ly clinical trials did not have a control group
(Table I). In the few that did have a control
group, control patients received standard care
- no control cell was injected. Further, only a
few studies were randomized.30, 32, 43, 46, 47
Even in the animal models, very few studies
compared the benefits of more than one cell
type. In humans, the TOPCARE-AMI trial 30-
32 compared 2 cell populations; the benefits
of cell therapy were similar in patients treat-
ed with BMMNCs and CPCs.30-32 To resolve
the issue of the ideal cell type, future basic
and clinical studies should be designed to
compare the beneficial effects of different
types of cells, especially different subpopu-
lations from the bone marrow and CSCs.
Although intramyocardial injection may be
employed during surgical procedures or
when an EMM system is available, perhaps
intravenous or intracoronary delivery would
be more suitable for general use. However,
myocardial homing/retention following injec-
tion into the blood stream has been subop-
timal. Four days after LV intracavitary injec-
tion, only 0.44% of the injected MSCs were
detected in the myocardium.7 This problem
is even greater with intravenous injection,
with only a small fraction of injected cells
homing to the myocardium.91, 94, 95 Even with
intracoronary injection, less than 3% of uns-
elected BMCs and 14-39% of CD34+ BMCs
559
DAWN CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION
were retained in the myocardium.91 To objec-
tively address cell kinetics, cells labeled with
enhanced green fluorescent protein, CM-DiI,
and radionuclides have been used. However,
each of these methods has specific limita-
tions and newer labeling techniques with
A
greater precision need to be devised to accu-
rately assess cell retention and viability fol-
lowing transplantation.
C
Low survival rate of grafted cells is a poten-
tial problem with cell transplantation in gen-
T ® DI
eral.96 The inflammatory milieu in the infarct-
ed myocardium 92 may cause death of trans-
planted cells and progressive apoptosis may
also contribute to cell loss.97 However, the
H E
true extent of this problem with bone mar-
row-derived cells and CSCs needs further
assessment. In an elegant study by Mangi et
IG M
al.,98 transplanted MSCs overexpressing the
antiapoptotic gene Akt restored four-fold
greater myocardial volume compared with
equal number of cells transduced with a
R A
reporter gene. Similar genetic modification
of CSCs may potentially offer significant addi-
tional benefits.
Y V
Augmentation of the beneficial effects of
cell therapy may also be achieved by express-
ing angiogenic factors in the transplanted
P R
cell. Tranfection of skeletal myoblasts with
vascular endothelial growth factor (VEGF)
O E
enhanced myocardial angiogenesis and
improved cardiac function.99 In another
study,100 transfection of endothelial progen-
C IN
itor cells (EPCs) with VEGF reduced the num-
ber of cells necessary to improve ischemic
limb salvage. CSCs are able to differentiate
into endothelial cells and vascular smooth
M
muscle cells, thereby regenerating the vas-
culature necessary to sustain cell survival.8, 20
However, since VEGF has been shown to
enhance the efficacy of EPCs,100 transfection
of CSCs with VEGF or other angiogenic fac-
tors may merit further investigation.
It is obvious that cell therapy for myocar-
dial regeneration is in its infancy and the
mechanism underlying the observed bene-
fits remains to be elucidated. From a clinical
standpoint, however, what is more impor-
tant is to achieve infarct size reduction, atten-
uation of LV remodeling, improvement in LV
function, and increase in myocardial perfu-
560 MINERVA CARDIOANGIOLOGICA
sion. If one considers new vessel formation
as a surrogate marker for improved perfu-
sion, in preclinical studies CSCs have been
shown to achieve all of these end-points.8, 20
Thus, the time seems ripe for testing the car-
diac regenerative efficacy of CSCs in patients
with AMI, ischemic cardiomyopathy, and oth-
er cardiac pathologies that result in heart fail-
ure.
Conclusions
Therapy with stem/progenitor cells holds
great promise for millions of patients with
AMI, ischemic cardiomyopathy, and heart
failure. Although many details regarding the
ideal cell type, route, and timing remain to be
optimized, the collective data from initial clin-
ical trials indicate that cell therapy can
improve LV function and perfusion without
significant adverse effects. In this regard, the
discovery of CSCs represents a major
advance. Based on the evidence generated
from extensive basic studies, CSCs appear to
be ideally suited for myocardial regeneration
as they exhibit many of the necessary pre-
requisites for successful cellular transplanta-
tion for cardiac repair. Although CSCs have
not been tested for cardiac regeneration in
humans, they may potentially be utilized in
ischemic and nonischemic cardiomyopathies,
myocardial infarction, and other pathologic
states that culminate in loss of myocytes.
However, the safety and efficacy of CSC ther-
apy in humans should be monitored with
stringent measures and compared with those
achieved with other forms of adult primitive
cells.
Riassunto
Terapia con cellule staminali per la rigenerazione
miocardica: una prospettiva clinica
Terapia con cellule staminali per la rigenerazione
miocardica: una prospettiva clinica L’infarto miocar-
dico ed altre patologie cardiache determinano perdita
netta di cardiomiociti, sviluppo di tessuto cicatrizia-
le, rimodellamento ventricolare ed infine scompenso
cardiaco. Poiche’ le strategie farmacologiche ed inter-
ventistiche non sono in grado di rigenerare il mio-
Dicembre 2005
CARDIAC STEM CELL THERAPY FOR MYOCARDIAL REGENERATION
cardio perduto, lo scompenso cardiaco rimane una
delle piu’ importanti patologie a livello mondiale.
Recenti studi su modelli animali di infarto miocardi-
co e di scompenso cardiaco hanno dimostrato come
vari sottotipi di cellule dell’adulto possano rigenera-
re cardiomiociti funzionali e tessuto vascolare car-
diaco, con miglioramento della struttura e della fun-
A
zione cardiaca. Piccoli trial clinici di terapia cellulare
in pazienti con infarto miocardico e cardiomiopatia
ischemica hanno confermato questi effetti benefici
C
anche nell’uomo, con riduzione delle dimensioni
infartuali e miglioramento di frazione di eiezione,
perfusione miocardica e cinetica di parete. Diverse
T ® DI
popolazioni cellulari fenotipicamente distinte sono
state impiegate per la rigenerazione miocardica e i
meriti relativi di ciascun tipo cellulare restano anco-
ra da determinare. La recente scoperta di cellule sta-
minali cardiache dell’adulto ha alimentato un’intensa
H E
speranza per la rigenerazione cardiaca con cellule
del cuore stesso e che sono pertanto intrinsecamen-
te programmate per la ricostituzione del tessuto car-
IG M
diaco. Lo scopo di questa revisione e’ di riassumere
le evidenze sulla fattibilita’ della riparazione tissuta-
le cardiaca nell’uomo mediante l’uso di cellule adul-
te staminali o progenitrici e di discutere la potenzia-
le utilita’ delle cellule staminali cardiache per la rige-
R A
nerazione miocardica.
Parole chiave: Cellule staminali cardiache -
Rigenerazione miocardica - Infarto miocardico -
Y V
Cardiomiopatie.
P R
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