Circulation 2006 Boyle 339 52 PDF

Stem Cell Therapy for Cardiac Repair: Ready for the Next Step
Andrew J. Boyle, Steven P. Schulman and Joshua M. Hare

Circulation. 2006;114:339-352
doi: 10.1161/CIRCULATIONAHA.105.590653
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CONTROVERSIES IN
CARDIOVASCULAR MEDICINE
Is stem cell therapy ready for patients?
Stem Cell Therapy for Cardiac Repair

Ready for the Next Step
Andrew J. Boyle, MBBS, PhD; Steven P. Schulman, MD; Joshua M. Hare, MD
oronary heart disease and heart failure continue to be

significant burdens to healthcare systems in the Western world. In the United States alone, there are 7.1
million survivors of myocardial infarction (MI) and 4.9
million people living with congestive heart failure (CHF).1
Despite recent advances in medical and device therapy for
heart failure, the incidence, hospitalization, and mortality
rates continue to rise. After receiving a diagnosis of CHF, 1
in 5 patients will be dead within 12 months.1 Therefore, any
new treatment modality that benefits heart failure patients has
the potential to result in a dramatic improvement in health
outcomes and substantial cost savings for the community.
Response by Oettgen p 352

The possibility of using stem cell based therapies for
people suffering an acute MI or living with CHF has captured
the imagination of both the medical and popular communities. Since early reports in animal models 10 years ago,2,3
the stem cell field has made enormous advances in moving
toward clinically applicable treatment options, and we now
stand at the dawn of a new therapeutic era. An abundance of
preclinical data demonstrate safety, feasibility, and efficacy,
justifying the current entry into clinical trials of stem cell
therapy in humans.4 8 This position, however, is extremely
controversial, with some arguing that trials are premature
because mechanistic insights are insufficiently addressed.9,10
Here, we argue that properly conducted rigorous clinical trials
are a key and appropriate next step not only to start the long
process of therapeutic development but also as an essential
component in the process of understanding the scientific

underpinnings of cardiac regeneration and its therapeutic
utilization. The field of regenerative medicine will advance
through the parallel conduct of in vitro/animal model studies
and clinical trials, the latter frequently guiding the former.
The publication of Menasche et al11 describing the first
patients to receive skeletal myoblasts spawned a profusion of
small clinical studies investigating cellular therapy for cardiac repair. At present, a number of early clinical studies have
been published and are summarized in Table 1. Several points
are immediately apparent from this table. First, 400 patients
have completed these published studies, yet most of them are
small pilot studies that lack randomization or control groups.
Second, despite the fact that several cell types have been
studied using different delivery methods, the overwhelming
message from all of these studies is that cell therapy is safe
and feasible. In addition, the results of these studies provide
encouraging, albeit preliminary, signs of efficacy. Finally,
although these trials represent the currently published data,
they have formed the basis for numerous larger, ongoing
trials accruing more patient data (Table 2).
Mechanism of Action of Stem Cells

One of the major obstacles in progressing to large-scale
clinical trials of cardiac stem cell therapy is the ongoing
debate regarding the mechanism of action by which stem cell
therapy leads to cardiac repair. The classic idea that provided
the primary motivation for stem cell therapy is that delivery
of the appropriate stem cells would repair a damaged heart via
The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.
From the Department of Medicine, Division of Cardiology, Institute for Cell Engineering, and Specialized Center for Cell-Based Therapy, The Johns
Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Joshua M. Hare, MD, Johns Hopkins Medical Institutions and Institute for Cell Engineering (ICE), Division of Cardiology, BRB
651, 733 N Broadway, Baltimore, MD 21205. E-mail jhare@mail.jhmi.edu
(Circulation. 2006;114:339-352.)
2006 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/CIRCULATIONAHA.105.590653
339
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Circulation
TABLE 1.
July 25, 2006
Clinical Studies of Stem Cell Therapy for Cardiac Repair
Study
Treated, n
Cell Type
Mode of Delivery
Randomized
Primary End Point
10
BMMNCs
Intracoronary
Safety
NA
TOPCARE-AMI51,52
59
BMMNCs/CPCs
Intracoronary
Safety and feasibility
NA
Stamm et al103,104
12
BMMNCs
Open heart surgery
NA
Tse et al105
BMMNCs
Endomyocardial
NA
Perin et al56
14
BMMNCs
Endomyocardial
Safety
NA
BOOST53
30
BMMNCs
Intracoronary
LVEF
0.0026
Fernandez-Aviles et al106
20
BMMNCs
Intracoronary
NA
NA
Strauer et al
Li et al
50
107
BMMNCs
Open heart surgery
IACT study108
18
BMMNCs
Intracoronary
Not stated
Janssens et al54
33
BMMNCs
Intracoronary
LVEF
0.36
Chen et al72
34
MSCs
Intracoronary
LVEF
0.01
Katritsis et al109
11
MSCsEPCs
Intracoronary
Scar size
0.02
Galinanes et al110
14
Bone marrow
Open heart surgery
Safety
NA
Fuchs et al111
10
Bone marrow
Endomyocardial
NA
MAGIC cell112
20
GCSF-mobilized PBMNCs
Intracoronary
NA
NA
GCSF-mobilized PBMNCs
Open heart surgery
13
GCSF-mobilized CPCs
Intracoronary
CFR and LVEF
GCSF-mobilized CD34
Intracoronary
NA
GCSF-mobilized AC133
Open heart surgery
NA
Dib et al116
30
Skeletal myoblasts
Open heart surgery
NA
POZNAN117
10
Skeletal myoblasts
Transcoronary-venous
NA
Ince et al118
Skeletal myoblasts
Endomyocardial
NA
10
Skeletal myoblasts
Open heart surgery
NA
Ozbaran et al
113
Erb et al78
Boyle et al114
115
Pompilio et al
Siminiak et al45
Smits et al119
Menasche et al44
Pagani et al120
Herreros et al121
NA
0.05
Skeletal myoblasts
Endomyocardial
NA
10
Skeletal myoblasts
Open heart surgery
NA
Skeletal myoblasts
Open heart surgery
Histology
NA
12
Skeletal myoblasts
Open heart surgery
NA
CPC indicates circulating progenitor cell; GCSF, granulocyte colony stimulating factor; PBMNC, peripheral blood mononuclear cell; CFR, coronary
flow reserve; and LVEF, LV ejection fraction.
active myocardial regeneration resulting from transdifferentiation of the administered stem cells.4,1214 However, new
data have led to the recognition of alternative mechanisms of
action (Figure 1): Exogenous stem cells may also stimulate
proliferation of endogenous cardiac precursors or stem cells
through neovascularization6,15 or paracrine signaling actions.16 In fact, observations in preclinical and clinical scenarios that all of these events occur allow us to generate a new
concept that cellular therapy contributes to the restoration of
stem cell niches, facilitating the ability of the heart to heal
itself.17 Still other mechanisms are proposed: Exogenous stem
cells may lead to cardiac repair via fusion of donor cells with
host cardiomyocytes.18 Finally, other investigators suggest
that the effects of stem cells are mediated by altering
mechanical properties to strengthen the MI scar, thereby
preventing deterioration in cardiac function19 (see Figure 1).
This ongoing debate about mechanism fuels the case for
slowing the pace of clinical trials; ie, we should not pursue
work in patients until we thoroughly understand, with a high
degree of scientific precision, the outcome of cell therapy in
in vitro systems and animal models.9,10 We would suggest

that we have reached the appropriate point in the development of cellular therapeutics to enter into the clinic; in fact,
entry into the clinic is the next step that will guide our
understanding of the mechanistic underpinning for effective
cellular therapeutics.
Types of Cells Contemplated for Cellular

Regenerative Therapy
Embryonic Stem Cell
Embryonic stem (ES) cells are derived from the inner cell
mass of the blastocyst-stage embryo, late in the first week
after fertilization. They are considered to be pluripotent, able
to give rise to many different cell lineages. For cardiac
regeneration therapy, there is a growing body of knowledge
from animal models regarding the steps of isolation, differentiation, and clinical application.
Human ES cells differentiate into spontaneously beating
cells with a cardiomyocyte phenotype. The morphology and
ultrastructure of these cells are organized with sarcomeric
Stem Cell Therapy
TABLE 2.
341
Clinical Trials of Stem Cell Therapy for Cardiac Repair Registered With ClinicalTrials.gov
Cell Type Used

