Rebuilding the Infarcted Heart Volume:
Author(s): Loren J. Field, Kai C. Wollert
germline stem cells may provide
a new source of pluripotent cells for regenerative
medicine. From mouse studies, we know that
germline stem cells can be cultured in vitro for an
indefinite period and are able to differentiate into
cells from all three germ layers including functional
cardiomyocytes. For stem cell-based therapy,
a major challenge will be to generate strategies that
enable the development of multipotent cells from
human adult SSCs and to develop further techniques
for their use in organ regeneration.
Modern reperfusion strategies and advances in
pharmacological management of acute myocardial
infarction (AMI) have resulted in an increasing
proportion of patients surviving the acute event.
Many of these patients eventually develop adverse
left ventricular remodeling and heart failure. None
of our current therapies addresses the underlying
cause of the remodeling process, i.e. the damage to
the cardiomyocytes and the vasculature in the
infarcted area. The alleged transdifferentiation
capacity of adult stem cells and the recent discovery
of endogenous cardiac repair mechanisms have
suggested that cardiac repair (i.e. replacement of
necrotic or scarred tissue with viable myocardium)
might be achieved in the clinical setting.
Stem cells are capable of self-renewal, transformation
into dedicated progenitor cells, and differentiation
into specialized progeny. Traditionally,
adult stem cells were believed to differentiate only
within tissue lineage boundaries (e.g. hematopoietic
stem cells giving rise to mature hematopoietic
cells). The fairly recent concept of adult stem cell
plasticity implies that stem cells can transdifferentiate
into cell types outside their original lineage.2
Along this line, it has been reported that
hematopoietic stem cells when transplanted into
infarcted mouse myocardium transdifferentiate into
cardiomyocytes and vascular cells.3 Another
hypothesis that has created some excitement
recently predicts that limited myocardial regeneration
can occur after tissue injury through the
recruitment of resident and cardiac stem cells.4
Ironically, while these new ideas have already triggered
clinical trials, fusion of transplanted stem
cells with resident cardiomyocytes has been offered
as an alternative explanation for previous claims of
transdifferentiation.5–8 Moreover, it has been proposed
that stem cells secrete cytokines and growth
factors which may promote angiogenesis, suppress
cell death of resident cardiomyocytes, modulate
interstitial matrix composition, and maybe even
recruit cardiac stem cells.9,10 Regardless of the mechanisms,
it appears that stem cell therapy has the
potential to improve perfusion and contractile performance
of the injured heart (Figure 13.1).
Potential Donor Cells
Conceptually, a variety of stem and progenitor cell
populations could be used for cardiac repair after
AMI, and many of these potential donor cells are
discussed in detail in this book. Each cell type has
its own profile of advantages, limitations, and
practicability issues. Studies comparing the regenerative
capacity of distinct cell populations are
scarce. Most investigators have therefore chosen a
pragmatic approach by using unfractionated
nucleated bone marrow cells, which contain different
stem and progenitor cell populations,
including hematopoietic stem cells, endothelial
progenitor cells, and mesenchymal stem cells.
Endothelial progenitor cells
Endothelial progenitor cells (EPCs) have been
defined by their cell surface expression of the
hematopoietic marker proteins CD133 and CD34
and the endothelial marker vascular endothelial
growth factor receptor 2, and their capacity to
incorporate into sites of neovascularization and to
differentiate into endothelial cells in situ. The cell
surface antigen CD133 is expressed on early
hematopoietic stem cells and EPCs, both of which
collaborate to promote vascularization of
ischemic tissues. There is increasing evidence that
culture-expanded EPCs contain a CD14+/CD34−/
CD133− -mononuclear cell population with “EPC
capacity”, which mediates its angiogenic effects
by releasing paracrine factors.11
Mesenchymal stem cells
Mesenchymal stem cells represent a rare population
of CD34−and CD133− cells present in bone
marrow stroma and other mesenchymal tissues.12
Mesenchymal stem cells can differentiate into
osteocytes, chondrocytes, adipocytes, and cardiomyocyte-
like cells under specific culture conditions.
When injected into infarct tissue,
mesenchymal stem cells may enhance regional
wall motion and prevent adverse remodeling. It
has been suggested that these effects may be
related to paracrine effects rather than differentiation
of mesenchymal stem cells into cardiomyocytes.
