What are stem cells?

Given that multiple cell types are generated from a single embryonic stem cell origin during embryonic development, the potential of such a cell for cardiac repair is self-evident. Embryonic stem cells (ES cells) can be isolated from the inner cell mass of the developing embryo at about 5 days after fertilisation. These cells are pluripotent and can produce all the tissue types needed to form a functional organism.

The adult organism has no ES cells, only adult stem cells (ASCs) and progenitor cells. ASCs like ES cells share important stem cell defining characteristics—ability to self-renew and to produce specialised daughter cells while maintaining the integrity of their own DNA and phenotype. Unlike ES cells, ASCs have a more limited menu of daughter cells appropriate to the tissue in which they reside that they can serve up when required—for example, haematopoietic stem cells being multipotent have the capacity to produce all blood cell lineages. In contrast, progenitor cells, such as endothelial progenitor cells, are not true stem cells, they are only able to undergo several cycles of cell division to produce one type of daughter cell, and are unable to undergo indefinite growth—that is, they lack clonogenicity.

ES cells have the greatest potential for regenerating the myocardium, as with the correct protocol their differentiation can be controlled to produce any cell type needed for therapeutic use. The potential of ES cells is counterbalanced by several significant drawbacks: collection of cells from embryos raises serious ethical considerations thus limiting their availability, implanted cells would be allogeneic and require immunosuppression to prevent rejection and lastly, the potential for growth and differentiation that makes the ES cells desirable as a cellular repair tool can become unregulated after transplantation, resulting in tumour formation.

Hence, although ES cells are an attractive option for use in cardiac repair, researchers have turned to ASCs to avoid the problems of ES cells noted above.

A wide range of adult cells have been reported to induce cardiac repair or functional improvement; these include bone marrow-derived mononuclear cells that consist of haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Other adult-derived progenitor cells include skeletal myoblasts, cardiac resident stem cells and umbilical cord blood stem cells (see table 1).

The potential for cell therapy to develop as a new treatment has created a new field of research—regenerative medicine.

Regenerative medicine: from bench to bedside

The foundation of stem cell therapy as a treatment for cardiac disease is based around the concept that myocardial infarction results in large-scale cell loss, and heart failure occurs as a direct result of this loss. The idea that stem cells could be used to replace lost or damaged cells to reverse the effects of cardiomyocyte loss has evolved over the past 10 years. The different cell types that may affect this repair and the evidence behind their potential use in man are considered below.

Embryonic stem cells

A major step towards the use of human ES cells for transplantation was taken in 1998 with the first report of embryonic cell line derivation in humans.1 Since this time, multiple independent centres have derived cardiomyocytes from ES cells that hold up to close scrutiny when examined histologically, physiologically and genetically. Further progress has been delayed owing to the problems previously discussed, including observation of a dose-dependent induction of teratomas with implantation of undifferentiated human ES cells, all be it at the same time as producing significant cardiac muscle regeneration.2 For the time being the problem of tumour formation has been dealt with by pre-differentiating ES cells into a cardiomyocyte phenotype before their transplantation.3 Currently, ES cell research is focusing on refining techniques for differentiation, selection and purification of cardiomyocytes and commercialisation of these techniques before attempting clinical trials.

Inducible pluripotent stem cells

Several independent investigators have produced a pluripotent autologous stem cell alternative to ES cells that retains the differentiation potential of ES cells but without the risk of tissue rejection after transplantation or causing ethical concerns (the cells can be produced from a patient's own cells—for example, from the mouth,4 or even from a few plucked hairs5). These cells have been termed inducible pluripotent stem cells (iPSCs). They were first identified in 2006 when mouse fibroblasts were reprogrammed back to an undifferentiated pluripotent state by inserting four genes, Oct3/4, Sox2, KL4 and c-Myc, into differentiated somatic cells.6 The treated cells subsequently took on the morphological phenotype of ES cells and were demonstrated in vivo and in vitro to have the same differentiation potential as ES cells (able to form all three germ layers—that is, mesoderm, endoderm and ectoderm).

Following the demonstration of iPSC production in mice, the results were replicated in humans. Just as in mice, when human adult cells were reprogrammed, by retroviral transduction of the same four genes, they returned to an undifferentiated pluripotent state with the ability to differentiate into all three germ layers.4 5 7

The potential of an autologous pluripotent cell derived from adult tissue is considerable. Research has already progressed towards practical applications of this emerging technology. So far, functioning cardiomyocytes8 and vascular cells9 have been produced demonstrating potential uses in cardiovascular regenerative medicine.

