Pending Challenges: Uncertainties and Limitations
As described above, recently more comprehensive, randomized-controlled trials evaluating the
therapeutic use of adult stem cell-containing cell populations in ischemic myocardial disease
have generated mixed results. Although it is conceivable that these mixed observations are
caused by technical differences amongst the trials using different cell types, cell sources, cell
doses, and delivery modes, it remains uncertain whether (1) cell-based therapy in general or
certain stem cell populations are truly efficacious, (2) we might not be able to detect present
effects by the chosen surrogate endpoints, (3) proposed dosing and timing of cell-transfer are
therapeutically ideal, and (4) what are the determinants of potential therapeutic benefit.
In order to fully exploit the cardiac repair potential of cell-based therapies and advance these
promising strategies towards the clinical setting, uncertainties need to be carefully addressed and
practical limitations to be overcome (figure 4). Notably, although the largest body of clinical
evidence is available for BMCs looking back on 5 years of follow-up, emerging answers might
not be conferrable to other cell populations.
Chasing the Ideal Cell Population
Several cell populations are presently under translational evaluation, and, almost weekly, other
cell (sub-) populations are promoted on the “regenerative market”. It is unknown, which
population is most potent or, respectively, most suitable for certain regenerative applications,
while it is conceivable that the regenerative effects vary amongst the populations due to their
distinct biological properties.103 For example, unselected BMCs and CD34-enriched BMCs may
regionally show different engraftment patterns of ischemic myocardium after intracoronary
infusion.15
In addition, in another pilot study proangiogenic progenitor cells preferentially
homed to extensive acute myocardial infarcts characterized by a low viability and reduced
coronary flow reserve.12 Further, considering that mechanistic understanding of adult
stem/progenitor cell-dependent repair (figure 3) has shifted towards a paracrine hypothesis (i.e.,
secreted factors positively affect ischemic tissue (for more details 104), it is unknown whether this
paracrine concept can be generalized for all cell populations. BMCs105, MSCs56 and resident
cardiac progenitor cells105a for example, are known for their paracrine capacity. Although the
mechanistic theorem of cell-based therapy has strongly tilted towards paracriny, there is also
evidence suggesting that physical integration of the cells is required (figure 3).106 In addition, it
has become clear that cardiovascular risk factor and disease may impair the repair capacity of
autologous, patient-derived stem/progenitor cells.116-119
Differences in clinical outcome can also be caused by a lack of a consensual definition and
varying preparations of cells. In this regard, a very prominent example are EPCs, since ideal
source, isolation and culture techniques, and particularly characteristics and phenotype remain
under debate.4,46 Finally, there is some evidence that there are potential advantages of cell
selection for subpopulations.44 Cell selection, on the other hand, comes to the cost of low cell
yield, which, in turn, might limit therapy. The suitability of certain cell (sub-) populations may
also depend on the character and severity of tissue damage we intend to treat. All these aspects
point towards a necessity of a targeted, population-sensitive use of stem and progenitor cells
dependent on the therapeutic purpose. Until now, clinical head-to-head comparisons are largely
lacking, and preclinical insights are scarce.
Endpoint Selection
From previous experience with the translation of novel therapies such as cardiovascular gene
therapy certain problems and pitfalls have been foreseeable.107-109 Thus, it might very well be
challenging to demonstrate bioactivity of cell-based therapy in men: The first aspect is the choice
2
of appropriate surrogate and clinical endpoints. In initial trials, global measures of LV function
(ejection fraction) and geometry (enddiastolic/endsystolic volumes) were chosen as endpoints to
determine cell-dependent efficacy. In light of the uncertainties how to ideally convey cell
therapy, a significant and sustained improvement in global LV measures on top of gold-standard
treatment may be difficult to achieve; particularly in small heterogenous populations. Partly
triggered by the lack of clear answers from these analyses of global LV function/remodeling,
regional contractility and perfusion, infarct size and diastolic function have more recently been
evaluated.110 As an example, Janssens et al. describe a reduction in infarct-size with no change
in ejection fraction in response to BMC injection.23 Consistently, accumulating evidence
underscores that infarct size is a strong predictor for clinical outcome in infarct patients.
Further, modality and accuracy of established diagnostic tools are not appropriate to assess the
microenvironment. For example, animal studies revealed that stem cells target the
microcirculation of ischemic tissue, but in patients our diagnostic tools barely picture this. 111 The
sensitivity of established imaging techniques such SPECT and MRI is limited and do not detect
the microvasculature (100μm) structurally. As an alternative, measures of tissue perfusion only
indirectly reflect the microcirculation. Notably, in a proof-of-concept study Erbs et al. reported a
restoration in flow reserve of the infarct-related coronary 4 months after cell transfer as assessed
by intracoronary Doppler in a REPAIR-AMI substudy.112 Along the same line, injection of
CD34+ cells into hibernating myocardium in patients with chronic angina in a phase I/IIa study
led to trends of a better perfusion in SPECT imaging.54
Technical advances of established diagnostic tools and the development of novel, high-resolution
imaging modalities will facilitate the advance and understanding of the field. Ultimately, larger
prospective randomised trials with clinical efficacy endpoints will be required.