13,14 Since mesenchymal stem cells can be
expanded in vitro, and reportedly have a low
immunogenicity, they might be used in an allogeneic
setting in the future.12
Skeletal myoblasts
Skeletal myoblasts (satellite cells) are progenitor
cells which normally lie in a quiescent state
under the basal membrane of mature muscular
fibers. Myoblasts can be isolated from skeletal
muscle biopsies and expanded in vitro.
Myoblasts differentiate into myotubes and retain
skeletal muscle properties when transplanted
into an infarct scar. Although grafted myotubes
may contract in response to electrical stimulation,
they do not express intercalated disc proteins,
indicating that the majority are not
electromechanically coupled to their host cardiomyocytes.
Nevertheless, myoblast transplantation
has been shown to augment systolic and
diastolic performance in animal models, possibly
through the release of paracrine factors.15
Resident cardiac stem cells
Recently, several groups have detected stem and
progenitor cells within the heart that are capable
of differentiating into cardiomyocytes and/or vascular
lineages. It has been suggested that these
cells can be clonally expanded and used for cardiac
repair in an autologous setting.4 Clearly, independent
confirmation of these provocative
findings is required. If confirmed, cardiac resident
stem and progenitor cells hold great promise for
clinical applications, although it is conceivable
that the bone marrow contains a pluripotent stem
cell population with similar properties.16,17
Embryonic stem cells
Embryonic stem cells (ESCs) are totipotent
stem cells derived from the inner cell mass of
blastocysts. Under specific culture conditions, ESCs
differentiate into multicellular embryoid bodies
containing differentiated cells from all three
germ layers including cardiomyocytes. Human
ESC-derived cardiomyocytes display structural
and functional properties of early-stage cardiomyocytes
that couple with host cardiomyocytes
when transplanted into normal or
infarcted myocardium.18,19 In theory, infinite
numbers of cardiomyocytes could be obtained
from human ESC clones. However, unresolved
ethical and legal issues, concerns about the
tumorigenicity of residual ESCs in ESC-derived
cardiomyocyte preparations, and the need to use
allogeneic cells for transplantation currently
hamper their use in clinical studies. Eventually,
nuclear transfer techniques may provide a
means of generating an unlimited supply of histocompatible
ESCs for the treatment of cardiac
disease.
Multipotent adult germline stem cells
A recent study has highlighted the pluripotency
and plasticity of murine spermatogonial stem
cells, which are responsible for maintaining spermatogenesis
throughout life in the male. In culture,
adult spermatogonial stem cells acquire ESC
properties. These multipotent adult germline
stem cells spontaneously differentiate into derivatives
of the three embryonic germ layers in
vitro, including cardiomyocytes and vascular
cells.20 Eventually, establishment of human multipotent
adult germline stem cells from testicular
biopsies may allow individual cell-based therapy
without the ethical and immunological problems
associated with human ESCs.20
Modes of Cell Delivery in the
Setting of Acute Myocardial
Infarction
The goal of any cell delivery strategy is to transplant
sufficient numbers of cells into the
myocardial region of interest and to achieve
maximum retention of cells within that area.
Retention may be defined as the fraction of
transplanted cells retained in the myocardium
for a short period of time (hours). The localmilieu is an important determinant of cell retention,
as it will influence short-term cell survival
and, if a transvascular approach is used, cell
adhesion, transmigration through the vascular
wall, and tissue invasion. Transvascular strategies
are especially suited for the treatment of recently
infarcted and reperfused myocardium when
chemoattractants are highly expressed.21 Direct
injection techniques have been used in patients
presenting late in the disease process when an
occluded coronary artery precludes transvascular
cell delivery (e.g. patients with chronic myocardial
ischemia) or when cell homing signals are
expressed at low levels in the heart (scar tissue).
However, cell delivery by direct injection may
not be safe in patients with AMI and friable
necrotic tissue.
