1. health and fitness
2. disease

REVIEWS
Cell therapy for the treatment of coronary
heart disease: a critical appraisal
Kai C. Wollert and Helmut Drexler
Abstract | Randomized, controlled clinical trials have demonstrated that cell therapy can improve the recovery
of cardiac function in patients after acute myocardial infarction (AMI). Trial results are inconsistent, however,
and uncertainty persists regarding the mechanism of action and prospect of cell therapy for patients with
heart disease. This Review examines the results from the first-generation trials and discusses procedurerelated variables that could have determined treatment outcomes. Obvious issues, including optimal timing
of cell transfer, dose, and delivery methods are being investigated in ongoing second-generation trials. These
studies aim to refine the protocols and identify the patients who will benefit most from cell therapy. Thirdgeneration trials will address the current limitations of cell therapy, such as cell retention and cell survival
after transplantation, and impaired cell functionality in patients with advanced cardiovascular disease. The
secretion of factors with paracrine effects by the transplanted cells is an increasingly recognized phenomenon.
Identification of these factors, by secretome analyses and bioinformatic approaches, could advance
protein-based therapies to promote healing and inhibit pathological remodeling of the heart after AMI. The
identification of reliable sources of pluripotent stem cells and their differentiation into mature cardiac cell
types could ultimately enable regeneration of the infarcted heart.
Wollert, K. C. & Drexler, H. Nat. Rev. Cardiol. 7, 204–215 (2010); published online 23 February 2010; doi:10.1038/nrcardio.2010.1
Introduction
Hans-Borst Center for
Heart and Stem Cell
Research, Department
of Cardiology and
Angiology, Hannover
Medical School, CarlNeuberg-Straβe 1,
30625 Hannover,
Germany (K. C. Wollert,
H. Drexler).
Correspondence to:
K. C. Wollert
wollert.kai@
mh-hannover.de
Acute myocardial infarction (AMI) is a leading cause of
death worldwide. Advances in treatment for patients after
AMI have led to a decrease in early mortality, but as a
result there is a higher incidence of heart failure (HF)
among survivors. 1 Current therapies do not address
the central problem associated with the aftermath of
AMI—the massive loss of cardiomyocytes, vascular cells,
and interstitial cells—so these patients continue to experience frequent hospitalizations and premature death.2 Cell
transplantation was conceptualized more than 10 years
ago as a means to augment cardiomyocyte numbers and
improve cardiac function after AMI.3,4 Regeneration of
the infarcted heart is a daunting task considering that the
cardiomyocyte deficit could be in the order of 1 billion
cells,5 that supporting cells as well as cardiomyocytes
have to be supplied, and that the environmental cues
required to guide transplanted cells into multicellular,
three-dimensional (3D) heart structures might be absent
from damaged myocardium.6
In many of the early experimental studies, fetal, neonatal, and adult cardiomyocytes were transplanted and
shown to form stable grafts in injured hearts.7 Owing to
the limited availability of differentiated cardiomyocytes,
however, it is unrealistic to use these cells for large-scale
clinical applications. Stem cells have, therefore, emerged
as the primary cell source for regenerative therapies
given the capacity of stem cells for self-renewal, infinite
Competing interests
The authors declare no competing interests.
ex vivo proliferation, and differentiation into specialized
cells. Pluripotent stem cells can differentiate into cells
derived from all three germ layers, a typical example
being embryonic stem cells (ESC), which are isolated
from the inner cell mass of blastocysts and can give rise
to all cardiac cell types. By contrast, adult stem cells
are multipotent and restricted in their differentiation
potential to cell lineages of the organ in which they are
located, such as hematopoietic stem cells (HSC) giving
rise to mature hematopoietic cells, or mesenchymal stem
cells (MSC) giving rise to osteoblasts, chondrocytes, and
adipocytes. Progenitor cells, for example, endothelial progenitor cells (EPC) or skeletal myoblasts, are even more
restricted in their differentiation potential and have a
limited capacity to self-renew. Experiments conducted
at the beginning of this millennium, however, appeared
to challenge the concept that adult stem and progenitor
cells are lineage restricted and suggested that these cells
can transdifferentiate into cell types outside their original lineage.8,9 This concept of so-called plasticity of adult
stem cells, combined with a large body of animal data
demonstrating that transplantation of adult stem and progenitor cells can improve contractile function after AMI,
provided the rationale to treat patients with adult stem
and progenitor cells.10
The field of cardiac cell therapy has made rapid progress. As discussed below, some clinical trials indicate that
cell therapy can improve cardiac function after AMI.
Parallel investigations of the mechanisms involved,
however, have shattered the concept of adult stem cell
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REVIEWS
plasticity and highlighted the possible importance of
paracrine effects. This scientific roller coaster has generated excitement and confusion. Further uncertainty
has been introduced by the lack of a universally accepted
nomenclature for stem and progenitor cells, the (occasional) imprecise use of terminology, for example, stem
cell therapy instead of bone marrow cell (BMC) therapy,
the large number of cell types that are undergoing clinical testing, and a lack of standardization in cell isolation
protocols, which would facilitate a comparison of clinical trial results from different institutions. To assess the
current status of this therapy, this Review will examine
the results from the first-generation trials and discuss
procedure-related variables that could have determined
treatment outcomes. It also explores continuing and
arising issues in the ongoing second-generation trials, and
what should be addressed in third-generation trials. We
use the nomenclature that has been applied by the respective investigators; the reader is encouraged to refer to the
original publications to learn more about the cell isolation
protocols that were used in the cited articles.
Lessons from the first trials
We start our discussion with a brief overview of the
largest randomized, controlled clinical trials that have
evaluated cell therapy in patients with coronary heart
disease (Table 1). Most of these studies used unfractionated BMCs as an easily accessible source of adult stem
and progenitor cells.