3
Timing and Dosing of Cell Transfer
According to earlier preclinical reports, there is evidence that efficacy of stem/progenitor cells is
dose-dependent,44,113 and, even more interesting, appear to follow a dose-effect relationship.113
The meta-analysis by Martin-Rendon, for example, suggests that a cell dose above 108
autologous, unselected BMCs was effective.43 The classical dose-effect curve with a parable-like
pattern may also be relevant in the clinical setting of cell-therapy, as we saw a dose-response
curve in our ACT34-CMI trial.55 This means, that threshold and optimal doses have to be
determined via dose titration, before a final call on the biopotency of stem/progenitor cells in
ischemic tissue repair can made. With regard to titration studies, cell population (including
differences in isolation/preparation techniques), but also targeted organ, disease entity and
severity may be relevant.
Furthermore, is it realistic to expect a significant impact on global cardiac measures after a onetime stem cell injection? Since biological processes are known to be highly complex, a singledose strategy may not result in sustained effects. A striking finding from the Frankfurt group
around Drs. Dimmeler and Zeiher is that cell-based efficacy was enhanced by repetitive
intraarterial infusions of BMCs (personal communication); that was observed in patients with
critical limb ischemia. It is very conceivable that repetitive cell applications boost efficacy.
In addition, timing of cell therapy is likely crucial to efficacy. Some clinical studies have
described an association of time point of cell transfer after myocardial ischemia and benefit. In
REPAIR-AMI the improvement of LV ejection fraction was accentuated when BMC transfer
took place at day five or later.20 Since these observations have only been reported for acute MI
but not in chronic MI or refractory angina so far. The explanation of that finding might be that
4
the harmful environment of acute ischemia eases over time and, in turn, harms cell viability and
functionality to a lesser degree. This notion has recently been contrasted by another pilot study
from the same group. Using proangiogenic progenitor cells, Schächinger et al. found
significantly better homing when these cells were injected in the early (≤14 days) compared to
the intermediate and late phase.12 It is uncertain, though, how much better homing relates to
retention, and, eventually, higher efficacy. Again, as mentioned above, this potential discrepancy
might be due to the use of different cell populations in both studies. Currently, the SWISS-AMI
study prospectively compares early vs. delayed BMC-therapy after large AMI.
Determinants of Therapeutic Benefit
Basic research suggested that age, cardiovascular risk factors and comorbidity influence stem
cell biology and its regenerative potency114-120 (recently reviewed in
121,122
). This aspect is
particularly relevant in autologous cell transfer for ischemic cardiovascular disease, where
eligible patients are elder, carry a high burden of cardiovascular risk factors, and are multimorbid. Autologous cells isolated from patients have subsequently been evaluated in preclinical
studies and in-vitro functional assays. For example, aging, anemia, renal failure, high levels of
triglyceride, C-reactive protein, interleukin-6 correlate with poor angiogenic potency of BMCs
isolated from these patients in-vitro.123 Further, female sex and source material were associated
with a higher yield of c-kit+ Islet-1 cardiac progenitor cells.124
From the clinical standpoint, levels of circulating CD34+/KDR+ EPCs predict the occurrence of
cardiovascular events and mortality.125 In-vitro migratory capacity of infused BMCs and CPCs at
the time of injection was closely related to cell-dependent reduction in Infarct size.126 In the same
line, BMCs with a high mitogenic capacity in-vitro determined by colony-forming units
5
significantly reduced markers of heart failure, namely NT-proBNP and NT-proANP, and
mortality within 3 months after injection.127 It is not clear, whether these factors determine
efficacy in clinical cell-therapy. Emerging substudies indicate that certain aspects impact
efficacy. As an example for a clinically relevant association between cardiovascular risk factors
and a therapeutic benefit, Roncalli presented (6th International Symposium on Stem Cell Therapy
And Cardiovascular Innovations, 2009) that non-smoking patients with microvascular
obstruction may benefit more from intracoronary BMC injection as compared to smokers as was
suggested from data of the BONAMI trial (clinicaltrials.gov: NCT00200707). In REPAIR-AMI,
patients with infarcts larger than the median benefited significantly, while the patients with
smaller infarcts did not.20 This observation was later reinforced by a MRI substudy, where the
improvement in ejection fraction was greater among patients with baseline EF less than median
of 48.9%.128 Closely related to infarct size, Meyer et al. described that infarct transmurality
above the median is associated with benefit on LV EF at 6, 18 and 61 months, while there was
no effect in patients with less infarct transmurality.16 These observations suggest that patients
with larger infarcts may potentially benefit more.