Intramyocardial autologous
MSCs
Randomized
Total Patients
to Be
Enrolled, n
Primary
Outcome
Sponsor
Region
Myocardial ischemia
40
Perfusion
Rigshospitalet
Copenhagen, Denmark
Trial
Design,
Phase
Condition
Treated
I/II
Autologous BMMNCs
CHF undergoing CABG
75
LV function
University of
Pittsburgh
Pittsburgh, Pa
Autologous BMMNCs
CHF undergoing VAD

implantation
10
LV function
University of
Pittsburgh
Pittsburgh, Pa
Intramyocardial BMMNCs
Ischemic cardiomyopathy
30
Safety
Texas Heart Institute
Texas
Intracoronary BMMNCs
Acute MI
60
Safety
Minneapolis Heart
Institute Foundation
Minneapolis, Minn
Intravenous MSCs
Acute MI
48
Safety
Osiris Therapeutics
Multicenter, United States
Autologous Intramyocardial
CD34
Chronic myocardial
ischemia
24
Not stated
St Elizabeths Medical
Center
Boston, Mass
I/II
Chronic myocardial
ischemia
10
Perfusion
Translational Research
Informatics Center,
Japan
Kobe, Japan
BMMNCs
II
35
LV function
Odense University
Hospital
Odense, Denmark
Intracoronary autologous
BMMNCs
II
Acute MI
100
Myocardial viability
Nantes University
Hospital
Nantes, France
skeletal myoblasts
15
Safety
Bioheart
skeletal myoblasts
II
Not stated
LV function
Genzyme
Multicenter, Europe/United
Kingdom
skeletal myoblasts
undergoing CABG
15
Safety
Bioheart
Autologous CD34
CABG indicates coronary artery bypass grafting; VAD, ventricular assist device.
structures, formation of intercalated disks, desmosomes, and

gap junctions, characteristic of cardiomyocytes,20,21 and they
demonstrate the presence of a functional syncitium with
action potential propagation.21,22 When transplanted into
infarcted myocardium, ES cell derived cardiomyocytes engraft and improve cardiac function in several rodent models.2326 In the failing heart, in addition to replenishing
cardiomyocytes by ES derived cells, a simultaneous increase
in the blood supply may be necessary for optimal and
prolonged engraftment. Hence, it is of interest that ES cells
differentiate to all cell lines necessary for formation of new
blood vessels. Both murine and human ES cells spontaneously differentiate to form endothelial and smooth muscle
cells in vitro27 and in vivo.28,29 To date, no human clinical
studies have been initiated because of both the ethical issues
surrounding access to embryos and the possibility of teratoma
formation, suggested by a study injecting ES cells in skeletal
muscle.30
Resident Cardiac Stem Cells

In recent years, compelling evidence has accumulated suggesting that the heart has endogenous regenerative potential.
Figure 1. Possible mechanisms for successful cardiac regenerative therapy. See

text for details.
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Circulation
July 25, 2006
Recent studies have isolated, from both human and murine

hearts, undifferentiated cells that are clonogenic, express
stem and endothelial progenitor cell (EPC) antigens/markers,
and appear to have the properties of adult cardiac stem
cells.31,32 These cells most likely mediate endogenous mechanisms for minor repair and for replacement of ongoing cell
turnover within the adult heart. More importantly, they may
represent a therapeutic target that, if enhanced, could induce
cardiac self-repair.
These cells have been phenotyped using different antigenic
approaches. In a seminal report published in 2003, Beltrami
and colleages31 separated c-kitpositive cells from the rat
heart and demonstrated their clonogenicity and multipotency
in vitro, as well as their capacity to participate in cardiomyocyte and blood vessel regeneration after MI. Subsequently,
Oh and coworkers32 separated resident murine cardiac stem
cells on the basis of the presence of stem cell antigen-1. They
also demonstrated in vitro and in vivo myocardial differentiation of these cells and provided evidence supporting the
fusion of these cells. In 2004, Messina and coworkers33
reported on the identification of cardiospheres, clusters of
self-adherent cells that grew from cultured adult cardiac
tissue derived from both human and murine hearts. These
cells were shown to be clonogenic and capable of transdifferentiation in vitro, and they induced both myocardial and
vascular regeneration after MI. Laugwitz and colleagues34
isolated a population of cardiac precursor cells from postnatal
mouse hearts using isl-1 transcription factor as a cell marker.
These cells are c-kit and stem cell antigen-1negative but
are capable of differentiation into cardiomyocytes with electrical and contractile properties. Finally, Martin et al35 identified a side population of cells (SP cells) in the developing
heart and adult heart that are capable of proliferating and
differentiating into cardiac and hematopoietic lineages in
vitro. These cells were identified on the basis of expressing
Abcg2, an ATP-binding cassette transporter, rather than by
detection of surface markers.
Cardiac stem cells can be harvested from patients and
expanded ex vivo to generate large numbers of cells. A recent
report by Urbanek and colleagues36 demonstrated that cardiac
stem cells increase in number immediately after MI, but in the
chronic phase, the numbers fall, and the remaining cardiac
stem cells have less regenerative potential. This suggests that
the left ventricular (LV) dysfunction in ischemic cardiomyopathy may be due to a defect in or deficiency of functionally
competent cardiac stem cells. In addition, Mouquet et al37
have recently shown in an experimental study that bone
marrow may represent a reservoir for cardiac stem cells and
suggested that depletion of this reservoir could contribute to
diminished reparative capacity.
To date, there are no clinical trials of human cardiac stem
cells. However, Smith et al38 demonstrated that cardiospheres
could be grown from human endomyocardial biopsy specimens. These cardiospheres represent an easily accessible
option for autologous stem cell therapy, making the possibil-
ity of clinical testing of this approach feasible. The Specialized Centers for Cell-Based Therapy initiative of the NHLBI
has funded clinical trials of cardiac stem cells that should
begin in the near future.
Skeletal Myoblasts
Autologous skeletal myoblasts are another potential source
for cardiac repair because of their biological properties and
lack of ethical and immunological problems. Skeletal myoblasts or satellite cells are the reservoir of regenerative cells
for skeletal muscle tissue; they have the ability for selfrenewal and differentiation if muscle injury occurs.39 They
have many desirable features as donor cells, including the
ability to be amplified in an undifferentiated state in vitro and
high resistance to tissue ischemia. They also have been shown
to continue proliferation in vivo to a certain extent, which
gives rise to a larger graft size.3 Satellite cells are committed
solely to the myogenic lineage. Therefore, regardless of
environmental influences, even if implanted into a scar made
up mainly of fibroblasts, myoblasts differentiate into functional muscle cells. A growing body of experimental data and
initial clinical studies has shown not only engraftment of
donor cells but also improvement in global cardiac pump
function.8,11,40 42 However, the exact mechanism by which
they improve LV function is still debated. There may be
beneficial effects of contracting noncardiac myocytes, some
paracrine actions and an effect on scar strengthening to
prevent LV dilatation and remodeling.43 The critical importance of progressing to clinical trials is underscored by the
early clinical experience with skeletal myoblasts. Myoblast
transfer in early studies44,45 was associated with sustained
ventricular tachycardia, a life-threatening arrhythmia. This
finding guided changes in future protocols involving skeletal
myoblasts, which now require prophylactic cardioverterdefibrillator implantation and/or amiodarone therapy to prevent ventricular tachycardia. This protocol change resulted in
less frequent clinically evident arrhythmias and is a powerful
demonstration of how clinical trials are integral in guiding
and refining the way in which stem cell therapy should be
administered in patients. Phase II studies of skeletal myoblast
therapy are presently underway.
Human Adult Bone MarrowDerived Stem Cells

