Regenerating cardiac cells: insights from the bench and the clinic

A major challenge in cardiovascular regenerative medicine is the development of novel therapeutic strategies to restore the function of cardiac muscle in the failing heart. The heart has historically been regarded as a terminally differentiated organ that does not have the potential to regenerate. This concept has been updated by the discovery of cardiac stem and progenitor cells that reside in the adult mammalian heart. Whereas diverse types of adult cardiac stem or progenitor cells have been described, we still do not know whether these cells share a common origin. A better understanding of the physiology of cardiac stem and progenitor cells should advance the successful use of regenerative medicine as a viable therapy for heart disease. In this review, we summarize current knowledge of the various adult cardiac stem and progenitor cell types that have been discovered. We also review clinical trials presently being undertaken with adult stem cells to repair the injured myocardium in patients with coronary artery disease.     

Adult stem cell therapy for the heart

The purpose of this review is to summarize current data leading to and arising from recent clinical application of cellular therapy for acute myocardial infarct (heart attack) and congestive heart failure. We specifically focus on use of adult stem cells and compare and contrast bone marrow and adipose tissue; two different sources from which stem cells can be harvested in substantial numbers with limited morbidity. Cellular therapy is the latest in a series of strategies applied in an effort to prevent or mitigate the progressive and otherwise irreversible loss of cardiac function that frequently follows a heart attack. Unlike surgical, pharmacologic, and gene transfer approaches, cellular therapy has the potential to restore cardiac function by providing cells capable of regenerating damaged myocardium and/or myocardial function. Skeletal muscle myoblast expansion and transfer allows delivery of cells with contractile function, albeit without any evidence of cardiomyogenesis or electrical coupling to remaining healthy myocardium. Delivery of endothelial progenitor cells (EPCs) which drive reperfusion of infarct zone tissues is also promising, although this mechanism is directed at halting ongoing degeneration rather than initiating a regenerative process. By contrast, demonstration of the ability of adult stem cells to undergo cardiomyocyte differentiation both in vitro and in vivo suggests a potential for regenerative medicine. This potential is being examined in early clinical studies.     

Growth and differentiation of human embryonic stem cells for cardiac cell replacement therapy

Due to the limited proliferation capacity of cardiac cells, cell replacement therapy has been proposed to restore cardiac function in patients suffering from ischemic heart disease and congestive heart failure. However, this approach is challenged by an insufficient supply of appropriate cells. Because of their apparent indefinite replicative capacity and their cardiac differentiation potential, human embryonic stem cells (hESCs) are potential candidates as sources of cells for cell replacement therapy. Significant progress has been made in improving culture conditions of undifferentiated hESCs, and using various methods, several laboratories have reported the generation of contracting cardiomyocytes from hESCs in vitro. Application of these cardiomyocytes to the clinic, however, still requires substantial experimentation to show that 1) they are functional in vitro; 2) they are efficacious in animal models of cardiac injury and disease; 3) they are safe and effective in human conditions, and 4) a sufficient amount of cardiomyocytes with expected characteristics can be generated in a reproducible manner. Here we review and discuss current findings on growth and differentiation of hESCs, and on characterization, enrichment and transplantation of hESC-derived cardiomyocytes.     

Arrhythmogenic risks of stem cell replacement therapy for cardiovascular diseases

Ischemic heart disease and congestive heart failure are major contributors to high morbidity and mortality. Approximately 1.5 million cases of myocardial infarction occur annually in the United States; the yearly incidence rate is approximately 600 cases per 100,000 people. Although significant progress to improve the survival rate has been made by medications and implantable medical devices, damaged cardiomyocytes are unable to be recovered by current treatment strategies. After almost two decades of research, stem cell therapy has become a very promising approach to generate new cardiomyocytes and enhance the function of the heart. Along with clinical trials with stem cells conducted in cardiac regeneration, concerns regarding safety and potential risks have emerged. One of the contentious issues is the electrical dysfunctions of cardiomyocytes and cardiac arrhythmia after stem cell therapy. In this review, we focus on the cell sources currently used for stem cell therapy and discuss related arrhythmogenic risk.     

Non-coding RNAs in cardiomyocyte proliferation and cardiac regeneration: Dissecting their therapeutic values

Cardiovascular diseases are associated with high incidence and mortality, contribute to disability and place a heavy economic burden on countries worldwide. Stimulating endogenous cardiomyocyte proliferation and regeneration has been considering as a key to repair the injured heart caused by ischaemia. Emerging evidence has proved that non-coding RNAs participate in cardiac proliferation and regeneration. In this review, we focus on the observation and mechanism that microRNAs (or miRNAs), long non-coding RNAs (or lncRNAs) and circular RNA (or circRNAs) regulate cardiomyocyte proliferation and regeneration to repair a damaged heart. Furthermore, we highlight the potential therapeutic role of some non-coding RNAs used in stimulating CMs proliferation. Finally, perspective on the development of non-coding RNAs therapy in cardiac regeneration is presented.     