Ideally we would be able identify likely responders, thereby exclude patients who are likely to
receive little or no benefit and, consequently, would unacceptably be at risk of treatment-related
complications (risk-benefit consideration), to adjust cell dose for each individual patient, and
allocate our regenerative resources to patients who benefit from the treatment (effectiveness).
For this purpose, computed stratification algorithms may be helpful.123
Limitations
From solid preclinical and clinical evidence, we have realized that current procedures of celltransfer are hampered by practical limitations, and that the therapeutic potency of
6
stem/progenitor cell therapy is not fully exploited. Early in-vivo cell tracking experiments
indicated that even hours after cell injection the vast majority of
111
In radiolabeled cells do not
remain in the therapeutic target zone14,129 In this context, it is important to understand, however,
that cell fate is influenced by different functional aspects, i.e. cell homing, viability, and
retention. Initially, homing of transferred cells is dependent on the delivery route; in contrast to
intracoronary delivery, cells that were injected intramyocardially detour the critical steps of
endothelial adhesion and vascular transmigration.14,129 In a case series reported by Hofmann and
Wollert, only ~2% of 18F-FDG-labeled BMCs but 14-39% CD34-enriched cells remained in the
myocardium within the first hour after intracoronary infusion, while the gross label activity was
localized in spleen and liver due to substantial biodistribution.15 Interestingly, the type of
injected cells resulted in different spatial distribution; CD34-enriched cells were prominent in the
border zone, while unselected BMCs engrafted homogenously throughout infarct and border
zone.15 More recently, Schächinger et al. detected ~7% of total radioactivity in the myocardium 1
hour after intracoronary application of
111
In radiolabeled proangiogenic progenitor cells; the
myocardial signal rapidly declined over the next 3 to 4 days to ~2%. Strikingly, the average
activity within the first 24 hours was highest in patients treated early after myocardial infarction
(≤14 days) versus the intermediate (>14 days to 1 year) and chronic infarction phase (>1 year).12
Similarly, in patients with chronic myocardial infarction ~4% of radiolabeled, CD34+ enriched
cells were detected in the myocardium by PET an hour after intracoronary injection. 130
Cumulatively, these pilot studies account to the fact, that cell homing and retention is low.
Notably, the infarct age, i.e. storm of secretion and inflammation acutely after MI, may
determine uptake and retention of cells, although the “hostile” microenvironment acutely after
MI is thought to be detrimental. It needs to be acknowledged that available studies do not allow
7
the clear distinction of homing from retention, as duration of the homing and beginning of the
retention phase are unknown: Most probably both phases overlap substantially. Up to now, only
a time frame of not earlier than ~1 hour and not later than 3-4 days after cell transfer has been
studied in patients due to technical reasons. For comprehensive insights, tracking studies need to
start minutes after cell transfer and last until label activity signal subsides. Furthermore, since
structural analyses such as histology are obviously not available in men, all present findings are
based on indirect measures of cell fate via labeling. However, label activity present in the
microenvironment does not exclusively reflect viable cells. Dyes can leak from the cells for
various reasons, e.g. binding instability and remnant (phagocytosed) dyes after cell death.
Preferentially, dye activity should be dependent on cell viability as it can be achieved by
genetically modifying cells using reporter genes. In the context of cell labeling, it must be
acknowledged that labeling dyes and procedures may affect cell functionality and viability, and,
in turn, affect results of cell tracking studies.
The “vanish” of injected cells, which is indirectly reflected by a diminishing label signal, cannot
exclusively be attributed to poor retention. Accumulation of label activity in spleen and liver
suggest that the viability of applied cells in the harmful environment of ischemic tissue is low.
Cell viability, in fact, is particularly relevant to autologous cell-therapy in patients with severe
cardiovascular disease and associated co-morbidities, because stem cells isolated from eligible
patients are already reduced in number, prone to apoptosis, and exhibit features of impaired
function.114,117,131,132 On the other hand, isolated cells injected into the harmful environment of
ischemic tissue characterized by ischemia, acidosis, oxidative stress and inflammation are
particularly prone to cell death. Detachment of cells from their native 3-dimensional extracellular
matrix because of the isolation process for therapeutic application favors cells death (anoikis).
8
Dependent on cell type and delivery route, the numbers of (viable) cells range from more than
30% to nearly 1% with a rapid decrease within the first 7 days after application in-vivo.133,134
This aspect is even more relevant, since the regenerative effect of cell-based therapies appears to
be dose-dependent.32,44
To comprehensively dissect aspects stem/progenitor cell fate, advances in dye technology and
imaging modalities, and carefully designed, mid-to-large scale studies will be needed (for more
details135-137). In light of these striking limitations, intense efforts have been undertaken to prime
stem/progenitor cells before the transfer in order to fully exploit their therapeutic potency.
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