Acute myocardial infarction
In the BOOST trial,11 60 patients were randomly assigned
to nucleated BMC transfer—on average 4.8 days after
acute percutaneous coronary intervention (PCI)—or
to standard therapy (control). Cardiac function was
assessed in a blinded fashion with MRI at serial time
points before cell transfer, and after 6, 18, and 61 months.
Patients in the nucleated BMC group showed improved
left ventricular (LV) contractility in the infarct border
zone and an improvement of global LV ejection fraction (LVEF) by 6 percentage points after 6 months when
compared with controls. Overall, the differences in LVEF
improvement between the nucleated BMC and control
groups were, however, no longer statistically significant
after 18 months (2.8 percentage points) and 61 months
(0.8 percentage points).12,13 In a post hoc analysis, patients
with larger infarcts and an infarct transmurality greater
than the median value in the study population appeared
to benefit from nucleated BMC transfer with sustained
improvements of LVEF also at the later time points.12,13
After 6 and 18 months, the control group had developed
echocardiographic signs of mild diastolic dysfunction,
which were attenuated in the nucleated BMC group.14
In the Leuven AMI trial,15 67 patients were randomly
assigned to receive mononucleated BMC or placebo
infusion within 24 h after acute PCI.15 MRI assessment
of LVEF at 3–4 days and 4 months after PCI did not
demonstrate a significant impact of mononucleated
BMC therapy on LVEF recovery, the primary end point
of the trial. Notably, however, the reduction of infarct
Key points
■ Clinical trials show that bone marrow cell therapy improves myocardial
perfusion and contractile performance in patients with acute myocardial
infarction, heart failure, and chronic myocardial ischemia
■ Trial results are not uniform, however, probably owing to the current lack of
standardization and optimization of cell isolation and delivery protocols
■ Ongoing clinical trials are addressing these limitations in an attempt to develop
robust and reproducible cell therapy protocols that can be applied more widely
and improve clinical outcome
■ Bone marrow cells are thought to have paracrine effects on neovascularization,
inflammation, wound healing and possibly resident stem and progenitor cells
■ Secretome analyses could lead to the identification of paracrine factors with
therapeutic potential for patients with coronary heart disease
■ Pluripotent stem cells provide an opportunity to generate patient-specific
cardiac cells, but tumorgenicity and poor engraftment after transplantation
currently limit their use for regenerative cell therapy and tissue engineering
volume after 4 months, as measured by serial contrastenhanced MRI, was greater in mononucleated BMCtreated patients than in controls. Moreover, a significant
improvement in regional contractility was observed on
MRI in the mononucleated BMC group with the greatest infarct transmurality at baseline.15 Echocardiographic
strain rate imaging confirmed that mononucleated BMC
infusions improved the recuperation of myocardial function in the infarct region, which suggests that quantitative
assessment of regional systolic function could be more
sensitive than measuring global LVEF for the evaluation
of cell therapy after AMI.16
In the REPAIR-AMI trial,17 204 patients were randomly
assigned to receive mononucleated BMC or placebo, on
average 4.4 days after acute PCI. LV function was assessed
by contrast angiography. Infusion of mononucleated BMC
promoted an increase in LVEF of 2.5 percentage points
after 4 months compared with placebo. In a subgroup of
54 patients who underwent serial MRI investigations,
the treatment effect of mononucleated BMC infusion on
LVEF amounted to 2.8 percentage points at 12 months.18
This finding is similar to that in the BOOST trial after
18 months.12
In the ASTAMI trial, 19 100 patients with anterior
AMI were randomized to receive mononucleated BMC
or standard therapy (control). Cells were infused on
average 6 days after acute PCI. After 6 and 12 months,
no significant effects of mononucleated BMC therapy
on LVEF, LV volumes, or infarct size were observed by
single-photon emission CT (SPECT), echocardiography,
or MRI.19,20 Fewer mononucleated BMC were infused in
the ASTAMI trial than in the REPAIR-AMI trial (median,
68 × 106 versus 198 × 106 cells).17,19 The cell isolation protocol used in the ASTAMI trial might also have recovered a
mononucleated BMC population with impaired functionality, as assessed by in vitro migratory and colony forming
capacities, and in vivo capacity to promote blood flow
recovery in a mouse model of hind-limb ischemia.21
In the FINCELL trial,22 80 patients with AMI treated
with thrombolytic therapy followed by PCI, were randomly assigned to mononucleated BMC infusions or
placebo. Cells were infused immediately after PCI, which
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Table 1 | Randomized trials in patients with acute myocardial infarction or ischemic heart failure
Trial name
Number
of patients
Cell type
Dose
Route of
delivery
Timing of
delivery
Primary end
point
Comments
nBMC
128 ml
i.c.
Day 6 ± 1
LVEF
Effect diminished after
18 and 61 months
Acute myocardial infarction
BOOST
60
REPAIR-AMI
187
mnBMC
50 ml
i.c.
Day 3–6
LVEF
NA
Leuven-AMI
66
mnBMC
130 ml
i.c.
Day 1
LVEF
Regional contractility
Infarct size
ASTAMI
97
mnBMC
50 ml
i.c.
Day 6 ± 1
LVEF
NA
FINCELL
77
mnBMC
80 ml
i.c.
Day 3
LVEF
NA
REGENT
117
mnBMC
(unselected
vs CD34+/
CXCR4+)
50–70 ml
(unselected)
100–120 ml
(selected)
i.c.
Day 3–12
LVEF with both
cell types
NA
HEBE
189
mnBMC
vs mnPBC
60 ml (mnBMC)
150 ml (mnPBC)
i.c.
Day 3–8
Regional
contractility
NA
Ischemic heart failure
MAGIC
97
SkM
400 or
800 × 106
i.m.