Despite the potential inherent in the ability to produce patient-specific iPSCs, problems remain that must be solved before they can be used in clinical trials. The main concern is tumour genesis. The majority of iPSC lines have been derived by inserting putative oncogenes using integrating retroviruses—for example, lentiviruses, into the host genome. One study demonstrated that up to 20% of offspring derived using iPSCs developed tumours.10 Advances have been made in circumventing this problem by use of non-integrating adenoviruses for gene delivery,11 or using small molecules to produce the same outcome without the need for gene insertion.12 One sophisticated study demonstrated the possibility of excising the inserted genes after the iPSCs had been formed with no impact on differentiation potential of the cells.13 Once the tumour genesis problem has been resolved, and the complete mechanism of reprogramming is understood, there are considerable expectations that iPSCs may provide a patient-specific source of cardiovascular cells for use in regenerative medicine.

Soon after the initial advances in human ES cells were made, and before the discovery of iPSCs, the first evidence for a contribution to cardiac repair by ASCs was made.

Adult stem cells and the path to clinical trials

The first steps taken towards the promotion of adult cell therapy for cardiovascular disease included animal experiments which demonstrated that circulating endothelial cells of bone marrow origin could contribute to neovascularisation in adult tissues.14 Evidence of bone marrow cells contributing to skeletal muscle,15 and for HSCs, contributing to cardiac muscle and vasculature, were also seen.16 Furthermore, human MSCs were shown to have multilineage potential17 and were able to form cardiomyocytes in vitro when chemically treated.18

Evidence from cardiac sex-mismatch transplants demonstrating cardiomyocyte and vascular chimerism supported the idea of a circulating progenitor able to contribute to cell repair of endothelium and cardiac muscle at a low frequency. Testing for the presence of the Y chromosome in explanted hearts of male patients who had received female donor hearts showed male-derived myocytes and endothelial cells. This finding was interpreted as representing cardiac repair affected by circulating progenitor cells.19 A second similar study in which the hearts of female subjects, who had received male bone marrow transplants, were examined for presence of the Y chromosome concluded that bone marrow progenitor cells were capable of transit to the heart and its subsequent repair.20 The concept of cellular repair of the heart using bone marrow stem cells was beginning to evolve.

Others were already demonstrating that exogenously derived cells—for example, fetal cardiomyocytes, could be transplanted into the heart, successfully integrate and improve cardiac function.21 22 Progress was also being made with skeletal myoblasts, the native stem cell of striated muscle,23 with functional benefits being shown when used to treat cryoinjured mouse hearts.24

Experiments with fetal and skeletal myoblasts had proved the concept of functional improvement after cell therapy. However, both cell types had drawbacks, fetal cell therapy for humans would be fraught with ethical issues and the need for immunotherapy after transplantation, while studies of skeletal myoblasts were demonstrating that transplanted cells were not able to integrate into the host myocardial syncitium.25

An autologous source of cells for transplantation was extremely desirable, and as all three stem cell or progenitor lines of the adult bone marrow had been shown to form muscle or vascular cells,14 16 18 20 bone marrow cells began to be investigated as a treatment for dysfunctional hearts.

Multiple animal studies initially suggested that all three cell lines from bone marrow could contribute to cardiac regeneration in models of myocardial ischaemia. MSCs were able to engraft into ischaemic myocardium, and express muscle proteins in models of myocardial infarction.26 27 Human EPCs were able to stimulate neoangiogenesis, within the infarcted vascular bed, reducing apoptosis and improving cardiac function in a mouse model.28

In one study, bone marrow-derived cells were shown to regenerate substantial amounts of myocytes, endothelial cells and smooth muscle cells when injected into the hearts of mice after myocardial infarction. The authors felt a subpopulation of HSCs regenerated myocardium in vivo, replacing dead tissue and promoted successful treatment of large myocardial infarcts.29 Strikingly positive animal studies such as this quickly spurred on the initial clinical studies.