The observation that bone marrow elements contribute to
cardiac repair in the infarcted heart served as the rationale for
adult bone marrow cell therapy after MI. Jackson and
coworkers46 transplanted wild-type mice with green fluorescent protein (GFP)positive bone marrow and then induced
MI through coronary artery ligation and reperfusion. They
demonstrated that bone marrow elements contributed to
cardiomyocyte and endothelial cell formation after MI by
finding GFP-positive cardiomyocytes and endothelial cells. It
appeared that there is an intrinsic repair mechanism for minor
cardiac damage within the bone marrow but that it is not
adequate to repair larger amounts of damage such as that after
MI. Substantial effort has been expended to try to enhance
Stem Cell Therapy
this endogenous repair mechanism and use bone marrow as a

potential source of stem cells for cardiac repair. Orlic and
coworkers4 have shown that lineage-negative, c-kitpositive
bone marrow derived cells differentiate into new cardiomyocytes after MI. This regenerative therapy can be harnessed by
either direct injection into the peri-infarct rim of functioning
myocardium or by using chemoattractant cytokines to mobilize the cells from bone marrow. In 1 experiment, bone
marrow was harvested from male mice, labeled with GFP,
and injected into the peri-infarct rim of female mice. This
resulted in a substantial increase in myocytes in the infarct
zone, and the myocytes were of donor origin (on the basis of
Y chromosome staining and GFP expression). There was a
corresponding improvement in hemodynamic parameters after only 9 days.
The same group showed that mobilization of lineagenegative, c-kit cells with granulocyte colony-stimulating
factor and stem cell factor before and after MI in mice
resulted in growth of new cardiomyocytes in the infarct zone
and improved ventricular function and led to substantial
improvement in survival of the treated group. Endothelial and
smooth muscle cells also were proliferating, but new myocyte
growth predominated.47 However, in contrast to these findings, Murry and colleagues48 and Balsam and coworkers49
reported that lineage-negative, c-kit cells did not differentiate into cardiomyocytes. In the latter study, bone marrow
cells transplanted into ischemic myocardium adopted hematopoietic fates rather than transdifferentiating into myocardium yet interestingly still prevented LV dilatation and dysfunction associated with postinfarction remodeling. Despite
the conflicting evidence regarding the ability of bone marrow derived cells to transdifferentiate, their efficacy in
preventing remodeling has been demonstrated in many laboratories; therefore, clinical efficacy trials have progressed in
parallel with ongoing mechanistic laboratory trials to determine the precise molecular mechanism by which these cells
exert their beneficial effects. Kamihata et al7 isolated mononuclear cells from swine bone marrow and injected them into
the infarct zone and peri-infarct region of pigs after MI
induced by left anterior descending coronary artery ligation.
This was associated with an improvement in myocardial
perfusion by contrast echocardiography, increased numbers
of capillaries, increased coronary collateral circulation on
angiography, improved ejection fraction, and reduction in
infarct size compared with controls. Of note, the endothelial
lining of newly formed capillaries was derived from the
donor bone marrow cells, but the fibroblasts in the fibrotic
area of the infarct were not. This suggests that the microenvironment of MI is proangiogenic rather than fibrosisinducing for this population of cells.
This evidence that precursors of both cardiomyocytes and
endothelial cells exist within the mononuclear cell fraction of
adult bone marrow forms the basis for the use of bone marrow
mononuclear cells (BMMNCs) in clinical trials (Table 1). In
just the past 3 years, BMMNC transplantation has become the
343
most widely studied cell-based therapy for human applications (Table 1). Several studies using autologous bone marrow warrant special mention. The initial studies used intracoronary delivery of BMMNCs in the post-MI setting
because animal studies demonstrate that tissue damage in MI
resulted in bone marrow stem cell homing to the infarcted
myocardium.6 In the first published human trial, Strauer et
al50 aspirated BMMNCs and reinfused them into the infarctrelated artery 7 days after MI in 10 patients and had a control
group comprising 10 patients who refused the treatment. This
method resulted in significantly improved myocardial perfusion and wall motion indexes. The Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI) investigators 51,52
randomized 59 patients after acute MI to receive intracoronary infusion of BMMNCs or ex vivo expanded circulating
progenitor cells. They delivered the cells into the infarctrelated artery 4 days after MI and showed improvement in LV
ejection fraction from 51% to 58% (P0.001), as well as
significantly enhanced myocardial viability and regional wall
motion in the infarct area. Interestingly, they were unable to
show a difference between the 2 active cell treatment groups.
In the BOne marrOw transfer to enhance ST-elevation infarct
regeneration (BOOST) trial, Wollert and coworkers53 randomized 60 patients after successful percutaneous coronary
intervention for acute MI to receive either intracoronary
BMMNCs or standard therapy. They demonstrated an improvement in LV ejection fraction of 6.7% in the treatment
group and 0.7% in the control group at 6 months (P0.0026).
Taken together, these studies with control groups suggest that
BMMNCs are safe and may improve cardiac function by a
substantial and clinically meaningful degree following MI.
However, more recently, Janssens and colleagues presented
their findings that intracoronary transfer of BMMNCs failed
to achieve their primary end point, improvement in global LV
function.54 They demonstrated a significant reduction in scar
size and an improvement in regional function, but there was
no improvement in LV ejection fraction (P0.36). Their
patient population differed from the BOOST trial in that they
were reperfused earlier and may therefore have gained only a
small benefit from cell therapy because they derived maximal
benefit from earlier reperfusion. In the 18-month follow-up to
the BOOST study, the improvement in LV ejection fraction in
the cell therapy group was sustained.55 However, the control
group also had improved by this time, and the difference
between the 2 groups was no longer significantly different.
This catch-up phenomenon in the control group suggests that,
rather than the effect of cell therapy being transient, cell
therapy may in fact accelerate the postinfarction LV functional recovery that is achievable with standard medical
therapy.
In contrast to the acute MI setting, patients with chronic
ischemic cardiomyopathy are unlikely to release signals from
damaged myocardium to induce stem cell homing. Therefore,
an alternative approach to intracoronary infusion of cells in
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Circulation
July 25, 2006
TABLE 3.
Clinical Studies Using MSCs

Treated,
n
Study
Autologous/
Allogeneic
Indication
Randomized
34
Autologous
MI
Bang et al122
Autologous
Stroke
Garcia-Olmo et al123
Autologous
Crohns disease fistula
Horwitz et al124
Allogeneic
Osteogenesis
imperfecta
LeBlanc et al125
Allogeneic
GVHD
72
Chen et al
GVHD indicates graft-versus-host disease.
this setting is endomyocardial injection of cells to deliver

them to the exact location where their effect is required. Perin
et al56 enrolled 14 subjects and 7 control subjects with
ischemic cardiomyopathy. The treatment group received
endomyocardial injection of 30 million BMMNCs. They
showed a significant reduction in reversible myocardial
perfusion defects and a significant improvement in overall
LV function. Notably, they enrolled subjects with significant
LV dysfunction at baseline; therefore, their results may be
more clinically relevant to the CHF patient group than
previous studies that enrolled patients with normal or mildly
impaired LV function after MI. Currently, a number of phase
II studies are ongoing with BMMNCs (Table 2).
Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are found in bone marrow,
muscle, skin, and adipose tissue and are characterized by the
potential to differentiate into any tissue of mesenchymal
origin, including muscle, fibroblasts, bone, tendon, ligament,
and adipose tissue.57 MSCs from adult bone marrow can be
separated by density gradient centrifugation and adhering-cell
culture in defined serum-containing medium.58 The cells
isolated after adherence in culture are negative for CD34 and
CD45, unlike hematopoietic progenitors from bone marrow,
and characteristically express CD29, CD44, CD71, CD90,
CD105, CD106, CD120a, CD124, SH2, SH3, and SH4.59 61
Some studies demonstrate that MSCs transdifferentiate into
cardiomyocytes and vascularlike structures.62 66 MSCs differentiate into cardiomyocytes and endothelial cells in vivo
when transplanted to the heart in both noninjury and MI
models. These cells have been strictly characterized by
immunohistochemistry and stain positively for cardiac and
endothelial specific markers, as well as gap junction proteins.64,65,67,68 Myocardial function and capillary formation
are significantly increased in experimental groups treated
with MSCs compared with controls.69,70 These results suggest
that MSCs act by regenerating functionally effective, integrated cardiomyocytes and possibly new blood vessels.
MSCs also have been injected into infarcted myocardium via
a catheter-based approach in pigs, resulting in regeneration of
myocardium, reduced infarct size, and improved regional and
global cardiac contractile function.5 Importantly, the latter
study used allogeneic MSCs, which did not produce evidence