>Week 4
LVEF
LVEDV
LVESV
TOPCARE-CHD
58
mnBMC
vs CPC
50 ml
i.c.
Month
81 ± 72
LVEF (mnBMC)
LVEF
(CPC)
NA
Only patients with complete imaging studies are considered here. Dose refers to the average amount of bone marrow or peripheral blood that was harvested, or
the number of transplanted skeletal myoblasts. Abbreviations: , decreased; , increased; , no significant change; CPC, circulating blood-derived progenitor
cells; i.c., intracoronary; i.m., intramuscular; LVEDV, left ventricular end-diastolic volume; LVEF, left ventricular ejection fraction; LVESV, left ventricular endsystolic volume; mnBMC, mononucleated bone marrow cells; mnPBC, mononucleated peripheral blood cells; NA, not applicable; nBMC, nucleated bone marrow
cells; SkM, skeletal myoblasts.
was performed 2–6 days after thrombolysis. LV contrast
angiography before and 6 months after cell transfer
showed that mononucleated BMC therapy improved
LVEF recovery by 5 percentage points compared with the
control group. Paired echocardiographic investigations
yielded similar results.22
In the REGENT trial,23 200 patients with anterior AMI
were randomized to receive an infusion of unselected
mononucleated BMC or CXCR4+/CD34+ mononucleated
BMC, on average 7 days after acute PCI, or to standard
therapy (control). Paired MRI images to assess LVEF
at baseline and after 6 months were available in 117
patients. Significant improvements in LVEF from baseline to follow-up were noted within the unselected and
selected mononucleated BMC groups (3 percentage
points each), but not in the control group (no change
in LVEF). Differences in LVEF improvements from
baseline to 6 months between the two BMC groups and
the control group, however, were not significant. This
trial was limited by imbalances in baseline LVEF and
incomplete follow-up. Nevertheless, the REGENT trial
indicates that a specific BMC population expressing
progenitor cell surface markers could be responsible for
most of the observed effects.
In the HEBE trial,24 200 patients with AMI were randomly assigned to receive an infusion of mononucleated
BMC or mononucleated cells isolated from peripheral
blood (PBMC), or to standard therapy (control). The
final results of the HEBE trial were presented at the AHA
Scientific Sessions in 2008, 25 and showed that intracoronary infusion of mononucleated BMC or PBMC did
not improve global or regional LV systolic function at
4 months, as assessed by MRI. Further discussion of these
data will have to await publication of the full trial report.
So far, no safety concerns relating to intracoronary
BMC infusions have emerged. An increased risk of instent restenosis was observed in a small, nonrandomized
study after intracoronary infusion of CD133 + mononucleated BMC.26 In the placebo-controlled FINCELL
trial, 22 no increased risk of in-stent restenosis was
observed by intravascular ultrasonography after 6 months.
In two meta-analyses, the risks of target-vessel restenosis
or repeat revascularization were not increased in patients
treated with BMC.27,28 Moreover, none of the clinical trials
reported an increased incidence of symptomatic arrhythmias after intracoronary BMC transfer. An electrophysiological study performed in the BOOST trial,11 and a
careful assessment of microvolt T-wave alternans and
signal-averaged electrocardiography measures in the
FINCELL trial22 provide further assurance of this.
Ischemic heart failure
Skeletal myoblasts were the first cell type to undergo
clinical testing in patients with HF.29 These are lineagerestricted progenitor cells that can be isolated from skeletal muscle biopsy samples and expanded in vitro. When
transplanted into an infarct scar, myoblasts differentiate
into myotubes that usually remain electromechanically
isolated from the host cardiomyocytes. Early, nonrandomized clinical studies confirmed the feasibility
of transplanting autologous skeletal myoblasts.30 The
MAGIC study 31 was the first randomized, placebocontrolled trial in this field. 97 patients with ischemic
HF received transepicardial injections of autologous
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skeletal myoblasts or placebo during CABG surgery,
in and around akinetic segments. After 6 months, no
difference was seen between myoblast transplantation
and placebo in the improvement of regional or global
LV function. Notably, significant reductions in LV enddiastolic and end-systolic volumes were observed after
myoblast therapy in the MAGIC trial. No significant
differences in major adverse cardiac events between
the placebo and cell-treated groups were observed.31 A
trend towards a greater incidence of arrhythmias was,
however, noted in myoblast-treated patients,31 thus confirming a safety concern that had already been raised
by earlier, nonrandomized trials. 32 Further investigations of skeletal myoblast therapy are needed to establish whether the potential benefits outweigh the risk of
increased arrhythmogenicity.32
In the TOPCARE-CHD trial,33 75 patients with ischemic HF were randomly assigned to receive no cell infusion, or infusions of mononucleated BMC or circulating
blood-derived progenitor cells into the patent coronary
artery supplying the most dyskinetic left ventricular
area. To obtain circulating blood-derived progenitor
cells, they were isolated by Ficoll density gradient centrifugation and cultured ex vivo in medium containing
vascular endothelial growth factor (VEGF), a statin,
and autologous serum. Three months after therapy, the
absolute change in LVEF, assessed by contrast angiography, was significantly greater among patients who
had received mononucleated BMC (+2.9 percentage
points) than among those receiving circulating bloodderived progenitor cells (–0.4 percentage points) or
no cell infusion (–1.2 percentage points).33 No adverse
effects were reported.
Chronic myocardial ischemia
An increasing number of patients with coronary heart
disease have chronic myocardial ischemia and experience
refractory angina that is not amenable to revascularization. Chronic ischemia can be associated with a
regional impairment of contractile function, which is
partially reversible when tissue perfusion is restored
(hibernating myocardium). New therapeutic strategies
aimed at delivering oxygenated blood to the myocardium
in these patients are needed. Three randomized, doubleblind, placebo-controlled cell therapy trials have been
completed.34–36 Blood or bone marrow-derived cells
were injected transendocardially into ischemic areas
that were identified by nuclear perfusion imaging and
electromechanical mapping. Apart from one episode of
ventricular tachycardia occurring during the mapping
procedure,34 and one pericardial infusion that was treated
with cardiocentesis,36 transendocardial injections caused
no adverse effects.
