STEM CELL THERAPIES FOR CARDIOVASCULAR DISEASE
Myocardial infarction and congestive heart failure are the leading causes of morbidity and mortality worldwide, despite great therapeutic achievements in the treatment of cardiovascular diseases. The inability of the heart to regenerate lost cardiac muscle, coupled with a robust fibrotic repair response, contribute to adverse ventricular remodeling and decline in postinjury cardiac function. Consequently, much of the research of the last three decades has focused on reducing the atherosclerotic burden of ischemic heart disease, reperfusion, and addressing the fibrotic changes associated with heart failure. After an ischemic insult and the formation of necrotic myocardium, the process of scar formation from the recruitment of activated cardiac fibroblasts leads to reduced cardiac pump function. However, in recent years, it has been convincingly shown that the heart has the ability to regenerate cardiomyocytes, albeit at a low rate (~0.3%–1% annually). These findings and our better understanding of stem cell biology are paving the way to a new area of research, with the main goal of regenerating cardiac tissue.
Two defining features of stem cells are their ability to self-renew and to differentiate to cells of a specific lineage under appropriate conditions. Recent observations have shed light on the existence of cardiac stem cells and extracardiac stem cells that are capable of leading to cardiomyocytes, smooth muscle cells, and endothelial cells. Such therapies are still experimental but hold great promise in potentially ushering in novel regenerative treatment strategies for heart disease.
Types of Stem Cells and Mechanisms of Benefit
Stem cells can be classified according to their level of potency. A totipotent stem cell, such as a fertilized zygote, can lead to an entire organ- ism. A pluripotent cell, such as an embryonic stem (ES) cell, leads to cells from all three germ layers but is unable to generate an organism. Multipotent stem cells, such as mesenchymal stem cells (MSCs), can lead to different types of cells from the same cell germ layer, such as adipocytes, bone, or cartilage cells. Skeletal muscle myoblasts, endothelial progenitor cells, and bone marrow mononuclear cells (BN-MNCs) are other examples of multipotent stem cells that have been studied in cardiac regeneration.
The signaling pathways that drive stem cells into cardiomyocyte fate are areas of intense research; better understanding of these pathways can be used as valuable therapeutic tools in enhancing cardiac regeneration. Differentiation is the process by which stem cells can become cardiomyocytes, whereas transdifferentiation is a process in which a somatic cell adopts alternative cell fates (e.g., a fibroblast adopting an endothelial cell fate). Another process by which stem cells can alter cardiac function is fusion. When stem cells fuse with somatic cells (e.g., cardiomyocytes), the resulting cells have characteristics of both cell types; however, the extent to which any clinical benefit can be achieved from this process is currently unclear. Another mechanism by which stem cells can enhance tissue regeneration is through a paracrine mechanism. After injection, stem cells are believed to release cytokines and/ or growth factors that have physiological effects on other cells in the injured environment and affect repair. For instance, injection of MSCs into the injured heart affects the balance of Wnt signaling in the injured environment to modulate angiogenesis and fibrosis. Identification of key stem cell secreted molecules mediating pro-reparative effects on the injured heart may lead to development of new therapeutic strategies in which injection of key stem cell secreted molecules, rather than stem cells, may be sufficient to augment cardiac healing and obviate issues such as immune response, dose titration, and availability.
ES cells have the ability to give rise to any cell type of the organism, and under the appropriate conditions, can differentiate into cardiomyocytes. They originate from the inner cell mass of the blastocyst during development. Studies have shown that injection of ES cells leads to successful engraftment of them into the surrounding cardiac tissue, making this approach appealing. However, large-scale generation of ES cell–derived cardiomyocytes currently remains unrealistic, because this is a field still in its infancy and filled with several ethical and political challenges. Differentiation of bone marrow stem cells into functioning cardiomyocytes has been more challenging.
Bone marrow contains different types of progenitor stem cells, among which BM-MNCs and MSCs have been extensively studied. Injection of BM-MNCs into the diseased cardiac muscle leads to improvement of cardiac function. Although initial studies suggested that BM-MNCs transdifferentiated into myocytes, later studies suggested an indirect effect likely related to the paracrine effects of cells. A paracrine effect contributing to salutary effects on cardiac repair was confirmed for MSC injection after cardiac injury. In vitro studies suggested that these cells could also differentiate into beating cardiomyocytes, and these findings generated a lot of excitement that led to several preclinical studies. These studies suggested that injection of MSCs into the injured myocardium resulted in improved cardiac function despite the low number of mesenchymal-derived cardiomyocyte cells, which suggested a multifactorial effect similar to BM-MNCs.