Vascular endothelial growth factor (VEGF) as a key therapeutic trophic factor in bone marrow mesenchymal stem cell-mediated cardiac repair

We recently demonstrated a novel effective therapeutic regimen for treating hamster heart failure based on injection of bone marrow mesenchymal stem cells (MSCs) or MSC-conditioned medium into the skeletal muscle. The work highlights an important cardiac repair mechanism mediated by the myriad of trophic factors derived from the injected MSCs and local musculature that can be explored for non-invasive stem cell therapy. While this therapeutic regimen provides the ultimate proof that MSC-based cardiac repair is mediated by the trophic actions independent of MSC differentiation or stemness, the trophic factors responsible for cardiac regeneration after MSC therapy remain largely undefined. Toward this aim, we took advantage of the finding that human and porcine MSCs exhibit species-related differences in expression of trophic factors. We demonstrate that human MSCs when compared to porcine MSCs express and secrete 5-fold less vascular endothelial growth factor (VEGF) in conditioned medium (40 ± 5 and 225 ± 17 pg/ml VEGF, respectively). This deficit in VEGF output was associated with compromised cardiac therapeutic efficacy of human MSC-conditioned medium. Over-expression of VEGF in human MSCs however completely restored the therapeutic potency of the conditioned medium. This finding indicates VEGF as a key therapeutic trophic factor in MSC-mediated myocardial regeneration, and demonstrates the feasibility of human MSC therapy using trophic factor-based cell-free strategies, which can eliminate the concern of potential stem cell transformation.     

Turning regenerative technologies into treatment to repair myocardial injuries

Regenerative therapies including stem cell treatments hold promise to allow curing patients affected by severe cardiac muscle diseases. However, the clinical efficacy of stem cell therapy remains elusive, so far. The two key roadblocks that still need to be overcome are the poor cell engraftment into the injured myocardium and the limited knowledge of the ideal mixture of bioactive factors to be locally delivered for restoring heart function. Thus, therapeutic strategies for cardiac repair are directed to increase the retention and functional integration of transplanted cells in the damaged myocardium or to enhance the endogenous repair mechanisms through cell-free therapies. In this context, biomaterial-based technologies and tissue engineering approaches have the potential to dramatically impact cardiac translational medicine. This review intends to offer some consideration on the cell-based and cell-free cardiac therapies, their limitations and the possible future developments.     

Cardiomyocyte proliferation,a target for cardiac regeneration

Cardiac diseases, characterized by cardiomyocyte loss, lead to dramatic impairment of cardiac function and ultimately to congestive heart failure. Despite significant advances, conventional treatments do not correct the defects in cardiac muscle cell numbers and the prognosis of congestive heart failure remains poor. The existence, in adult mammalian heart, of low but detectable cardiomyocyte proliferative capacities has shifted the target of regenerative therapy toward new therapeutical strategy. Indeed, the stimulation of terminally differentiated cardiomyocyte proliferation represents the main therapeutic approach for heart regeneration. Increasing evidence demonstrating that the loss of mammalian cardiomyocyte renewal potential shortly after birth causes the loss of regenerative capacities, strongly support the hypothesis that a detailed understanding of the molecular mechanisms controlling fetal and postnatal cardiomyocyte proliferation is essential to identify targets for cardiac regeneration. Here, we will review major developmental mechanisms regulating fetal cardiomyocyte proliferation and will describe the impact of the developmental switch, operating at birth and driving postnatal heart maturation, on the regulation of adult cardiomyocyte proliferation, all these mechanisms representing potential targets for cardiac repair and regeneration.     

Partial reprogramming as a therapeutic approach for heart disease: A state-of-the-art review