Losordo et al. explored the therapeutic potential of
CD34+ cells in 24 patients.34 CD34+ cells were collected
from the peripheral blood after five daily injections of
granulocyte colony-stimulating factor. Angina frequency
and exercise time showed trends in favor of CD34+ cell
therapy, but perfusion imaging at 3 and 6 months yielded
no clear-cut evidence for a greater reduction in myocardial
ischemia in the cell-treated group compared with control.
A larger phase IIb study is now under way.34
In the PROTECT-CAD trial,35 which included 28
patients, injections of mononucleated BMC were associated with improvements in NYHA class, exercise time,
LVEF, and wall thickening over the target regions by
6 months. Angina class decreased similarly in cell-treated
and placebo groups. On myocardial perfusion imaging,
stress-induced perfusion defects tended to decrease more
in patients treated with mononucleated BMC.35
Van Ramshorst et al. randomly assigned 50 patients
to intramyocardial injections of mononucleated BMC or
placebo.36 A twofold to fivefold higher number of mononucleated BMC were administered in this study than in
the PROTECT-CAD trial. At 3 months, the cell-injected
group showed significantly greater improvements in
LVEF and myocardial perfusion, and a more pronounced
improvement in angina class, exercise capacity, and
quality of life than the placebo group. Considering that
previous adjunctive therapies have failed to improve perfusion in patients with chronic myocardial ischemia,37,38
these data are the strongest so far that cell therapy can
improve myocardial perfusion and anginal symptoms.
Whether these effects are sustained over time and are
associated with reduced morbidity and mortality needs
to be investigated, although symptomatic benefit could
be the primary goal in these patients.
Ongoing clinical trials
Considering the heterogeneity of cell isolation protocols,
trial design, and the methods to evaluate outcome, it is
unsurprising that mixed results have emerged from the
first clinical trials in this field. Furthermore, autologous
cell preparations represent a medical product whose complexity far exceeds that of any drug currently prescribed
to patients with coronary heart disease. That said, the
overall data suggest that BMC transfer after AMI has the
potential to improve the recovery of LV systolic function
beyond what can be achieved by current inter ventional
and medical therapies. The early effects achieved with
BMC therapy is comparable to what is achieved by
established therapies including acute PCI, angiotensinconverting-enzyme inhibition, or β-blocker therapy.39 The
effects of BMC transfer on LV function and remodeling
beyond an observation period of 4–6 months, however,
remain poorly characterized, emphasizing the need to
obtain long-term follow-up data in clinical trials.
On the basis of the favorable safety profile and promising efficacy data, several clinical trials are underway to
further explore the prospect of cell therapy in patients
with various manifestations of coronary heart disease.
As discussed below, important issues are addressed in
these second-generation trials in an attempt to maximize patient benefit (Table 2). Given the variation in
outcomes with apparently similar cell isolation protocols
in earlier trials,17,19,22,25 it is absolutely critical to establish
assays that assess cell functionality and the quality of the
cell product. Developing such assays will require a better
understanding of which cellular functions determine
clinical benefit.
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Table 2 | Ongoing cell therapy trials in patients with coronary heart disease
Study identifier
Trial name
Number
of patients
Cells
Primary end
point
Route of cell delivery
100
Bone marrow-derived progenitor
cells
Coronary flow
reserve
Intracoronary
Non-ST-elevation acute coronary syndrome
Clinical trial
NCT00711542
REPAIR-ACS
Acute myocardial infarction
Controlled trial
ISRCTN17457407
BOOST-2
200
Bone marrow cells
Low vs high cell number
Nonirradiated vs irradiated cells
LVEF
Intracoronary
Clinical trial
NCT00355186
SWISS-AMI
150
Bone marrow-derived stem cells
LVEF
Intracoronary
Day 5–7 vs day 21–28
Clinical trial
NCT00684021
TIME
120
Bone marrow mononuclear cells
LVEF
Intracoronary
Day 3 vs day 7 post AMI
Clinical trial
NCT00684060
Late TIME
87
Bone marrow mononuclear cells
LVEF
Intracoronary
2–3 weeks post AMI
Clinical trial
NCT00501917
MAGIC Cell-5
116
Peripheral blood stem cells
mobilized with G-CSF vs G-CSF
with darbepoetin
LVEF
Intracoronary
Clinical trial
NCT00877903
–
220
Allogeneic mesenchymal stem
cells
LVESV
Intravenous
Clinical trial
NCT00677222
–
28
Allogeneic mesenchymal stem
cells
Safety
Perivascular
Ischemic heart failure
Clinical trial
NCT00526253
MARVEL
390
Skeletal myoblasts
6 min walk test,
QOL, LVEF
Transendocardial
Clinical trial
NCT00824005
FOCUS
87
Bone marrow mononuclear cells
MVO2, LVESV,
ischemic area
Transendocardial
Clinical trial
NCT00747708
REGENERATEIHD
165
G-CSF-stimulated bone
marrow-derived stem/progenitor
cells
LVEF
Transendocardial vs
intracoronary
Clinical trial
NCT00326989
Cellwave
100
Bone marrow mononuclear cells
LVEF
Extracorporal shock
wave, then intracoronary
cell therapy
Clinical trial
NCT00285454
–
60
Bone marrow mononuclear cells
Safety, perfusion
Systolic function
Retrograde coronary
venous delivery
Clinical trial
NCT00462774
Cardio133
60
CD133+ bone marrow cells
LVEF
Transepicardial during
CABG
Clinical trial
NCT00810238
C-Cure
240
Bone marrow-derived
cardiopoietic cells
LVEF
Transendocardial
Clinical trial
NCT00768066
TAC-HFT
60
Bone marrow cells vs
mesenchymal stem cells
Safety
Transendocardial
Clinical trial
NCT00644410
–
60
Mesenchymal stem cells
LVEF
Transendocardial
Clinical trial
NCT00587990
PROMETHEUS
45
Mesenchymal stem cells
Safety
Transepicardial during
CABG
Clinical trial
NCT00721045
–
60
Allogeneic mesenchymal
precursor cells
Safety
Transendocardial
Clinical trial
NCT00474461
–
40
Cardiac stem cells harvested
from right atrial appendage
Safety
Intracoronary
Unless otherwise stated, autologous cell sources are used. Abbreviations: AMI, acute myocardial infarction; CABG, coronary artery bypass grafting; G-CSF,
granulocyte colony-stimulating factor; LVEF, left ventricular ejection fraction; LVESV, left ventricular end-systolic volume; MVO2, maximal oxygen consumption;
QOL, quality of life.