The limited supply of ES cells and associated social challenges have led to other pathways to develop pluripotent stem cells. A type of cell that has attracted a lot of interest recently is the resident cardiac pro- genitor cell. The existence of these cells and their ability to differentiate into cardiomyocytes, as well as endothelial and smooth muscle cells, has shaken the long-standing belief that the heart is a fixed organ unable of regeneration. Several challenges remain to fully derive the potential benefits of these cells. First, different cell markers have been used to characterize these cells, and it is presently unclear if there are any biological differences between cells with different cell markers. Second, the number of these cells is small, and their role in normal cardiac function is not clear. However, injection of these cells into an infarcted heart leads to improvement of cardiac function. What makes these cells particularly attractive is their ability to differentiate into other cell types (e.g., endothelial and smooth muscle cells) because the regenerating cardiomyocytes will need new blood vessels and supporting cells to properly function. Also, use of these cells appears to avoid some of the ethical challenges that can arise with the ES cells. Their small number and the technical difficulties associated with successfully multiplying them have led to the development of other types and techniques, among which induced pluripotent stem cells merit special mention. These are ES cells derived from skin fibroblasts through genetic manipulation (through the overexpression of certain transcription factors). What makes this approach unique and revolutionary is the ability of skin fibroblasts to be reprogrammed into induced pluripotent stem cells that could be injected into an injured heart. Although this field and its technology are in their infancy, it holds great promise for future cardiovascular regeneration therapy.
Another type of stem cell that deserves special mention is the endothelial progenitor cell, especially for vascular regeneration. Injury of the vascular endothelium triggers a cascade of events that aims to reconstitute the endothelium via the proliferation of the remaining endothelial cells and differentiation of the endothelial progenitor cells. It is based on these premises that administration of endothelial progenitor cells might indirectly improve cardiac function by enhancing vasculature repair and angiogenesis.
Cardiospheres have also been tested in heart regeneration after myocardial infarction. Cardiospheres are three-dimensional multicellular structures from cardiac explant cultures in nonadhesive surfaces. It is currently believed that injected cardiospheres modulate scar formation via paracrine effects or secreted exosomes containing active molecules such as microRNA. The phase I clinical trial testing cardiospheres in humans did not show any left ventricular ejection fraction improvement; however, this trial did show a reduction of scar mass at 6 months and an increase in viable myocardium.
During this first wave of excitement, skeletal myoblasts have also been studied, with promising results in early tests, but with no significant benefit in clinical trials; therefore the use of these cells in cardiovascular regeneration is uncertain (Table 6.1).
The main two approaches of stem cell administration have been catheter- based intracoronary injection and surgical-based epicardial injection; each delivery method presents unique advantages and disadvantages. One factor to keep in mind when selecting the appropriate approach is to have an optimal environment for successful stem cell engraftment.
For example, an intracoronary injection approach after revascularization is disadvantageous because the myocardial areas that need the most number of cells might have reduced blood flow and thus would receive less stem cells. Another factor to consider when selecting the optimal delivery method is the type of cells delivered. For example, certain stem cells (e.g., skeletal myoblasts) are larger; therefore they could lead to microvasculature obstruction and subsequent decreased blood flow. The epicardial injection delivery approach solves the problem of reduced delivery in poorly perfused areas, but cardiac perforation is a real risk, especially in the setting of inflamed and necrotic myocardium tissue. The inflamed cardiac microenvironment is also not optimal for engraftment of injected cells, and the first 4 days after infarction are associated with the least amount of cell engraftment. Focal delivery of stem cells also may not be sufficient in cases of global myocardial dysfunction, such as in nonischemic-dilated cardiomyopathy. Thus it is likely that the decision on what route of delivery to choose will depend on the cardiac condition that needs to be treated and the type of cells to be delivered.
From a safety standpoint, stem cells have been shown to be safe, but long-term data are lacking. Beyond the technical challenges and potential procedure-related complications, there are several specific concerns that need to be taken into consideration when discussing stem cell–based therapy. First, ventricular arrhythmias are a real concern with the newly differentiated and engrafted cells. This specific adverse effect has been observed with skeletal myoblasts, which are believed to be secondary to a lack of electromagnetic coupling with native cardiomyocytes. The second concern is noncardiac engraftment; if the route of administration is through the systemic circulation, then stem cells can also populate other organs; this has been seen in patients who have undergone stem cell injection. Localization of stem cells to other organs can have unpredictable effects, especially tumor angiogenesis or enhancement of malignancy.
Clinical Evidence of Stem Cell Therapy
There have been many trials involving thousands of patients and different types of stem cells injected into patients with different cardiac pathologies (for a more comprehensive list of selected trials, see Table 6.2). BM-MNCs have been evaluated in acute myocardial infarction and chronic myocardial ischemia. In post–acute myocardial infarction patients, the benefits of bone marrow cells were studied in randomized- controlled trials such as the BOne marrOw transfer to enhance ST- elevation infarct regeneration (BOOST) and Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trials. Both studies showed improvements in left ventricular ejection fraction, and 1-year follow-up in REPAIR-AMI patients also showed a reduction in major adverse cardiovascular events. Patients with more severely impaired left ventricular function appeared to benefit the most according to subgroup analysis of these studies. Bone marrow stem cell therapy for chronic myocardial ischemia was also evaluated in several studies, including the Prospective Randomized Trial of Direct Endomyocardial Implantation of Bone Marrow Cells for Therapeutic Angiogenesis in Coronary Artery Disease (PROTECT- CAD) trial, as well as in a randomized-controlled, double-blind trial with improvement in the quality of life and cardiac perfusion. Although these findings are promising, for most of the studies, the left ventricular ejection fraction might not be the most accurate endpoint, and larger phase III clinical trials with significant primary endpoints (such as mortality, recurrent myocardial infarction, or stroke) will reveal the true impact of bone marrow stem cell therapies and their long-term safety.