of rejection.
Whereas autologous cell-based therapy poses no risk of
rejection, an off the shelf allogeneic cell product would be
much more cost effective and much easier to administer and
could potentially allow delivery of greater numbers of cells
than autologous cell therapy. MSCs appear to avoid the
problem of rejection by being hypoimmunogenic. These cells
lack MHC-II and B-7 costimulatory molecule expression,
thereby preventing T-cell responses, and induce a suppressive
local microenvironment through the production of prostaglandins and other soluble mediators.58,71 As such, MSCs
may allow allogeneic cell therapy while avoiding rejection.
MSCs also have been studied clinically. Chen and colleagues72 randomized 69 patients after MI to receive intracoronary autologous MSCs or placebo. They demonstrated a
significant improvement in global and regional LV function
and a significant reduction in the size of the perfusion defect,
suggesting that MSC therapy can regenerate infarcted myocardium or protect against LV remodeling.
Currently, several studies have been published using MSCs
for noncardiac disorders (see Table 3), and allogeneic MSCs
are in clinical trials for myocardial regeneration in the United
States under the sponsorship of Osiris Therapeutics (Table 2).
In addition, the Specialized Centers for Cell-Based Therapy
program also plans to conduct clinical trials of MSCs for
patients with CHF.
Endothelial Progenitor Cells

Cells with phenotypic and functional characteristics similar to
the fetal angioblast also are present in adult human bone
marrow.6 These cells, known as EPCs, express some, but not
all, cell surface markers characteristic of mature endothelium,
certain surface markers of hematopoietic cells, and transcription factors that identify them as precursor cells.27,7375 In
addition to endothelial cell surface markers, EPCs also
express markers of immature cells, including AC133, a novel
hematopoietic stem cell marker76 not expressed on mature
endothelial cells.77 After MI, intravenously injected EPCs
homed to the infarct region within 48 hours.6 At 14 days,
there was a marked increase in the number of capillaries in
the infarct zone and the peri-infarct rim resulting from the
induction of both vasculogenesis and angiogenesis, but there
Stem Cell Therapy
was no change in the noninfarcted regions of the heart. There

was a significant reduction in collagen deposition and apoptosis of cardiomyocytes and an improvement in cardiac
function on echocardiography.6 It appears that neovascularization induced by these cells leads to the prevention of
apoptosis and LV remodeling and may lead to some degree of
cardiomyocyte regeneration.15
Erb and colleagues78 randomized patients with recanalized,
chronically occluded coronary arteries to receive intracoronary progenitor cells or placebo. They mobilized bone marrow cells using granulocyte colony-stimulating factor, harvested them from peripheral blood, expanded them ex vivo,
and reinfused them via the coronary artery. This treatment
resulted in significant improvements in coronary flow reserve
and cardiac function and a significant reduction in infarct
size. Currently, clinical trials of EPC therapy for angiogenesis
and myocardial regeneration are in progress that use CD34
cells from bone marrow that are enriched for EPCs. These
cells can be immunoselected from the mononuclear fraction
of bone marrow (Table 2).
Umbilical Cord Blood Stem Cells

Umbilical cord blood (UCB) contains both hematopoietic
stem cells and mesenchymal precursor cells.79 Because stem
cells in UCB exist in higher numbers than in adult human
blood or bone marrow,80 several populations of cells derived
from UCB are possible sources of stem cells for cardiac
repair. Kogler and colleagues81 have described a population
of cells from human UCB called unrestricted somatic stem
cells. These cells, which are fibroblastlike in appearance,
adhere to culture dishes; are negative for c-kit, CD34, and
CD45; and are capable of differentiating, both in vitro and in
vivo, into a variety of tissues, including cardiomyocytes.
Human unrestricted somatic stem cells,82 when delivered by
direct injection at thoracotomy in immunosuppressed pigs
after MI, improved perfusion and wall motion, reduced
infarct scar size, and enhanced global cardiac function.
Ma et al83 injected human mononuclear UCB cells, a small
fraction (1%) of which were CD34, intravenously 1 day
after MI in NOD/scid mice. The cells homed to the infarcted
hearts, reduced infarct size, and enhanced neovascularization
with capillary endothelial cells of both human and mouse origin.
Interestingly, they found no evidence of myocytes of human
origin, arguing against cardiomyogenic differentiation.
In a rat model of MI,84 UCB CD34 improved cardiac
function when injected into the peri-infarct rim immediately
after MI compared with control animals that received injection of medium.
At present, no clinical studies of UCB have been reported.
Guiding Principle for Adopting

New Therapies
As outlined above, a number of promising cell types for
cardiac regenerative therapy have accumulating preclinical
and early clinical data supporting their potential. Neverthe-
345
less, controversy abounds as to whether ongoing clinical trials

of these novel strategies are warranted. We believe that
fundamental criteria supporting ongoing trials have been met.
The World Medical Association in the Declaration of Helsinki has set down principles for clinician researchers to
follow.85 These state: In the treatment of a patient, where
proven prophylactic, diagnostic and therapeutic methods do
not exist or have been ineffective, the physician, with
informed consent from the patient, must be free to use
unproven or new prophylactic, diagnostic and therapeutic
measures, if in the physicians judgment it offers hope of
saving life, re-establishing health or alleviating suffering. In
the case of CHF, it is clear that there are unmet needs for new
treatments to save lives and alleviate suffering. Accordingly,
this principle supports clinical trials of new cell-based
therapies.
In assessing the possibility of adopting a new treatment
into clinical practice, rigorous scientific and ethical standards must be followed. Stem cell therapy has presented us
with a number of unique challenges. Not only is the
optimal cell type not clear, but how to best deliver these
cells also is not immediately apparent, and these questions
can be addressed only by prospectively designed clinical
trials. Our understanding of the exact mechanisms by
which stem cells exert their beneficial effects on cardiac
function has evolved substantially, initially assuming
transdifferentiation as the only mode of action and subsequently expanding to include, among other possibilities,
effects on strengthening the MI scar19 and paracrine
effects.16 Importantly, we propose that all of these features
may be important for an integrated and orchestrated
mechanism of repair that involves restoring endogenous
stem cell niches. Despite incomplete understanding of
mechanism, many independent laboratories have consistently demonstrated the beneficial effects on cardiac function in preclinical and clinical studies. These beneficial
findings cannot be ignored and ongoing clinical studies
delayed on the basis of needing better understanding of the
molecular underpinnings of the observed effects because
these functional improvements may translate to clinical
benefit for patients. These ethical and logistic issues, not
encountered with most previous drug or device trials, may
appear daunting. However, despite the difficulties of stem
cell research, the basic tenets of clinical research still
apply:
Patient safety is paramount.

The risk of depriving patients of a potential new therapy
must be balanced against the inherent risk of testing the
new therapy.
It is with these guiding principles in mind that we must

decide whether stem cell therapy is ready for clinical trials.
Patient Safety Is Paramount