Patient selection
For safety reasons, initial studies of cell therapy mainly
included patients who had experienced an AMI and who
had a moderately depressed baseline LVEF. Such patients,
however, have a favorable prognosis and might not be in
need of cell therapy.40 By comparison, in patients with
more extensive infarct damage, identified by severely
depressed baseline LVEF or stroke volumes,17,23,33 or
substantial transmural extent of the infarct,12,13,15 BMC
transfer seems to improve LVEF to a greater extent. Many
of the ongoing trials focus on these higher-risk patients.
Conversely, the presence of microvascular obstruction
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in the reperfused infarct territory, as identified by late
enhancement MRI, could identify a patient subpopulation
who do not respond to intracoronary BMC therapy.15
Procedural details
The timing of cell transfer, dose, cell type, isolation protocol, and mode of delivery must be tailored to the specific
disease setting. This approach results in hundreds of possible permutations, which highlights the complexity of
optimizing cell therapy protocols. A subgroup analysis
of the REPAIR-AMI trial17 and one systematic review 28
suggest that intracoronary BMC transfer in the first days
after AMI is associated with less improvement in LVEF
than later delivery. The same review also suggested that
the improvement in LVEF correlates with BMC dose.28
The dose–response relationship of BMC therapy is
currently being tested in the BOOST-2 trial.
Existing cell therapy protocols might be improved
by head-to-head comparisons of cell delivery strategies
(REGENERATE-IHD trial), and cell types (TAC-HF
trial), exploration of new delivery methods, for example,
transcoronary venous infusion or transcoronary arterial
injection into the perivascular space, and improvements
in intramyocardial injection needle design (for example,
needles that limit immediate washout and promote cell
dispersion).41–44 Progress can also be expected by use
of more comprehensive imaging techniques that help
to characterize the target tissue and facilitate delivery
of cells to tissue sites on the basis of their physiological
characteristics and anatomic location.44,45 As shown in a
swine model of AMI, 3D MRI can be fused with twodimensional fluoroscopy to target transendocardial cell
injections precisely to the correct infarct location. This
technique can be applied without the need for a combined
X-ray/MRI suite and could be used in conjunction with
electroanatomic mapping.45 State-of-the-art imaging
techniques and end point evaluation by external core
laboratories are required to unequivocally demonstrate
moderate functional effects of cell therapy. LV dimensions
and systolic function, for example, should be evaluated by
MRI rather than echocardiography or angiography.
Clinical end points
The first trials were not powered to assess the impact of
cell therapy on mortality and other clinical end points.
In the REPAIR-AMI trial,46 the cumulative end point
of death, recurrent AMI, or necessity for revascularization was significantly reduced in the mononucleated
BMC group compared with that in the placebo group
after 12 months. Likewise, the combined end points of
death, AMI, and hospitalization for HF were significantly reduced after transfer of mononucleated BMC.46
In another study, intracoronary mononucleated BMC
transfer after AMI was associated with a significant reduction in mortality after 5 years.47 Patients who declined
cell treatment served as controls in this nonrandomized
study.47 Trends in favor of BMC therapy with regard to the
end points of death, risk of recurrent AMI, and hospitalization for HF, have also emerged from meta-analyses.27,28
Ultimately, outcome trials will have to be conducted.
Current limitations of cell therapy
Cell therapy is currently limited by low rates of cell
engraftment after intracoronary delivery and poor cell
survival after intramyocardial injections.5,44,48–51 Moreover,
advanced patient age, cardiovascular risk factors (in particular diabetes), and HF appear to have a negative impact
on the functional activity of BMC and blood-derived progenitor cells.52–55 Mononucleated BMC and EPC isolated
from patients with diabetes or HF display reduced activity
in promoting re-endothelialization of denuded arteries
and blood flow recovery after ischemia when transplanted
into nude mice.55–59 The functional deficits that cause
these reduced in vivo activities are poorly characterized,
but markers of reduced functionality, such as impaired
migration in vitro or diminished colony formation, have
been associated with decreased functional benefit in cell
therapy trials.60,61 Cell enhancement strategies are, therefore, needed to realize the full therapeutic potential of cell
therapy. Third-generation clinical trials are expected to
explore such cell enhancement strategies.
Strategies to enhance cell engraftment
Radiolabeling studies show that only a small fraction of
nucleated BMC or blood-derived progenitor cells are
retained in the infarcted area after intracoronary delivery, especially in patients with old infarcts.44,48,49 Priming
ischemic or infarcted tissue has been proposed as a way
to address this problem. Preconditioning of tissue with
low-energy shock waves improved EPC recruitment in a
hind-limb ischemia model by stimulating the expression
of chemoattractants.62 Moreover, ultrasound-mediated
destruction of microbubbles in the coronary circulation
improves the recruitment of mononucleated BMC and
MSC after transvascular delivery, possibly by creating
capillary pores.63,64 Extracorporal shock wave treatment
in combination with intracoronary transfer of mononucleated BMC is already undergoing clinical testing in
patients with ischemic HF in the Cellwave trial.