Selective intracoronary application delivers a
maximum concentration of cells homogeneously
to the site of injury. Unselected bone
marrow cells, circulating blood-derived progenitor
cells, and mesenchymal stem cells have been
delivered via the intracoronary route in patients
with AMI. In these studies, cells were delivered
through the central lumen of an over-the-wire
balloon catheter during transient balloon inflations
to maximize the contact time of the cells
with the microcirculation of the infarct-related
artery.22 In experimental models, intravenous
delivery of endothelial progenitor cells and mesenchymal
stem cells has been shown to improve
cardiac function after AMI. However, cell homing
to non-cardiac organs limits the applicability
of this approach. Indeed, in a recent clinical
study, homing of unselected bone marrow cells
to the infarct region was observed only after
intracoronary stop-flow delivery but not after
intravenous infusion (Figure 13.2).23 Considering
that the acutely infarcted myocardium recruits
circulating stem and progenitor cells to the site
of injury, mobilization of stem and progenitor
cells by cytokines may offer a non-invasive strategy
for cardiac regeneration. Indeed, it has been
reported that the stem cell mobilizing cytokines
stem cell factor (SCF) and granulocyte colony
stimulating factor (G-CSF) improve cardiac function
after AMI in mice.24,25 Notably, G-CSF can
accelerate infarct healing directly by enhancing
macrophage infiltration and matrix metalloproteinase
activation, and suppressing cardiomyocyteapoptosis, suggesting that stem cell-independent
mechanisms may contribute to the favorable
effects of G-CSF post-AMI.26,27
Current Status of Cell Therapy in
Patients with Acute Myocardial
Infarction
Inspired by experimental data suggesting that
functional recovery after AMI can be augmented
by stem cell transfer, clinical trials were initiated
to assess the therapeutic potential of cell therapy
in patients post-AMI. All clinical studies have
included patients who had undergone primary
angioplasty and stent implantation to reopen the
infarct-related artery and who received optimal
medical treatment during the acute phase and
follow-up, and cells were infused intracoronarily
by using the stop-flow balloon-catheter approach.
Current trials may be categorized into studies
using unselected bone marrow cells or selected
stem cell populations.
Unselected bone marrow cells
Following initial safety and feasibility studies,
four larger randomized trials of bone marrow cell
therapy after AMI have now been completed
(Table 13.1).28–31 The combined experience from
these trials indicates that intracoronary delivery
of unselected bone marrow cells is feasible and
safe in the short and mid-term (up to 18 months).
Bone marrow harvest and intracoronary cell
delivery are not associated with bleeding complications
or ischemic damage to the myocardium.
No increased rates of in-stent restenosis have
been observed. Clinical surveillance, Holter
monitoring, and data from an electrophysiological
study indicate that bone marrow cell transfer
is not associated with an increased propensity to
ventricular arrhythmias; moreover, intramyocardial
calcifications or tumor formation were not
observed after intracoronary bone marrow cell
delivery.28–32
In the BOne marrOw transfer to enhance STelevation
infarct regeneration (BOOST) trial,
intracoronary transfer of nucleated bone marrow
cells resulted in an improvement of global left
ventricular ejection fraction of 6 percentage
points after 6 months as compared to the control
group. This improvement of ejection fraction
was due mostly to improved regional wall
motion in the infarct border zone.28 For comparison,
improvements of 3–4 percentage points are
achieved by primary angioplasty and stent
implantation in AMI, suggesting that the further
improvement of ejection fraction by cell therapy
may be clinically meaningful.
The beneficial effects of intracoronary bone
marrow cell transfer were confirmed in the
recent Reinfusion of Enriched Progenitor cells
And Infarct Remodeling in Acute Myocardial
Infarction (REPAIR-AMI) trial. In this study,
mononucleated bone marrow cell transfer promoted
an increase in left ventricular ejection
fraction of 2.5 percentage points after 4 months
as compared to a control group that also underwent
bone marrow aspiration but received an
intracoronary infusion of a placebo.29
In contrast to BOOST and REPAIR-AMI, two
other randomized studies did not report significant
improvements of left ventricular ejection
fraction after intracoronary cell transfer.30,31
While the exact reasons for the differing results
remain elusive, it is worthwhile to take a closer
look at the design of these studies (Table 13.1). In
one of the negative studies, cells were delivered
already 24 hours after coronary reperfusion.30 In
BOOST and REPAIR-AMI, cells were transplanted
several days later. Subgroup analyses in REPAIRAMI
indicate that the timing of cell delivery may
be important, and that the beneficial effects on
ejection fraction are lost when the cells are delivered
too early.29 The Autologous Stem cell
Transplantation in Acute Myocardial Infarction
(ASTAMI) trial also did not find a significant
effect of bone marrow cell transfer on left ventricular
ejection fraction recovery.31 While the
cells were delivered late after reperfusion in
ASTAMI (after 4–6 days), a particular cell preparation
method that leads to an enrichment of
lymphocytic bone marrow cells was employed. It
is not clear whether this method actually recovers
the bone marrow cell populations that are
required to achieve functional improvements
after AMI. Together, these data remind us thatprocedural issues, such as the cell preparation
method and timing of cell transfer, need to be
further refined.