Heart disease such as myocardial infarction is the first cause of mortality in all countries. Today, cardiac cell-based therapy using de novo produced cardiac cells is considered as a novel approach for cardiac regenerative medicine. Recently, an alchemy-like approach, known as direct reprogramming or direct conversion, has been developed to directly convert somatic cells to cardiac cells in vitro and in vivo. This cellular alchemy is a short-cut and safe strategy for generating autologous cardiac cells, and it can be accomplished through activating cardiogenesis- or pluripotency-related factors in noncardiac cells. Importantly, pluripotency factors-based direct cardiac conversion, known as partial reprogramming, is shorter and more efficient for cardiomyocyte generation in vitro. Today, this strategy is achievable for direct conversion of mouse and human somatic cells to cardiac lineage cells (cardiomyocytes and cardiac progenitor cells), using transgene free, chemical-based approaches. Although, heart-specific partial reprogramming seems to be challenging for in vivo conversion of cardiac fibroblasts to cardiac cells, but whole organism-based in vivo partial reprogramming ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in mouse. Notably, cardiac cells produced using partial reprogramming strategy can be a useful platform for disease modeling, drug screening and cardiac cell-based therapy, once the safety issues are overcome. Herein, we discuss about all progresses in de novo production of cardiac cells using partial reprogramming-based direct conversion, as well as give an overview about the potential applications of this strategy in vivo and in vitro.     

Cardiac tissue engineering: regeneration of the wounded heart

New solutions are needed to regenerate hearts damaged by myocardial infarction, to overcome bad prognosis of patients with heart failure, and to address the shortage of heart donors. In the past few years, cardiac tissue engineering has emerged as a new and ambitious approach that combines knowledge from material chemistry with cell biology and medicine. In this short review, we present an overview on the most promising materials and cell-therapy strategies used in the past few years for the regeneration of the wounded heart.     

Injectable cardiac tissue engineering for the treatment of myocardial infarction

Heart disease is a leading cause of morbidity and mortality worldwide. Myocardial infarction leads to permanent loss of cardiac tissue and ultimately heart failure. However, current therapies could only stall the progression of the disease. Thus, new therapies are needed to regenerate damaged hearts to overcome poor prognosis of patients with heart failure. The shortage of heart donors is also a factor for innovating new therapies. Although the cardiac performance by cell-based therapy has improved, unsatisfactory cell retention and transplant survival still plague this technique. Because biomaterials can improve the cell retention, survival and differentiation, cardiac tissue engineering is now being explored as an approach to support cell-based therapies and enhance their efficacy for cardiac disease. In the last decade, cardiac tissue engineering has made considerable progress. Among different kinds of approaches in the cardiac tissue engineering, the approach of injectable cardiac tissue engineering is more minimally invasive than that of in vitro engineered tissue or epicardial patch implantation. It is therefore clinically appealing. In this review, we strive to describe the major progress in the flied of injectable cardiac tissue engineering, including seeding cell sources, biomaterials and novel findings in preclinical studies and clinical applications. The remaining problems will also be discussed.     

Extrinsic regulation of cardiomyocyte differentiation of embryonic stem cells

Cardiovascular disease is one of leading causes of death throughout the U.S. and the world. The damage of cardiomyocytes resulting from ischemic injury is irreversible and leads to the development of progressive heart failure, which is characterized by the loss of functional cardiomyocytes. Because cardiomyocytes are unable to regenerate in the adult heart, cell-based therapy of transplantation provides a potential alternative approach to replace damaged myocardial tissue and restore cardiac function. A major roadblock toward this goal is the lack of donor cells; therefore, it is urgent to identify the cardiovascular cells that are necessary for achieving cardiac muscle regeneration. Pluripotent embryonic stem (ES) cells have enormous potential as a source of therapeutic tissues, including cardiovascular cells; however, the regulatory elements mediating ES cell differentiation to cardiomyocytes are largely unknown. In this review, we will focus on extrinsic factors that play a role in regulating different stages of cardiomyocyte differentiation of ES cells.     

Myocardial tissue engineering: toward a bioartificial pump

Regenerative therapies, including cell injection and bioengineered tissue transplantation, have the potential to treat severe heart failure. Direct implantation of isolated skeletal myoblasts and bone-marrow-derived cells has already been clinically performed and research on fabricating three-dimensional (3-D) cardiac grafts using tissue engineering technologies has also now been initiated. In contrast to conventional scaffold-based methods, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cell sheets to construct 3-D cell-dense tissues. Upon layering, individual cardiac cell sheets integrate to form a single, continuous, cell-dense tissue that resembles native cardiac tissue. The transplantation of layered cardiac cell sheets is able to repair damaged hearts. As the next step, we have attempted to promote neovascularization within bioengineered myocardial tissues to overcome the longstanding limitations of engineered tissue thickness. Finally, as a possible advanced therapy, we are now trying to fabricate functional myocardial tubes that may have a potential for circulatory support. Cell sheet-based tissue engineering technologies therefore show an enormous promise as a novel approach in the field of myocardial tissue engineering.     