Some of the key factors involved in progenitor cell
homing to ischemic and injured tissue have been
identified. These mechanisms offer a potential strategy to improve cell delivery.65 AMI leads to the release
of chemotactic factors from necrotic cells, for example
high mobility group box 1 (HMGB-1), which increase
the recruitment of progenitor cells from the bloodstream.66 An initial increase is seen in the local expression of chemokines, such as stromal cell-derived factor 1
(SDF-1), although levels rapidly decline.67 Increase and
prolongation of SDF-1 expression in the heart, or expression of its receptor CXCR4 on stem and progenitor
cells, increases the recruitment of cells in experimental
models.68–70 Chemokine-induced activation of β2 integrins represents another crucial step in the cascade of
molecular events leading to progenitor cell homing after
AMI.71,72 Ex vivo activation of β2 integrins by antibodies,
HMGB-1, or small molecules that act on intracellular
pathways leading to β2 integrin activation, increases the
neovascularization capacity of EPC.73,74 Endothelial nitric
oxide synthase could also increase the rate of homing of
EPC to sites of tissue ischemia as reduced nitric oxide
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bioavailability appears to impair progenitor cell function in patients with diabetes or advanced coronary
heart disease.56–59 Stimulation of EPC with statins before
transfer increases their migratory, invasive, and neovascularization capacity—effects that are mediated, at
least in part, by activation of endothelial nitric oxide synthase.75–77 Similarly, prestimulation with the endothelial
nitric oxide synthase transcription enhancer AVE9488
improves the migratory and neovascularization potential
of EPC and mononucleated BMC obtained from patients
with ischemic HF.58 Finally, prestimulation of EPC from
diabetic individuals with the peroxisome proliferatoractivated receptor γ-agonist rosiglitazone, enhances nitric
oxide availability and the in vivo re-endothelialization
capacity of these cells.59
Strategies to prolong cell survival
Cell survival after transplantation is challenged by ischemia, which is most pronounced in the setting of AMI,
and the reduced perfusion environment of scar tissue; by
inflammation associated with oxidant stress and amplification of cytotoxic cytokines; and by loss of extracellular
matrix attachment, which triggers programmed cell death
(anoikis). A number of strategies have been explored to
improve long-term engraftment after intramyocardial
transplantation. Besides increasing resistance to cell
death, pharmacological preconditioning can also boost
the paracrine or differentiation potential of transplanted
cells.78–81 One such protocol that uses a combination of
growth factors to stimulate the expression of cardiomyocyte genes in MSC is undergoing clinical testing in
ischemic HF (C-Cure trial).
Heat shock has emerged as an attractive strategy
to increase the resistance of cells to external stressors
because of its simplicity, low cost, and ability to activate
multiple protective signaling pathways. 82 For skeletal
myoblasts and ESC-derived cardiomyocytes, heat shock
pre conditioning alone, however, might not support
long-term engraftment in cardiac injury models. 83–85
Pretreatment of ESC-derived cardiomyocytes with heat
shock and a cocktail of survival factors improved the formation of stable and functional grafts after transplantation in a rat model of myocardial ischemia–reperfusion
injury. 83 The survival cocktail included Matrigel ®
(Collaborative Biomedical Products, Bedford, MA,
USA), which is a mixture of extracellular matrix proteins, to prevent anoikis, a cell-permeant Bcl-xL peptide
to block mitochondrial death pathways, ciclosporin to
attenuate cyclophilin-D- dependent mitochondrial
pathways, pinacidil (a compound that opens ATPdependent K+ channels) to mimic ischemic preconditioning, insulin-like growth factor 1 (IGF-1) to activate
cytoprotective Akt pathways, and the caspase inhibitor
ZVAD-fmk.83 A simpler protocol combining heat shock
with carbamylated erythropoietin increased the rate of
ESC-derived cardiomyocyte engraftment in a similar
model.5 Preconditioning skeletal myoblasts with the ATPdependent K+ channel opener diazoxide improves cell
survival and graft size after transplantation into acutely
infarcted rat hearts. 78 Other investigators have used
SDF-1 to activate Akt-dependent signaling pathways and
prolong the survival of transplanted MSC.79
Preconditioning of cells with small molecules and
growth factors might only provide transient protection
after transplantation, and genetic modification has been
proposed as a means to lengthen the duration of engraftment and maximize clinical benefit.86 For example,
overexpression of antiapoptotic genes, such as heme
oxygenase 1, Bcl-2, or Akt, increases the survival and
the functional effects of MSC after transplantation into
ischemic hearts.87–89 Genetic modification could also be
used to maintain the functionality of cells, including their
capacity to secrete paracrine mediators,90 connect with
host cardiomyocytes,91 or differentiate into specialized
cardiac cell types.6,92 While the concept of genetic cell engineering is appealing, it obviously raises the general safety
and regulatory issues associated with gene therapy.