It should be noted that none of the trials so far
has revealed a significant effect of bone marrow
cell transfer on left ventricular end-diastolic
volumes, an index of left ventricular remodeling.
However, larger studies may be required to settle
this issue. Moreover, few data are available
regarding the long-term effects of bone marrow
cell transfer after AMI. Follow-up data from the
BOOST trial indicate that the improvements of
left ventricular ejection fraction are maintained
18 months after cell transfer;32 however, ejection
fraction also increased somewhat in the control
group during long-term follow-up,32 which
would be expected in AMI patients on chronic
angiotensin converting enzyme inhibitor and
β-blocker therapy. Tissue Doppler echocardiography
analyses in BOOST indicate that bone marrow
cell transfer may prevent the development
of diastolic dysfunction during long-term followup.
Significant effects of cell transfer on E/A ratio
(peak early diastolic/late diastolic velocities) and
tissue Doppler Ea/Aa (annular velocities) ratio,
but not on isovolumic relaxation time, suggest
that cell therapy positively affects left ventricular
stiffness but not active relaxation.33
Selected bone marrow cell populations
The Transplantation Of Progenitor Cells And
Regeneration Enhancement in Acute Myocardial
Infarction (TOPCARE-AMI) trial compared
mononucleated bone marrow cells with circulating
blood-derived progenitor cells (mostly EPCs)
in post-AMI patients a few days after reperfusion.
Both cell types appeared to have similar safety
and efficacy profiles.34 However, since TOPCAREAMI
was not randomized, firm conclusions
regarding the efficacy of endothelial progenitor
cells post-AMI cannot be drawn at the present
time. In the recent randomized Transplantation Of
Progenitor Cells And Regeneration Enhancement
in Chronic Heart Failure (TOPCARE-CHF) trial,
patients received an intracoronary infusion of
unselected mononucleated bone marrow cells or
circulating blood-derived progenitor cells months
or years after an AMI. Notably, a significant
improvement in left ventricular ejection fraction
was observed only in patients receiving mononucleated
bone marrow cells in this setting.35 The
effects of culture-expanded mesenchymal stem
cells after AMI have been investigated in one randomized
clinical trial.36 While no serious sideeffects
were reported, it is not known whether
intracoronary mesenchymal stem cell delivery
promoted ischemic damage to the myocardium,36
a complication that has occurred after intracoronary
mesenchymal stem cell infusions in dogs.37
Six months after cell transfer, regional wall
motion and global left ventricular ejection fraction
were improved and left ventricular enddiastolic
volumes were decreased.36 These striking
effects need to be confirmed by additional studies.
In another trial, CD133+ enriched bone
marrow cells were infused into the infarct-related
artery in post-AMI patients. Higher luminal losses
within the stented segment and the distal,
non-stented segments of the infarct-related artery
were observed, raising the concern that CD133+
cells may promote in-stent restenosis and atherosclerosis
progression.38 As these data were
obtained from retrospective analysis and lacked
randomized controls, future studies need to
define the risk and mechanisms of such adverse
effects on the epicardial coronary circulation
(which were not observed in any of the trials
using unfractionated nucleated bone marrow
cells).38
Stem and progenitor cell mobilization
The Front-Integrated Revascularization and Stem
Cell Liberation in Evolving Acute Myocardial
Infarction by Use of Granulocyte-Colony-
Stimulating Factor (FIRSTLINE-AMI) trial randomized
50 patients with AMI to a control group
or a 6-day open-label course of G-CSF that was
initiated within 1–2 hours after primary angioplasty
and stenting.39 G-CSF therapy after stent
implantation was not associated with an
enhanced rate of in-stent restenosis or other serious
adverse events, and promoted significant
improvements in left ventricular ejection fraction
and metabolic activity in the infarct territory.