The main issue in going forward into clinical trials is
patient safety. Primum non nocere (first do no harm) is,
346
Circulation
July 25, 2006
as always, our overriding concern. But how much safety

data are enough to justify advancing to the next stage of
clinical investigation? Autologous stem cell therapy begins
with the premise that cellular repair occurs naturally in the
human body and that enhancing that process should be
safe. In fact, numerous animal studies have demonstrated
that this is the case, with no adverse effects of cellular
therapy for cardiac repair being observed; this has served
as the platform for launching into human safety studies.
Several postulated safety issues about stem cell therapy
were raised. There was concern regarding the intracoronary delivery route, fearing that cells administered this
way may result in blockage of the coronary arteries and
further damage to the myocardium. One study using
autologous MSCs from dog bone marrow injected into
normal coronary arteries demonstrated microinfarction in
the region supplied by that artery, suggesting that these
cells aggregate in vivo, leading to arterial or arteriolar
obstruction.86 Nevertheless, intracoronary MSC therapy in
1 human trial resulted in improved cardiac function and
was safe.72 Differences in cell numbers or handling or
delivery techniques may account for these differing clinical outcomes. A number of studies have now demonstrated
safety of the intracoronary route using BMMNCs obtained
by direct bone marrow aspiration50,52,53 and MSCs.72 Another theoretical safety concern was dysregulated angiogenesis or angioma formation within the myocardium seen
with some angiogenic gene therapies.87 Could this occur
with stem cell therapy designed to enhance neovascularization? Or could myogenic therapy result in the formation
of myoma or myosarcoma? Tumor formation has not been
reported in animal or human studies with stem cell
therapies to date when passaged for standard periods such
as 6 to 8 weeks. However, there has been 1 report of
late-passaged MSCs (16 to 20 weeks) developing chromosomal abnormalities and spontaneous cancerous transformation in vitro.88 It is therefore essential that human trials
use rigorous safety analysis of transplanted cells to detect
any possible malignant change or chromosomal abnormalities. Additionally, the problems encountered with gene
therapy may relate to factors not associated with stem
cells. These include the need for viral or other vectors to
deliver the genes, problems with short biological half-lives
of the gene products, and problems delivering the precise
dose required. Although there may be similarities between
gene and stem cell therapies as novel biological therapeutics, the practicalities and logistic difficulties are different
(and substantially less) with stem cell therapy.
Regardless of the type of stem cell used in clinical trials,
it is critically important to be vigilant against ex vivo
contamination of the specimen. Infected or contaminated
cells injected into the heart could potentially lead to
devastating consequences of endocarditis and myocarditis.
As such, all stem cell trial protocols should use good
manufacturing practice facilities to minimize the risk of
such outcomes. Rigorous follow-up also is essential after

stem cell therapy to monitor for complications. Arrhythmias must be actively looked for both by continuous ECG
monitoring around the time of the procedure and by
ambulatory monitoring in follow-up. Additionally, patients
enrolled in stem cell trials using allogeneic cells must be
closely followed up for signs of allograft rejection.
A number of cells have been tested so far in preliminary
trials (Table 1). More than 400 patients have been tested,
and the totality of evidence from these studies supports
safety of each cell therapy and the general approach. This
evidence strongly supports ongoing efforts to advance to
large, randomized, placebo-controlled, double-blind studies with clinical end points.
Balance of Risk
The risk of exposing patients to possible adverse outcomes
of a new treatment must be weighed against the risk of
depriving all patients of a new and possibly effective
treatment to alleviate suffering or to prolong life. There is
now sufficient preclinical and clinical data to warrant
larger randomized controlled trials evaluating stem cell
therapy. The argument that these trials should be delayed
until mechanisms are further understood will unnecessarily
deprive large numbers of patients of therapeutic approaches that may improve their clinical outcome. Only
clinical trials can lead to optimization of stem cell therapy
with recognition of the best type of cell and the best
delivery method for our patients.
Another compelling argument for initiating clinical
research is that the results of these investigations often
provide pivotal insights that allow a new field to advance.
Several examples in cardiovascular medicine illustrate this
concept. ACE inhibitors89 and 3-hydroxy-3-methylglutaryl
coenzyme A reductase inhibitors (statins),90 widely used
drugs with solid mechanistic underpinning supporting their
use, were both appreciated after entry into clinical practice
to have pharmacological effects that extended beyond their
initially intended design. The additional mechanistic effects were suggested by results obtained in clinical trails,
demonstrating the important synergy between in vitro/
animal studies and clinical trials conducted in parallel in
guiding clinical therapies.
Another important example of how mechanistic, novel,
pathophysiological, and therapeutic insights can progress
in parallel during clinical development is percutaneous
transluminal coronary angioplasty (PTCA). In 1977, pioneer Andreas Gruntzig published results obtained in 8
dogs, then progressed to human postmortem experiments91
and next to human clinical trials, publishing his first series
of 5 patients only 1 year later.92 The first study of 50
patients followed in 1979.93 This translational research
program introduced PTCA as a new clinical treatmen, but
was not free of serious complications. Soon the problem of
restenosis, the Achilles heel of PTCA, was appreciated,
Stem Cell Therapy
347
Figure 2. Clinical development of novel therapies.

Pivotal role of clinical trials in developing stem cell
therapeutics. Current phase I and II clinical trials
of cell-based cardiac regenerative therapy are
based on basic science observations and preclinical observations. As indicated, the basis for proceeding with clinical trials includes adequate
safety data and a rational scientific basis. Trials
themselves offer novel mechanistic insights and
may be negative even if a mechanistic basis is
well understood or, as frequently occurs, may be
positive for reasons other than that posited by the
initial hypothesis. Even when a therapy proves to
be of clinical value, unanticipated adverse side
effects may be detected, eliminating the therapy
from the therapeutic armamentarium. In either
case, results of clinical trials offer critical insights
and guide new scientific hypotheses. Clinical trials
are rationally and ethically conducted experiments
that are a necessary step in the development of
any new therapeutic principal. The majority of
clinical trials are negative.
prompting substantial new basic research and the development of the coronary artery stent, which reduced but did
not eliminate the incidence of restenosis. As is now well
known, drug-coated stents that nearly eliminate restensosis
have been developed.94 Before the publication of this
seminal article, safety and feasibility data had been published on 100 patients,9597 substantially less than the
number who have been enrolled in preliminary stem cell
trials. This stepwise progression to clinical trials was
appropriate and timely, with patients receiving the benefits
of the proven therapies, while clinical trial results guided
further basic and preclinical research to eventually refine
and improve these clinical therapies. We anticipate a
similar iterative pathway for the development of stem cell
therapy (see Figure 2).
Importantly, clinical trials do not always produce positive outcomes, yet unexpectedly negative results are often
pivotal to the understanding of disease pathophysiology.
For example, in the Cardiac Arrhythmia Suppression Trial
(CAST), administration of antiarrhythmic drugs for
asymptomatic nonsustained ventricular tachycardia after
MI resulted in an excess of deaths in the treatment arms.98
No animal study had suggested that this would happen. In
fact, further laboratory investigations and animal studies
would have pursued a wrong path and would have wasted
valuable resources. Although the outcome was unfortunate
and unexpected, a properly designed prospective clinical
trial discovered this deadly complication of antiarrhythmic
therapy and changed future therapeutic approaches. The
appropriate progression into prospective, randomized, controlled clinical trials will prevent exposing more patients to
potentially dangerous therapies in uncontrolled trials or in
the proliferation of delivering the therapy out of the
clinical trial context.99 Additionally, the transition from
preliminary studies to randomized controlled trials will

demonstrate efficacy and safety of therapies that are
indeed safe and effective, thereby allowing them to become incorporated into routine clinical practice. The time
to take that step is now.
Clinical Trial Design

In the United States, the Food and Drug Administration
(FDA) Modernization Act, section 113 (FDAMA 113),
mandates the registration of all clinical trials that test
effectiveness for serious or life-threatening conditions
submitted to the FDA under investigational-new-drug
applications.100 A Web-based registry, clinicaltrials.gov,
was established in 2000 by the National Library of
Medicine on behalf of the National Institutes of Health as
a result of this law. This database permits registration of all
clinical trials, including phase I studies that are not
mandated to be registered; the number of registered trials
has been increasing.101 The International Committee of
Medical Journal Editors now demands registration of
clinical trials as a prerequisite for publication.102 Currently, a number of clinical trials103125 of stem cell therapy
for cardiac repair are registered with clinicaltrials.gov;
they are listed in Table 2. All of these trials are phase I or
II trials rather than large phase III trials, and a number
continue to be nonrandomized. We have not included trials
of cytokines/growth factors alone.
Although current data do not support routine clinical use
of stem cell therapy for cardiac repair, the results of
preliminary studies are promising. Many treatments that
improve cardiac function after MI in animal models have
failed to translate into clinical benefit. A number of
reasons may account for this disparity between preclinical
and clinical results, but the most important difference
348
Circulation
July 25, 2006
between the 2 situations is the complex biology and the