Combined injection of cells and biomaterials represents another strategy to protect transplanted cells from
anoikis and improve their regenerative potential because
cell survival and functionality critically depend on the
cardiac microenvironment. For example, BMC encapsulation within a scaffold of peptide nanofibers displaying
the fibronectin-derived RGD (Arg-Gly-Asp) cell adhesion epitope, supports cell survival after transplantation.93 Conceptually, biomaterials could be designed to
release growth factors in a controlled manner that promotes survival and engraftment of cells, and also guides
cell phenotype decisions.94,95
The future of cardiac cell therapy
While bone marrow and blood-derived cells seem to
have a favorable impact on the function of the infarcted
heart, they cannot replenish lost cardiomyocytes and vascular cells to a meaningful extent. Human bone marrow
is thought to contain multipotent stem cell populations
with the potential to differentiate into cells that express
vascular and cardiomyocyte markers; isolation of these
rare stem cells, however, requires serial culture steps and
clonal expansion.96,97 Studies done to map genetic fate
indicate that freshly isolated unfractionated BMC do not
transdifferentiate into cardiomyocytes when transplanted
into infarcted mouse hearts.98 The benefits of MSC transplantation in rodent infarct models are independent of
the differentiation of these cells into cardiomyocytes.99–102
Moreover, only a small subpopulation of culture-expanded
EPC behave as true progenitor cells that can differentiate into mature endothelial cells in situ.103 Finally, skeletal
myoblasts are lineage restricted and unable to differentiate
into cardiomyocytes or vascular cells.51
Paracrine effects
A large body of evidence indicates that the beneficial
effects of cell therapy are related to the secretion of
soluble factors acting in a paracrine manner.104 Human
peripheral blood-derived EPC, nucleated BMC, and
skeletal myoblasts secrete large arrays of bioactive molecules that are distinct from the secretory profiles of other
cell types, such as blood leukocytes or fibroblasts.105–107
Potential effects of paracrine factors include myocardial
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REVIEWS
protection, neovascularization, modulation of inflammatory and fibrogenic processes, cardiac metabolism and
contractility, increase in cardiomyocyte proliferation, and
activation of resident stem and progenitor cells. The relative importance of these proposed paracrine actions will
depend on the age of the infarct. Cytoprotective effects,
for example, might be most important early after reperfusion. Cytoprotective and proangiogenic effects have
been studied most extensively in experimental models
and could salvage cardiomyocytes at risk and lead to a
reduction in infarct size.90,104,108,109 Increased angiogenesis
has been postulated to improve infarct healing, energy
metabolism, and contractility in the infarct border
zone.96,110–112 Notably, cell transplantation could induce
secondary humoral effects in the infarcted heart, which
are sustained by the host tissue after the transplanted cells
have been eliminated.109
Data from the REPAIR-AMI trial indicate that mononucleated BMC transfer leads to an improvement in
regional microvascular function and tissue perfusion.113
The BOOST investigators have shown that conditioned
nucleated BMC supernatants promote proangiogenic
effects in cultured human coronary artery endothelial
cells and protect cultured cardiomyocytes from ischemia–
reperfusion-induced apoptosis.106 Use of antibody array
and oligonucleotide microarray analyses showed that
nucleated BMC secrete more than 100 soluble factors,
some with known proangiogenic and cytoprotective
activities.106 While these data indicate that intracoronary
infusion of BMC delivers a cocktail of cytokines and
growth factors to the infarcted heart, experimental studies
suggest that individual soluble factors, when applied at
sufficient dosages, might be responsible for much of
the therapeutic effects. For example, the Wnt-signaling
modulator secreted frizzled-related protein 2 (Sfrp-2)
plays a key part in mediating the cytoprotective effects
of Akt-transduced rat MSC,114 and interleukin 10 contributes significantly to the anti-remodeling effects of mouse
mononucleated BMC.115
The human genome encodes more than 1,400 secreted
proteins, many with as yet unknown biological functions.116 A comprehensive functional analysis of the
BMC, MSC, or EPC secretomes might lead to the identification of new paracrine factors with cytoprotective and
proangiogenic potential. Individual soluble factors are
thought to stimulate cardiomyocyte proliferation or
reactivate tissue-resident (epicardial) progenitor cells in
the adult heart,117,118 so secretome analyses might also lead
to the identification of factors promoting tissue regeneration. These efforts may eventually enable therapeutic
approaches based on the application of specific paracrine
factors for patients after AMI (Figure 1). Advantages of
such protein-based therapies include: the relative ease of
standardization and large-scale production; the potential
for off-the-shelf, systemic, noninvasive, and repetitive
administration, thus avoiding the logistic challenges of
cell therapy; the potential to design growth factor cocktails tailored to specific disease settings; and the potential
to obtain patent protection, which could attract industry support for large clinical trials. The most obvious
Skeletal muscle
SkM
Secretome analyses
Oligonucleotide microarray
Antibody array
Bioinformatic approach
n = 100–1,000
EPC
Peripheral blood
CD34+
CD133+
Bone marrow
Candidate factors
Consider current state of knowledge
Obtain/generate recombinant protein
n = 10–100
Functional in vitro analyses
Cardiac myocytes
Endothelial cells
Inflammatory cells
Fibroblasts
Progenitor cells
n = ~10
BMC
Adipose tisue
Disease models
Protein therapy
Gene transfer
Overexpression in transgenic animals
Small large animals
A few
MSC
Clinical
development
ADSC
Figure 1 | Secretome analyses to identify new cardioactive paracrine factors.