The first in man feasibility study published in 2001 documented the successful administration of intracoronary autologous bone marrow cells to a 46-year-old man after primary PCI for an anterior infarct.30 Two larger case series, with a combined total of 30 subjects and a year's follow-up, demonstrated that selective intracoronary cell transplantation was safe and effective under clinical conditions.31 32

After the initiation of clinical trials, basic science studies began to question the proposed mechanism of action of the transplanted cells (See figure 1). Muscle generation or transdifferentiation was not seen in two high profile studies of bone marrow progenitor therapy, although functional benefit was still seen.33 34 New vessel and muscle formation were only noted as rare events and were attributed to donor and host cell fusion.35 36

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Figure 1

Potential mechanisms of action of adult stem cells. Several mechanistic pathways have been implicated in the process of cardiac repair after experimental myocardial injury in animal models and are shown here. Initially direct myogenesis and vasculogenesis driven by stem cell plasticity were considered to be the primary mechanisms of benefit. Subsequently, fusion has been demonstrated to account for many instances of presumed de novo blood vessel and muscle formation. Many now believe that a paracrine effect of transplanted cells on the host organ is the major driver of benefit. It is unknown which of these, either alone or in combination, are relevant to man.

The need for a new explanation for the benefits gained with stem cell treatment, led to the proposal that local release of paracrine factors was the key mechanism of effect of transplanted cells rather than myogenesis. In support of this idea, it is clear that ASCs can secrete a wide range of cytokines.37 The released paracrine factors have been shown to induce a number of beneficial effects, including induction of angiogenesis in host tissues,28 reduced apoptosis37 and immunomodulation of injury.38 A further documented paracrine benefit is the proposed stimulation of the recently described resident cardiac progenitor cells.39

Cardiac progenitor cells (CPCs)

Until recently, the adult heart was understood to be a postmitotic organ—that is, it consists of a constant number of cells from birth to death unless cells are lost to pathological insults. The only adaptive cellular response was considered to be cardiomyocyte hypertrophy. This view is now being challenged. The first suggestion of a new understanding of cardiac cellular homoeostasis was the discovery of continuing cell division in adult hearts, which appeared to be stimulated by cardiac injury such as myocardial infarction, left ventricular hypertrophy and heart failure.40

The discovery of chimeras, as discussed above, further increased the suspicion that there may be a local pool of cardiac progenitor cells within the adult heart. Several independent investigators have now located and defined a CPC that meets standard stem cell defining characteristics and is capable of differentiating to multiple cardiac cell lineages such as cardiomyocytes and vascular cells.41–43 In animal models CPC treatment has been shown to repair and improve cardiac function after myocardial ischaemia.42 43

CPCs are an attractive option for use in clinical trials, as they are intrinsically more aligned to producing all the cells needed to repair the damaged heart. However, they are significantly more difficult to harvest and isolate than bone marrow-derived mononuclear cells, and the way ahead may be activation and stimulation of the patient's own intrinsic CPCs rather than transplantation. Currently, the results of early safety studies using CPCs are awaited.

The body of evidence from basic science studies suggests that a significant benefit to left ventricular function can be produced with the use of almost any type of ASC in the setting of myocardial ischaemia. Despite doubts over the mechanism of action of ASCs, clinical trials have continued as major concerns over safety have not been raised, and the clinical imperative to improve treatment for heart failure is compelling.

The problems with translation

The translation of basic research described above into clinical trials occurred relatively early on in the evolution of this new field of research. The nature of this step—a complexity of ethics approvals, regulations and cross-discipline working—meant that the early studies dealt with safety and feasibility and were never of sufficient power to assess efficacy. This did not, however, stop the authors of this work from commenting on what appeared to be a beneficial effect of stem cell therapy in their small patient studies. This early optimism based on small studies has in part led to a degree of scepticism within the medical community that has set a high bar for the clinical studies that followed. What should not be forgotten is that these early studies did demonstrate that cell therapy was feasible and relatively safe given the degree of illness within the patient groups and the highly invasive procedures that are necessary to deliver cells.

What has been lost in translation is our understanding of the value of surrogate end points in these clinical trials. Surrogate end points are useful ways of looking for effects that may have clinical relevance. Invariably, their use is to decrease the number of patients needed to demonstrate a potential benefit. In trials of stem cell therapy surrogate end points enable the cost of the study to be kept down thereby leaving some of these trials within the control of academia. The problem that has arisen is that the clinical relevance of small changes in surrogate measures with respect to their overall consequence is poorly understood. Hence numerically small changes even when statistically significant are deemed to be clinically insignificant. Early reports of adult bone marrow-derived cell therapy to treat patients after myocardial infarction31 32 did indeed show that the processing and transplantation of cells from the bone marrow into the heart was feasible and safe in the small number of patients studied. These reports went on to speculate over efficacy by comparison with historical data to suggest that the patients benefited from the cell therapy and saw improvements in their heart function (invariably measured as ejection fraction). The literature is now full of similar small trials that were designed to test for safety but report effect despite a lack of statistical power.44 The point that must not be missed is that the meta-analysis of these trials using different cell types and methods of delivery suggest that cell therapy appears to be feasible. The procedures themselves appear to be safe but increasing numbers of patients and long-term follow-up are needed to draw a robust conclusion about safety.45