multitude of factors in clinical situations that can never
fully be replicated in experimental animal models. At some
point in the discovery of any new treatment modality, only
translation into clinical trials will provide the necessary
data to move forward. There is also a need for ongoing
basic science research in parallel to advance stem cell
therapy toward clinical application.
Clinical studies of stem cell therapy should use rigorous
trial designs and appropriate built-in safeguard mechanisms (Data Safety and Monitoring boards). In this regard,
they should be prospective, double-blind studies with
randomization on a background of best conventional therapy. Clinical trials should be designed to go beyond
surrogate end points and should have adequate power to
detect differences in relevant clinical end points such as
survival, hospitalization, or reinfarction. Furthermore, future studies should be designed not only to assess safety
and efficacy but also to gain further insight into mechanism of action of stem cells. One method of achieving this
is to take advantage of state-of-the-art imaging modalities
to assess myocardial function and to assess myocardial
regeneration or neovascularization.126
Many issues in the burgeoning field of regenerative
medicine still need to be addressed, and clinical trials will
participate in a fundamental way. First, it is necessary to
determine the optimal cell number required to gain maximal clinical effect; this will, in turn, guide how to access
the cells (eg, bone marrow aspirate versus cytokine mobilization) and whether they need to be expanded ex vivo.
Second, the optimal cell type for different patient populations remains unknown. Finally, effective delivery mechanisms must be determined and will likely be cell and/or
disease specific. From these types of studies, not only will
it be possible to determine whether cell therapy for cardiac
repair is effective and safe in humans, but it is also
probable that a great deal of mechanistic information will
be derived that will guide further basic science and
protocol design for subsequent clinical trials.
Conclusions
Although at this time no data support either cell-based or
specific stem cell therapies as standard clinical practice for
cardiac applications, there is a wealth of preclinical and early
clinical data showing safety, feasibility, and early efficacy of
adult cell-based therapy. Adult stem cell therapies should
therefore proceed into randomized, placebo-controlled,
double-blind clinical trials. The ongoing rigorously designed
trials will contribute greatly to this emerging and exciting
new therapeutic approach for diseases of the cardiovascular
system.
Funding
This work was supported by The Johns Hopkins Specialized Center
for Cell-Based Therapy (U54 HL081028), The Johns Hopkins
University School of Medicine Institute for Cell Engineering (ICE),
the John Hopkins University, and University of Texas, Southwestern

Donald W. Reynolds Centers, and NIH grants HL-65455, HL-72185,
and AG078915.
Disclosures
Dr Hare received a research grant from Osiris Therapeutics. The
other authors report no conflicts.
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Response to Boyle et al
Peter Oettgen, MD
Boyle et al raise several important points about the current status of stem cell therapy for cardiac repair and nicely review
the results of multiple clinical trials to date. The vast majority of these trials were conducted using small numbers of patients
with mixed results. Larger, recently completed, prospective, randomized, clinical trials have had similarly mixed results.
Significant gaps remain with respect to our understanding of which stem cells may ultimately be the most therapeutically
useful to promote cardiac tissue regeneration and how best to deliver the stem cells. Because the vast majority of stem cells
do not remain within the heart, even after local injection, it is critical that better ways be developed to promote retention
of the delivered cells within the heart and to be able to track and monitor the stem cells that do not remain within the heart
after delivery. Other safety concerns include the progression of vascular disease and the development of arrhythmias. Do
the results of the completed studies warrant initiation of larger additional clinical trials? The financial resources available
for conducting stem cell research are limited. Large clinical trials, in particular, are costly. Although a significant unmet
clinical need remains in patients with cardiovascular disease, we must carefully weigh how much of these resources should
be devoted to additional basic research in stem cell biology and studies aimed at optimizing their therapeutic potential in
animal models of cardiovascular disease versus conducting additional large clinical trials.
CONTROVERSIES IN
CARDIOVASCULAR MEDICINE
Cardiac Stem Cell Therapy

Need for Optimization of Efficacy and Safety Monitoring
Peter Oettgen, MD
ardiovascular disease remains the number one cause

of morbidity and mortality in the United States and
Europe. In the United States alone, 1 million
patients suffer a myocardial infarction every year, with an
associated mortality of 25% at 3 years.1 A more sobering
statistic is the fact that there are 5 million Americans with
congestive heart failure, with an associated 20% mortality per
year. This remains the case despite advances in pharmacotherapy, cardiac resynchronization therapies, and the use of
implantable cardioverter-defibrillators.2 Some patients with
end-stage congestive heart failure are considered for cardiac
transplantation, but the demand for this therapeutic approach
greatly outweighs the availability of donor hearts. Over the
past few years, several animal studies and a few clinical trials
have supported the use of stem cells as a potential therapeutic
modality to address this unmet clinical need.
Response by Boyle et al p 358
Type of Cells Used for

Cardiac Transplantation
Several different types of cells have been used in both animal
studies and patients to promote the repair of damaged
myocardium. The 2 main sources of stem cells are adult stem
and embryonic stem (ES) cells.
ES Cells
ES cells are derived from the inner mass of developing embryos
during the blastocyst stage. Characteristic features of ES cells
include their proliferative and self-renewing properties and their
ability to differentiate into a wide variety of cell types, including
cardiac myocytes.3 The major concerns with their use in human
trials include the formation of teratomas when ES cells are
injected into immunocompromised animals. This is particularly
important because the ES cells currently available for use in
humans would be of allogeneic origin and therefore would
require immunosuppression. As nuclear transfer techniques improve, they will provide a way of generating an unlimited supply
of histocompatible ES cells using the nuclei of cells obtained
directly from the recipient patients with heart disease.4
Adult Stem Cells

Bone MarrowDerived Stem Cells
Several different types of stem cells can be isolated from adult
bone marrow. Examples of some of these subpopulations of
cells include hematopoietic stem cells, endothelial progenitors,
and mesenchymal stem cells. Several investigators have chosen
to deliver unfractionated bone marrow derived cells, a technique that has the advantage of minimizing extensive ex vivo
manipulation of the cells to isolate and expand a selected
population of cells. The potential disadvantage of delivering a
mixture of cells is that the percentage of cells that are therapeutically useful may be small. An alternative strategy is to isolate
The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.
From the Division of Cardiology, Beth Israel Deaconess Medical Center, Boston, Mass.
Correspondence to Peter Oettgen, MD, Division of Cardiology, Beth Israel Deaconess Medical Center, Boston, MA 02115. E-mail
joettgen@bidmc.harvard.edu
(Circulation. 2006;114:353-358.)
2006 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/CIRCULATIONAHA.106.639385
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July 25, 2006
purer populations of cells that express specific antigens. For

example, endothelial progenitors express the cell surface marker
CD133. These cells have a greater potential to promote angiogenesis but are more technically challenging to isolate in
significant quantities.5 Mesenchymal cells represent a rare population of bone marrow derived cells that do not express CD34
or CD133. Mesenchymal cells can differentiate into bone,
cartilage, adipocytes, and, under certain culture conditions,
cardiac myocytes.6 One advantage of using mesenchymal cells
is that clones of these cells can easily be expanded in vitro,
exhibit relatively low immunogenicity, and might be particularly
useful when autologous stem cells are not readily available.7
Skeletal Myoblasts
Skeletal myoblasts are a population of progenitor cells that
can be isolated from skeletal muscle biopsies and expanded in
vitro. These myoblasts can differentiate into myotubes and
exhibit skeletal muscle phenotype after transplantation, leading to improvements in left ventricular (LV) systolic and
diastolic function.8 However, the transplanted skeletal myocytes are not electrically coupled to surrounding cardiomyocytes and thus may lead to the development of arrhythmias.
Resident Cardiac Stem Cells
Several investigators have recently identified a population of
stem cells within the myocardium that are capable of differentiating into cardiac myocytes. It has recently been reported
that these cells can be harvested from cardiac biopsies.
Injecting these cells in the setting of myocardial infarction
can promote cardiomyocyte formation with associated improvements in systolic function.9 At present, these cells are
limited in number and require ex vivo separation and expansion over several weeks.
Methods of Stem Cell Delivery