39 Critics have pointed out that the
beneficial effects of G-CSF in FIRSTLINE-AMIwere magnified by an unexpected decrease in
ejection fraction in the control group.40 The
favorable safety profile of G-CSF post-AMI was
confirmed in two other recent trials which, however,
did not observe a beneficial effect on left
ventricular ejection fraction.41,42 It remains to
be seen whether differences in study design, e.g.
later start of G-CSF injections in the negative
trials, account for the discrepant results.
The Foreseeable Future of Cell
Therapy in Patients with Acute
Myocardial Infarction
We advocate to no longer perform studies
involving small numbers of patients, but rather
to conduct larger, double-blind, randomized
controlled clinical trials to firmly establish the
effects of cell therapy on surrogate markers, such
as left ventricular ejection fraction and remodeling,
myocardial perfusion, or exercise capacity.43
Most important, upcoming trials should address
procedural issues such as the optimal cell type,
cell dosage, and timing of cell transfer. While
these trials may also look at combined morbidity
and mortality endpoints, they may be too small
to be conclusive in this regard. Some of the
ongoing trials of intracoronary bone marrow cell
transfer after AMI are summarized in Table 13.2.
Ultimately, outcome trials and cost–benefit
analyses will be required.
Notably, the absolute number of transplanted
bone marrow cells did not correlate with subsequent
improvements in ejection fraction in previous
post-AMI studies. This may be because the
cell numbers infused were within a narrow
range, or because differences in the functional
capacity of the cells, such as the ability to home
to and engraft in the infarcted area, to undergo
transdifferentiation, and/or to produce paracrine
factors, may override differences in cell numbers.
Intriguingly, cell labeling studies indicate that
fewer than 5% of unselected nucleated bone
marrow cells are retained in the infarcted area
after intracoronary delivery in patients (Figure
13.2).23 Although this rate of cell retention was
sufficient to improve left ventricular systolic and
diastolic function in the BOOST trial, it is
conceivable that pharmacological strategies
might be used to enhance the homing capacity
or other functional parameters of the cells.
Experimental studies are already pointing in this
direction.44 Post-hoc analyses of the BOOST trial
database suggest that the effects of bone marrow
cell transfer are consistent across several subgroups
defined according to sex, age, infarct territory,
and time from symptom onset to
reperfusion.28 However, patient subgroups that
derive the greatest benefit from cell transfer need
to be identified prospectively in future trials (e.g.
patients presenting late after symptom onset in
whom little myocardial salvage can be expected
from reperfusion therapy). In this regard, data
from REPAIR-AMI indicate that the effects of
bone marrow cell transfer may be more pronounced
in patients with more severely
depressed baseline left ventricular ejection fraction.
29 Cytokines with stem-cell mobilizing
and/or direct cardioprotective properties
should be further evaluated as stand alone therapy
or in combination with cell transfer after
AMI. Eventually, cytokines may emerge as a
non-invasive alternative or as an adjunct to cell
therapy.
Meanwhile, fundamental questions need to be
addressed experimentally. What is the fate of the
injected cells after transplantation? Genetic and
transgenic markers should be employed to determine
lineage commitment of engrafted cells.
Cell labeling and imaging techniques need to be
developed to track stem cell fate in patients and
correlate cell retention and engraftment with
functional outcomes. Can the regenerative
capacity of transplanted stem cells be enhanced
by drugs, cytokines, or gene therapy approaches?
Pharmacological and genetic strategies may help
to enhance stem cell retention, engraftment, differentiation,
and paracrine capability.45,46
For the time being, cardiac repair remains the
holy grail of cell therapy. While unselected bone
marrow cells may have a favorable impact on
systolic function, they probably do not make
new myocardium. This should stimulate further
basic research into the prospects of cell types
with transdifferentiation capacity, such as ESCs,multipotent adult germline stem cells, and,
possibly, multipotent bone marrow-derived stem
cells and resident cardiac stem cells.
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Primary Principal
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REGENT Poland Randomized, 200 Mononucleated Global LVEF, Michal Tendera
multicenter BMCs vs LV volumes
CD34+/CXCR4+ cells (by MRI)
HEBE The Netherlands Randomized, 200 Mononucleated Regional Alexander Hirsch
multicenter BMCs vs peripheral function in Jan J Piek
mononucleated dysfunctional
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therapy (by MRI)
BOOST-2 Germany, Randomized, 200 Placebo vs Global LVEF Kai C Wollert
Norway, multicenter, nucleated BMCs (by MRI) Gerd P Meyer
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