The first randomised control studies of bone marrow-derived stem cells used cardiac function (specifically ejection fraction) as a surrogate measure of effect (see figure 2). The results demonstrated small improvements in ejection fraction that have widely been interpreted as clinically irrelevant, even though some of the numerical changes reached statistical significance. Furthermore, in those studies that performed additional analysis at a later time point (1 year or more), the small improvement seen in cardiac function in patients treated with cells compared with placebo was lost. Although these results would overall appear to suggest that cell therapy after myocardial infarction is not effective at improving cardiac function, meta-analysis of trial data does show statistically significant improvement in markers of cardiac function.46 Since it is unclear as to how surrogate end-point measures translate into clinical benefit, debate continues over whether these small changes in cardiac function have therapeutic relevance. As there are no large outcome studies to answer this question it may be of use to consider whether there is historical information, from other interventional studies, which established a link between changes in surrogate and hard end points. A recent review published in 200947 summarised a sample of key clinical trials, the results of which changed medical practice (see table 2).

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Figure 2

Relationship between delay in administration of cell therapy and outcome. Schematic diagram showing the changes in ejection fraction in relation to time after infarction that cells were injected. Four randomised control trials in acute myocardial infraction (AMI) are shown and one in chronic cardiac failure (CCF). Similar analyses have led to the suggestion that the optimal time to deliver cells after myocardial infarction (MI) is between days 5 and 10. *Denotes 18-month follow-up and loss of initial statistical significance. BMC, bone marrow cells.

Although these trials demonstrated significant improvements in clinical outcome, they were associated with very small changes in cardiac function. This suggests that subtle improvements in cardiac function in man may lead to substantial clinical benefits, and that a better understanding of ejection fraction as a surrogate end point is needed. Furthermore the use of symptom class (eg, New York Heart Association) is also closely linked with long-term outcome but seemingly poorly correlated with cardiac function as measured by ejection fraction.54

We also do not have a good understanding of how changes in surrogate end points relate to changes in outcome. Most of the survival and quality-of-life data using these markers relies on a single reading not a change after treatment. It is not until we better understand the significance of changes in symptoms or cardiac function and their bearing on outcome that we will be able to understand the significance of the results of surrogate end-point translational studies.

While the debate over surrogate markers and the results of the clinical studies continues, consideration must also be given as to why the trials have failed to translate the preclinical expectations into man. Various explanations have been suggested:

Too few cells injected—The translation from the results of animal experiments to man does not allow for the same number of cells injected per unit of heart muscle. This is because there is a limit to the number of cells obtained from a bone marrow aspirate in a clinical situation (animal experiments allow the pooling of cells from several donors) and the inability to expand the cells sufficiently (using tissue culture techniques to increase the cell number) in the time between harvest and injection. It may well be that this exponential difference in cell number can explain the loss of effect, and in future the expansion of the most efficacious cell type or combination of cells will be necessary to reproduce the conditions that lead to the successes seen in animal models.

Wrong cell type—The use of unselected populations of cells based on a pragmatic approach of translation into man, and some interesting animal data, again opens a debate about whether the potentially beneficial effect of a given cell type, or combination of cells, within this unselected population is diluted by the other cells found therein. The problem with the preclinical experiments is that the animal models do not give a clear lead as to which cell type will work best. This has led to parallel approaches from different groups, which have ranged from using unselected bone marrow-derived cells to select populations such as CD34+ and AC133+ cells.55 56 It remains unclear as to what type of cell these markers actually represent,57 but preliminary results56 would suggest that cell selection carries no additional benefit to that seen with the unselected fraction. This may suggest that in man the effect if any is due to a non-specific cell interaction, that may be related to the environment from which the cells were taken (relative hypoxia within the bone marrow), rather than the potential of an individual cell type in itself.