A major goal of cardiac stem cell therapy is to transplant enough
cells into the myocardium at the site of injury or infarction to
maximize restoration of function. Several different approaches
currently are being used to deliver stem cells.
Transvascular Route
A transvascular approach is particularly well suited to treat
patients with acutely infarcted and reperfused myocardium.
Stem cells can be infused directly into the coronary arteries
and have a greater likelihood of remaining in the injured
myocardium as a result of the activation of adhesion molecules and chemokines.10 The advantage of an intracoronary
infusion is that the cells can be directed to a particular
territory. An alternative approach is to inject stem cells
intravenously.11 In the setting of myocardial infarction, circulating stem cells have been shown to home to sites of
injury, but the number of cells that home to the heart in this
way is significantly less than by local injection.
Direct Injection Into the Ventricular Wall

Direct injection of stem cells is used in patients presenting with
established cardiac dysfunction in whom a transvascular ap-
proach may not be possible because of total occlusion or poor

flow within the vessel of the affected territory. There are 3
different approaches to direct injection. A transendocardial
approach can be used in which a needle catheter is advanced
across the aortic valve and positioned against the endocardial
surface.12 Cells can then be injected directly into the left
ventricle. Electrophysiological mapping can be used to differentiate sites of viable, ischemic, or scarred myocardium. In a
transepicardial approach, cells are injected during open heart
surgery. The advantage of this approach is that it allows direct
visualization of the myocardium and easier identification of
regions of scar and border zones of infarcted tissues. A third
approach involves the delivery of cells through one of the
cardiac veins directly into the myocardium.13 The limitation of
this approach is that positioning the catheter within a particular
coronary vein may be considerably more time consuming and
technically challenging.
Safety Concerns
Arrhythmias
Over the past few years, some of the early-phase clinical
studies have suggested the possibility of a proarrhythmic
effect associated with stem cell transplantation. In 1 study,
skeletal myoblasts were injected transepicardially at the time
of coronary artery bypass surgery. Four patients had documented ventricular tachycardia at 11, 12, 13, and 22 days
after stem cell implantation.14 Interestingly, these events
occurred early and were not observed in treated patients later
after several months of follow-up. A similar proarrhythmic
effect was observed when autologous skeletal myoblasts were
delivered via a transvascular route.15 Other studies have
similarly reported an increased frequency of nonsustained
ventricular tachycardia in patients treated with skeletal myoblasts, peaking 11 to 30 days after stem cell transplantation.16,17 A proposed mechanism for the increased incidence
of arrhythmias is that the injected stem cells do not communicate electrically with neighboring cardiac myocytes and/or
result in slowed conduction, thereby promoting reentrant
arrhythmias. It has recently been suggested that skeletal
myoblasts that have been genetically engineered to express
gap junction protein connexin 43 exhibited decreased arrhythmogenicity.18 Although proarrhythmic effects have been
observed predominantly in patients receiving skeletal myoblast transplantation, they also have been observed recently in
2 patients shortly after transplantation of CD133 cells.19,20
Restenosis, Accelerated Atherosclerosis, and

Coronary Obstruction
There have been conflicting reports regarding the potential
for increased restenosis after stem cell transplantation. In 1
study, a high rate of restenosis was observed after intracoronary delivery of peripheral blood stem cells mobilized with
granulocyte colony-stimulating factor in the setting of myocardial infarction and stent placement.21 In another study,
CD133 cells were delivered via intracoronary injection in
Stem Cell Therapy
the setting of myocardial infarction, with in-stent restenosis

rates of 37% and reocclusion rates of 11%.19 Relatively low
rates of restenosis were observed in earlier studies using bone
marrow derived stem cells.10,20 In addition to restenosis, it is
also possible that stem cell transplantation may promote the
formation of de novo lesions or atherosclerotic plaque progression. In 2 recent studies, there was a fairly high proportion of new lesions identified in the nonstented vessels after
stem cell transplantation.19,22 It is also possible that if the cells
are delivered at a high enough concentration via the coronary
circulation, they may adhere to each other, form aggregates,
and thereby lead to the occlusion of microvessels. In 1 study
in which mesenchymal stem cells were administered by
intracoronary injection in pigs, there was associated occlusion
of microvessels and macrovessels.23
Abnormal Cellular Differentiation

Fortunately, no clinical trials to date that have used stem cells
to promote cardiac tissue regeneration have demonstrated an
increased frequency of tumor formation. However, most of
the clinical trials have been conducted on small numbers of
patients. Furthermore, it is not clear how adequate testing
would be conducted to monitor for this potential side effect.
Because stem cells are known to migrate to several other
organs after delivery to the heart, it is conceivable that
aberrant cellular differentiation with the potential of tumor
formation could occur in any of these organs.
Tracking of Stem Cells

One of the major concerns regarding the delivery of stem
cells is determining which cells remain in the heart and which
cells ultimately end up in other organs as a result of a washout
effect.24 Within a few hours after transplantation, stem cells
injected locally within the heart also are observed within the
lungs, spleen, liver, and kidney. One day after transplantation
of neonatal cardiac myocytes into rat hearts, only 24% of the
originally injected cells remained in the heart.25 Given the
small fraction of stem cells that remain within the heart after
injection and the multiple organs to which the stem cells
migrate, it is imperative that better methods of tracking stem
cells be developed to determine the fate of these cells after
transplantation. Several potential methods have been developed to label and track stem cells in animal models, including
scintigraphy, PET, and MRI.26 28 PET scanning and MRI also
have been tested recently in humans to track stem cells.29,30
One hour after injection of 18F-fluorodeoxyglucoselabeled
CD34 cells, only 5.5% of the cells were detectable in the
heart by PET scanning. Unfortunately, because of the short
half-life of 18F-fluorodeoxyglucose, other isotopes with a
longer half-life may need to be evaluated for optimal longterm tracking of stem cells.
Evidence for Tissue Regeneration

The ultimate goal of stem cell therapy is to promote cardiac
tissue regeneration so that the regenerated cardiac tissue leads
355
to improvements in cardiac function in a fashion that is

synchronized with the rest of the functioning heart in the
absence of proarrhythmic or other adverse effects. More
recently, however, there is evidence that stem cells may lead
to improvements in cardiac function that are independent of
tissue regeneration. Although early studies supported the
ability of bone marrow derived mononuclear cells to differentiate into cardiac myocytes, subsequent studies failed to
support these initial observations.3133 It has been suggested
that the locally injected cells can act in a paracrine fashion to
improve ventricular function through the release of growth
factors or other paracrine mediators. These mediators may act
to directly augment systolic function, prevent apoptosis of
ischemic myocardial cells, or limit injury by promoting
angiogenesis.34 The locally injected stem cells would promote
the salvage of injured myocardium rather than tissue regeneration. Additional long-term studies are needed to determine
whether the improvements observed after weeks to a few
months are generally sustained over longer periods of time.
These paracrine effects are more likely to be useful in patients
with acute myocardial ischemia or with hibernating myocardium and less likely to be beneficial in patients with chronically infarcted myocardium with significant scar formation.
Significant challenges remain with regard to cardiac tissue
regeneration. Future studies are needed to identify the best
stem cell type to use. To promote cardiac tissue regeneration,
sufficient numbers of cells will need to be delivered and
maintained within the heart at the site of LV dysfunction,
and the new tissue needs to be vascularized, electrically and
mechanically coupled with the rest of the myocardium. The
hope is that the strategies will include ways of replacing
scarred or fibrotic tissue in regions of LV dysfunction. Unless
autologous cells can be used to generate the cardiac tissue,
potential graft rejection needs to be addressed. Real progress
toward this goal will require the collaborative interaction of
investigators with expertise in tissue engineering, molecular
biology, electrophysiology, cardiac physiology, immunology,
and vascular biology.
Recent Clinical Trials

Several small clinical studies using a variety of different cell
types have shown some initial promise regarding the benefit of
stem cell therapy, but these small clinical studies have several
limitations. In addition to being small, some of the studies lacked
adequate controls or randomization in a blinded fashion. Furthermore, some of the studies failed to assess infarct size or
ventricular function before administration of the stem cells, and
the follow-up period often was short.
Results of recent randomized clinical trials evaluating the
therapeutic effect of administering bone marrow derived
mononuclear cells via intracoronary injection at the time of
myocardial infarction have recently been reported (Table).
Results of these studies have been mixed. The primary end
point of these studies was LV ejection fraction. The administration of stem cells in 2 studies, BOne marrOw transfer to
356
Circulation
July 25, 2006
Randomized Clinical Trials of Stem Cell Therapy for MI