MSCs also have been tested for stem cell therapy, but clinical data are scarce. The initial phase I clinical data used intravenous injection of these cells, which established their safety but did not show any cardiovascular benefit. However, the passage of these cells through the pulmonary circulation might have hampered any potential benefit of these cells in the coronary circulation and cardiac seeding. Subsequently, early nonrandomized studies showed that intracoronary injection of MSCs resulted in improvement of the left ventricular ejection fraction and symptoms. Another source of mesenchymal cells are adipose tissue derived stem cells. The AdiPOse-derived stem ceLLs in the treatment of patients with ST-elevation myOcardial infarction (APOLLO trial assessed the safety of adipose tissue–derived cells and showed a significant reduction in infarct size and improved perfusion at 6 months in patients with acute myocardial infarction. The AdiPose-deRived stEm Cells In the treatment of patients with nonrevaScularizable ischEmic myocardium (PRECISE) trial evaluated the safety and benefits of these cells in chronic myocardial ischemia, which suggested an improvement in the functional status in patients treated with adipose tissue–derived stem cells.
After skeletal muscle injury, resident stem cells (satellite cells) contribute to skeletal muscle regeneration. Hence, there was an initial interest in the usefulness of skeletal myoblasts in cardiovascular regeneration. Although the early nonrandomized small studies suggested a clinical benefit, the most comprehensive trial was the randomized, double-blind, phase II Medical Research Council Adjuvant Gastric Infusional Chemotherapy (MAGIC) trial, which showed no benefit at 6 months. These cells were injected around the scar tissue in patients who underwent bypass surgery. The potential proarrhythmic and microembolization effects of myoblasts, although not fully understood, limited the excitement for these cells as potential source of stem cells in cardiovascular regeneration.
The recent findings that human cardiomyocyte renewal occurs naturally at a rate of 1% in the young and 0.3% in older adults led to the quest of identifying and characterizing these resident cardiac stem cells. The first challenge was to fully evaluate these cells; several subtypes of cells were described, based on the cell markers used to identify them. It is still not clear if these are cells of different subpopulations with distinct phenotypes or if they are part of the same subpopulation. The second challenge was to expand them, because their number is low in the heart. Despite these challenges, early-stage clinical studies, such as the Stem Cell Infusion in Patients with Ischemic cardiOmyopathy (SCIPIO) and CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction (CADUCEUS) trials, established their safety and feasibility, as well as showed a reduction in infarct size. Further ongoing studies are needed to further elucidate any clinical benefit of these cells.
FUTURE DIRECTIONS OF STEM CELL THERAPY
After an exciting decade full of both reassuring and disappointing results, the future of stem cell therapy in cardiovascular disease is expected to grow even more. With the discovery of inducible pluripotent cells and their ability to differentiate into functional cardiomyocytes, a new field has been born in stem cell therapy. However, before applying these cells in clinical trials, certain crucial hurdles have to be resolved, especially safety concerns regarding their tumorigenicity (which has been observed in animal models). Furthermore, technology for upsizing to an effective number of induced pluripotent stem cells is needed.
In addition to the previously described stem cell approaches, the concept of pretreating the stem cells to enhance their therapeutic activity before injection has been tested in several trials. The idea is to improve any function that influences migration, engraftment, survival, differentiation, and other functions that can improve efficiency. The Enhanced Angiogenic Cell Therapy in Acute Myocardial Infarction (ENACT-AMI) trial tested the usefulness of overexpression endothelial nitric oxide synthase in endothelial progenitor cells, because it had previously been shown that a reduction of endothelial nitric oxide synthase limits the repair capability of endothelial progenitor cells in patients with hypertension and diabetes. Another trial is the Mesenchymal Stem Cells and Myocardial Ischemia (MESAMI) II trial, which is designed to pretreat MSCs with melatonin before injection. This approach is based on data that suggest that the melatonin hormone improves survival and the paracrine effects of MSCs. It is expected that many more trials will test different pretreatment methods to enhance the effects of stem cell therapy in cardiovascular regeneration.
Another technology that has not been applied to any clinical trials yet is nanotechnology. The harmful microenvironment after myocardial infarction has detrimental effects on survival and function of the newly transplanted stem cells. Therefore there has been a growing interest in designing biomaterials that can provide a supportive role for the transplanted cells within infarcted myocardium. Before any human trials can be planned, several potential questions need to be addressed, such as dosing, biodegradability, excretion of the byproducts, and any immunological incompatibility of these bio-nanomaterials.