Autologous stem and progenitor cells from a variety of sources have been used
in clinical trials and are thought to mediate their effects, at least in part, through
the secretion of paracrine factors. Genome-wide screening coupled with
bioinformatic approaches is used to characterize the secretome of these cell
types. Some of the identified factors are screened for functional activities in
miniaturized, high-throughput cell culture assays. Candidate factors with
presumed therapeutic potential are then further explored in cardiovascular
disease models in vivo. Eventually, a few of these newly discovered secreted
factors could enter clinical trials and be developed for therapeutic use in
patients with cardiovascular disease. n refers to the numbers of factors entering
the next step of exploration. Abbreviations: ADSC, adipose-tissue-derived stem
cell; BMC, bone marrow cell; EPC, endothelial progenitor cell; MSC,
mesenchymal stem cell; SkM, skeletal myoblast.
challenge for protein therapy is the necessity to maintain therapeutic concentrations for the necessary length
of time.104 New strategies are emerging to address this
problem and to allow sustained therapeutic delivery of
recombinant proteins.119–121
Pluripotent stem cells
The identification of reliable sources of pluripotent stem
cells has revitalized the dream of regenerating the failing
heart (Figure 2). ESC were the first pluripotent cell type
to be tested in this context. They can differentiate into
most cell types, including endothelial cells, vascular
smooth muscle cells, and cardiomyocytes. These three
major cardiac cell types seem to emerge from a common
multipotent cardiac progenitor cell that is characterized by the expression of the transcription factor Isl1. 92
Cardiomyocytes derived from ESC display structural and
functional properties of early stage cardiomyocytes. The
first reports demonstrated their potential to act as biological pacemakers, providing evidence for their functional
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Skin fibroblasts
Cell therapy
Tissue engineering
Cardiac
myocytes
Endothelial
cells
Ovary
Testis
PSC
SGSC
Smooth
muscle
cells
Reprogramming
(viruses/proteins/
small molecules) Blastocyst
iPS cells
ESC
Resident CPC
Cardiac
progenitor cells
Figure 2 | Sources of cardiac progenitor cells. Pluripotent stem cells from different sources can be expanded in vitro and
differentiated into cardiac progenitor cells and mature cardiac cell types, thus enabling cell replacement therapy or tissue
engineering. Abbreviations: CPC, cardiac progenitor cell; ESC, embryonic stem cell; iPS cell, induced pluripotent stem cell;
PSC, parthenogenetic stem cell; SGSC, spermatogonial stem cell.
coupling with host cardiomyocytes when transplanted into
normal or infarcted myocardium.122 Additional data have
shown that transplantation of cardiac-committed murine
ES cells into infarcted sheep myocardium can improve
systolic function.123 Unfortunately, ethical issues and the
need to use ESC in an allogeneic setting hamper their
use for cardiac cell therapy in humans. Spermatogonial
stem cells could offer a potential solution. These cells,
which are responsible for maintaining spermatogenesis
throughout the life of the male, can acquire ESC-like properties in culture and possess the capacity to differentiate
into cardiac cell types.124,125 In premenopausal women,
ESC-like cells can be derived by parthenogenesis from
oocytes.126 Moreover, several groups have identified putative multipotent stem cells and progenitor cells in the adult
myocardium, but the true nature and cardiomyogenic
potential of these cells remains to be established.127–129
Reprogramming of skin fibroblasts represents possibly the most promising source of autologous pluripotent stem cells. These cells can be reprogrammed to a
pluripotent state by retroviral transduction of so-called
‘stemness’ transcription factors.130,131 By genetic and
developmental criteria, these induced pluripotent stem
cells are very similar to ESC: they can be maintained in
culture for several months and can be induced to differentiate into derivatives of all three germ layers, including
cardiomyocytes, with electrophysiological properties and
a gene expression profile that is similar to ESC-derived
cardiomyocytes.132,133 To reduce the risk of insertional
mutagenesis following infection with retroviral vectors,
polycistronic retroviral vectors and nonviral vectors that
can be removed after reprogramming have been developed.134,135 Eventually, small-molecule cocktails might
become available to reprogram somatic cells into induced
pluripotent stem cells.136,137
Tumor formation is a general concern associated with
the use of pluripotent cells for regenerative purposes. The
risk can be reduced by transplanting only fully differentiated and highly purified progeny, such as differentiated
cardiomyocytes.138 An additional strategy involves the
genetic modification of cells with suicide genes before
transplantation, which can be turned on in the case of
tumor development.139
Conceptually, spermatogonial stem cells, parthenogenetic stem cells, and induced pluripotent stem cells
would allow the generation of patient-specific differentiated cardiac cell types. The costs involved in producing
these cells under good manufacturing practice conditions,
and the assurance of nontumorgenicity, however, could be
prohibitive when applied on a per-patient basis. An alternative approach would be to compile cell banks of pluripotent
stem cell lines ready for off-the-shelf use.140,141
Assuming that these emerging techniques will ultimately allow the safe production of cardiac cell types in
sufficient numbers, direct transplantation of these cells
into infarcted and failing hearts will face the same challenges that have been encountered with first-generation
cell types—limited cell survival and the lack of signaling cues to promote the formation of 3D heart tissue.
Although these problems might be addressed, for example,
by combined injection of cells with small molecules,
growth factors, or biomaterials, pluripotent stem cellderived cardiac cells could eventually turn out to be more
useful for tissue engineering than cell transplantation.
Conclusions
The realization that endogenous and transplanted adult
stem and progenitor cells promote functional adaptation
and repair after ischemic injury, has led to a new understanding of the pathobiology of cardiovascular disease.
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REVIEWS
Translation of these concepts from disease models into
the clinic could lead to the development of completely new
therapeutic approaches for patients with AMI, HF and
chronic myocardial ischemia. Early clinical trials provide
a signal that cell therapy can enhance tissue perfusion and
contractile performance of the infarcted human heart. The
field is currently plagued by inconsistent trial results that
might arise from a lack of standardization and optimization of cell isolation and delivery protocols, and an incomplete understanding of which patient subgroups should
be targeted for cell therapy. Ongoing and future trials
will have to establish robust and reproducible protocols
that can be offered to patients, and that improve clinical
outcome. Parallel investigations into the mechanisms of
adult stem and progenitor cell therapy may result in the
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Acknowledgments
We thank Dr Kerstin Bethmann for assistance with
the tables and figures. This work was supported
by the Deutsche Forschungsgemeinschaft (KFO 136).
Professor Helmut Drexler died during the writing of
this article. His premature death is a tragic loss to all
those who knew him and to the field of Cardiology.
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