Study
Treated, n
Cell Type/Treatment
Mode of Delivery
End Point
Janssens et al37
67
BMMNCs
Intracoronary
LV ejection fraction
0.36
BOOST35
60
BMMNCs
Intracoronary
0.0026
ASTAMI
100
BMMNCs
Intracoronary
0.05*
REPAIR-AMI36
204
BMMNCs
Intracoronary
0.021
Zohlnhoefer et al35
114
G-CSF
Subcutaneous
Infarct size
0.79
62
G-CSF
Subcutaneous
LV systolic wall thickening
1.00
36,38
STEMMI42
BMMNCs indicates bone marrow mononuclear cells; G-CSF, granulocyte colony-stimulating factor; and STEMMI, Stem Cells in
Myocardial Infarction.
*LV ejection fraction was higher in the control group.
enhance ST-elevation infarct regeneration (BOOST) and

Reinfusion of Enriched Progenitor cells And Infarct Remodelling in Acute Myocardial Infarction (REPAIR-AMI), resulted in significant increases in LV ejection fraction.35,36 The
differences in ejection fraction of the treated and control
groups at 6 months in the BOOST trial were 56.7% and
52.0%; in the REPAIR-AMI treated and control groups, the
differences were 54% versus 50%. In contrast, in the study by
Janssens et al,37 no difference between control and treated
groups was observed, and in the Autologous Stem cell
Transplantation in Acute Myocardial Infarction (ASTAMI)
trial, the LV ejection fraction was higher in the control
group.36,38 The cause of these differences is unclear but may
relate to how the cells were prepared before delivery or to the
fact that the baseline ejection fractions at the time of
myocardial infarction were only mildly diminished. There are
ongoing additional randomized trials. BOOST-II will enroll
200 patients with large myocardial infarctions and depressed
ejection fractions to receive bone marrow derived mononuclear cells or placebo. The Myoblast Autologous Grafting in
Ischemic Cardiomyopathy (MAGIC) trial will evaluate the
effect of autologous skeletal muscle myoblasts in patients
with chronic heart failure who are undergoing coronary
bypass and defibrillator implantation.
One attractive alternative to delivering autologous stem cells
via local injection is to identify the mechanisms by which stem
cells are recruited to the heart and try to augment mobilization of
stem cells, particularly in the setting of acute infarction. Small
clinical studies suggested that cytokines such as granulocyte
colony-stimulating factor could promote the mobilization of
bone marrow derived stem cells in the setting of myocardial
infarction, leading to improvements in myocardial function.39,40
The advantage of this approach is that the cells are autologous
and can be mobilized via systemic injections of granulocyte
colony-stimulating factor. Two larger randomized trials, consisting of 78 and 114 patients, similarly used granulocyte colonystimulating factor to mobilize stem cells in the setting of
myocardial infarction.41,42 Unfortunately, neither of these studies
demonstrated a significant benefit with respect to cardiac function after 6 months. Furthermore, another smaller study in which
intracoronary injections of stem cells isolated after stimulation
with granulocyte colony-stimulating factor were administered in

the setting of myocardial infarction resulted in an increased rate
of restenosis.21
Conclusions
Stem cells remain a highly promising therapeutic modality
that could address the large, unmet clinical need of treating
patients throughout the world with significant cardiac dysfunction that cannot be adequately treated with conventional
therapeutic approaches or cardiac transplantation because of
the limited availability of this resource. On the basis of the
mixed results of more recent larger clinical trials, we should
err on the side of caution before committing precious resources to conduct additional large clinical trials. Several
questions need to be addressed. First, have we identified
which stem cell type to use? Second, have we determined the
mechanisms by which stem cells promote myocardial function or repair? At present, there is limited evidence to support
that stem cells used thus far in patients promote significant
cardiac tissue regeneration. Third, can the stem cell be
retained efficiently within the heart? Finally, can clinical
trials be done in such a way that important safety issues will
be adequately addressed? What methods will be used to
monitor the development of life-threatening arrhythmias and
to track injected stem cells throughout the body? Because the
financial resources available for clinical and basic stem cell
research are not unlimited and because of the high cost
associated with conducting larger clinical trials, it is particularly important that we address the aforementioned questions
before proceeding with larger clinical trials. It is clear that
additional basic research is needed to optimize ways to
promote cardiac tissue regeneration, to improve methods by
which delivered stem cells will remain in the heart, and to
optimize the way in which stem cells are tracked after
delivery.
Funding
This work was supported by National Institutes of Health grant
PO1-HL-76540.
Disclosures
Dr Oettgen has received a received a research grant from the NIH.
Stem Cell Therapy
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Response to Oettgen
Andrew J. Boyle, MBBS, PhD; Steven P. Schulman, MD; Joshua M. Hare, MD
We agree with Dr Oettgen on many issues surrounding cell therapy. Preclinical studies demonstrate considerable promise for
many cell types to effect cardiac repair, yet the initial promise from small animal work is far more difficult to demonstrate in
humans. Moreover, early clinical trials of skeletal myoblasts and granulocyte colony-stimulating factor have discovered
unexpected side effects not apparent in animal models. These rigorously designed early clinical studies and their unanticipated
findings have now guided not only the design of next phase of clinical studies but also the future of basic and animal studies.
Most importantly, we agree that understanding the mechanistic underpinnings of cell-based therapy is essential. Our area of
fundamental disagreement relates to the role of the clinical trial in advancing the burgeoning field of cell-based therapy. We
believe that these trials must proceed but must be conducted by responsible investigators concerned with patient safety and
committed to incorporating mechanistic studies into the trials. Clinical development of any new therapy begins with a phase I
study aimed at proving safety, and major trials contain independent Data Safety and Monitoring boards. Every detail regarding
the mechanism of action of these cells cannot be appropriately determined in animal models because, by definition, that which
happens in controlled animal experiments may not translate into humans, who are much more heterogeneous and have many
comorbidities that affect responsiveness to novel therapies. There exists, by necessity, a synergy between basic science and
clinical research. An incomplete understanding of molecular/cellular mechanisms should not halt the progression of clinical trials;
quite the opposite is the case. The success or failure of clinical trials guides future basic science research, which, in turn, informs
future clinical trials to achieve improvements in health outcomes for patients.

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Stem Cell Therapy for Cardiac Repair: Ready for the Next Step

Andrew J. Boyle, Steven P. Schulman and Joshua M. Hare

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Stem Cell Therapy for Cardiac Repair

oronary heart disease and heart failure continue to be

Response by Oettgen p 352

component in the process of understanding the scientific

Mechanism of Action of Stem Cells

July 25, 2006

Clinical Studies of Stem Cell Therapy for Cardiac Repair

Primary End Point

Safety and feasibility

Open heart surgery

Safety and feasibility

Safety and feasibility

Safety and feasibility

Safety and feasibility

Open heart surgery

Open heart surgery

Safety and feasibility

Safety and feasibility

Open heart surgery

Safety and feasibility

CFR and LVEF

Safety and feasibility

Open heart surgery

Safety and feasibility

Open heart surgery

Safety and feasibility

Safety and feasibility

Safety and feasibility

Open heart surgery

Safety and feasibility

Safety and feasibility

Open heart surgery

Safety and feasibility

Open heart surgery

Open heart surgery

Safety and feasibility

in vitro systems and animal models.9,10 We would suggest

Types of Cells Contemplated for Cellular

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Stem Cell Therapy

Cell Type Used

CHF undergoing CABG

CHF undergoing VAD

Texas Heart Institute

Multicenter, United States

Multicenter, United States

Multicenter, United States

structures, formation of intercalated disks, desmosomes, and

Resident Cardiac Stem Cells

Figure 1. Possible mechanisms for successful cardiac regenerative therapy. See

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July 25, 2006

Recent studies have isolated, from both human and murine

Human Adult Bone MarrowDerived Stem Cells

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Stem Cell Therapy

this endogenous repair mechanism and use bone marrow as a

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July 25, 2006