Wrong timing relative to infarct age— The issue of translation from animal models to man raises the question of understanding the similarities and differences that exist within the common biology. Recognition that the time course for ischaemic injury and subsequent remodelling is different depending upon the species means that care must be taken in translating the results of these experiments into the clinical setting. For example, in our clinical trial of adult stem cells in patients with heart failure, the average time from evidence of the last myocardial infarct to inclusion in the trial is 1000 days.58 It is not clear which comparative animal model is representative of this clinical time point, or if indeed such a model exists. Other discrepancies between the animal models and man include the absence of coronary artery disease in animal models (most are mechanical models of myocardial injury in healthy, young animals), the absence of concomitant pharmacotherapy that may modulate the response to treatment, the effect of ageing and other illnesses (eg, diabetes) and the effect of the environment. The animal models can only therefore be considered as a preliminary tool to establishing new treatments in man and, ultimately (maybe sooner rather than later), translation to man has to occur through well-designed clinical trials. The belief that animal models should fully answer mechanistic questions relevant to human disease before translation to the clinic is clearly incorrect.

Wrong delivery method—Important advances in our understanding of how to deliver cell therapy are often overlooked in the pressure to demonstrate efficacy. In man, cell therapy has been delivered by a number of methods that are designed to target specific areas of the heart (myocardial necrosis or hibernation) with the ability to administer the highest number of cells. These include intracoronary infusion, epicardial and endocardial direct injection, retrograde venous (coronary sinus) infusion and indirect methods (either mobilisation of cells from the bone marrow using growth factors or application of cells to the myocardium using bioengineered patches (figure 3)). Indirect methods have also been used in animals that include intravenous infusion of cells and intra-aortic delivery (aortic root near the coronary ostia).

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Figure 3

Routes for delivery of cell therapy. Delivery methods that have already been used in man are not underlined while those that have been successfully used in animal models but have yet to be used in clinical trials are underlined. Reproduced with permission of Nature publishing group (licence via rightslink, licence number 2397610744512).

The fact that these various routes have been tried in man is commendable. The problem is that the approaches have used different cell types with no real standardisation to allow comparison of delivery method alone (it is really always the combination of delivery method and cell type). Direct comparisons using animal models tend to suggest that the intramyocardial route of delivery always leads to a higher retention of injectate.59

The problem with intramyocardial injection is that the injection process itself can cause myocardial injury and therefore raise the bar even higher in respect to what the cell therapy needs to achieve. Ultimately head-to-head comparison of delivery methods will be needed in man to assess the trade-off between effective delivery and minimal myocardial injury in the process.

Animal models are not representative—This concept has already been touched on in the timing section but is summarised in the commentary that human disease is complex and that the outcome of coronary artery disease is determined by a combination of biological, pathological, environmental and pharmacological processes in man which are hard to reproduce in animal models. It is therefore little surprise that the promise of stem cell therapy for cardiac repair first seen in rodent models has found difficulty in translating to similar gains in man. The problem that needs to be examined is to understand the exact contribution that animal models can make to advance the application of cell therapy into man.

What next?

Refining the use of cell therapy for cardiac repair in man will require a thoughtful and measured approach. Consideration must be given to deciding where this promising field of research, which has led to high expectation, should go next. Some would suggest that a return to the laboratory is needed, but this path avoids the problem of deciding when translation into man should occur and how this is best achieved. Given the progress that has already been made in clinical trials it is important to remember that the field of cell therapy is at an early stage, and comparisons with drug development would equate to several more years of clinical research and development. There is clearly a long and difficult path ahead that is not helped by the lack of tools that we have for assessing mechanistic questions posed by use of cell therapy in man. Given these difficulties it is important that a consensus approach (such as that proposed by a European Society of Cardiology Task Force, see box 1) is used, based on the best evidence available at the time and standardised so that the results of new trials can be directly compared.60 Indeed following these recommendations results of randomised controlled trials currently underway in London (Barts and the London and University College London Hospital) will soon be available to guide future progress in this important area of research. Without an answer to the mechanistic question the field of cell therapy for cardiac repair in man will always be open to attack from those who demand a back-to-basics approach. There are many examples in clinical practice of new treatments that have been implemented with only a provisional understanding of the mechanism, an understanding that has been modified over time following clinical experience. With this in mind the question arises as to how the clinical trials of cell therapy for cardiac repair should be judged? If clinical medicine is to be consistent the opportunity to continue to develop cardiac stem cell therapy in man should not be missed. Ultimately, it is benefit to the patient that must remain the goal of this research—perhaps before a full scientific understanding of mechanism has been achieved.