Speckle variance optical coherence tomography of blood flow in the beating mouse embryonic heart

Efficient separation of blood and cardiac wall in the beating embryonic heart is essential and critical for experiment‐based computational modelling and analysis of early‐stage cardiac biomechanics. Although speckle variance optical coherence tomography (SV‐OCT) relying on calculation of intensity variance over consecutively acquired frames is a powerful approach for segmentation of fluid flow from static tissue, application of this method in the beating embryonic heart remains challenging because moving structures generate SV signal indistinguishable from the blood. Here, we demonstrate a modified four‐dimensional SV‐OCT approach that effectively separates the blood flow from the dynamic heart wall in the beating mouse embryonic heart. The method takes advantage of the periodic motion of the cardiac wall and is based on calculation of the SV signal over the frames corresponding to the same phase of the heartbeat cycle. Through comparison with Doppler OCT imaging, we validate this speckle‐based approach and show advantages in its insensitiveness to the flow direction and velocity as well as reduced influence from the heart wall movement. This approach has a potential in variety of applications relying on visualization and segmentation of blood flow in periodically moving structures, such as mechanical simulation studies and finite element modelling. Picture : Four‐dimensional speckle variance OCT imaging shows the blood flow inside the beating heart of an E8.5 mouse embryo.     

Cardiac regeneration therapy: connections to cardiac physiology

Without heart transplantation, a large number of patients with failing hearts worldwide face poor outcomes. By means of cardiomyocyte regeneration, cardiac regeneration therapy is emerging with great promise as a means for restoring loss of cardiac function. However, the limited success of clinical trials using bone marrow-derived cells and myoblasts with heterogeneous constituents, transplanted at a wide range of cell doses, has led to disagreement on the efficacy of cell therapy. It is therefore essential to reevaluate the evidence for the efficacy of cell-based cardiac regeneration therapy, focusing on targets, materials, and methodologies. Meanwhile, the revolutionary innovation of cardiac regeneration therapy is sorely needed to help the millions of people who suffer heart failure from acquired loss of cardiomyocytes. Cardiac regeneration has been used only in limited species or as a developing process in the rodent heart; now, the possibility of cardiomyocyte turnover in the human heart is being revisited. In the pursuit of this concept, the use of cardiac stem/progenitor stem cells in the cardiac niche must be focused to usher in a second era of cardiac regeneration therapy for the severely injured heart. In addition, tissue engineering and cellular reprogramming will advance the next era of treatment that will enable current cell-based therapy to progress to "real" cardiac regeneration therapy. Although many barriers remain, the prevention of refractory heart failure through cardiac regeneration is now becoming a realistic possibility.     

Progressing a human embryonic stem-cell-based regenerative medicine therapy towards the clinic

Since the first publication of the derivation of human embryonic stem cells in 1998, there has been hope and expectation that this technology will lead to a wave of regenerative medicine therapies with the potential to revolutionize our approach to managing certain diseases. Despite significant resources in this direction, the path to the clinic for an embryonic stem-cell-based regenerative medicine therapy has not proven straightforward, though in the past few years progress has been made. Here, with a focus upon retinal disease, we discuss the current status of the development of such therapies. We also highlight some of our own experiences of progressing a retinal pigment epithelium cell replacement therapy towards the clinic.     

Heart rate variability analysis in general medicine

Autonomic nervous system plays an integral role in homeostasis. Autonomic modulation can frequently be altered in patients with cardiac disorders as well as in patients with other critical illnesses or injuries. Assessment of heart rate variability is based on analysis of consecutive normal R-R intervals and may provide quantitative information on the modulation of cardiac vagal and sympathetic nerve input. The hypothesis that depressed heart rate variability may occur over a broad range of illness and injury, and may inversely correlated with disease severity and outcome has been tested in various clinical settings over the last decade. This article reviews recent literature concerning the potential clinical implications and limitations of heart rate variability assessment in general medicine.     

Chronomics of pressure overload-induced cardiac hypertrophy in mice reveals altered day/night gene expression and biomarkers of heart disease

There is critical demand in contemporary medicine for gene expression markers in all areas of human disease, for early detection of disease, classification, prognosis, and response to therapy. The integrity of circadian gene expression underlies cardiovascular health and disease; however time-of-day profiling in heart disease has never been examined. We hypothesized that a time-of-day chronomic approach using samples collected across 24-h cycles and analyzed by microarrays and bioinformatics advances contemporary approaches, because it includes sleep-time and/or wake-time molecular responses. As proof of concept, we demonstrate the value of this approach in cardiovascular disease using a murine Transverse Aortic Constriction (TAC) model of pressure overload-induced cardiac hypertrophy in mice. First, microarrays and a novel algorithm termed DeltaGene were used to identify time-of-day differences in gene expression in cardiac hypertrophy 8 wks post-TAC. The top 300 candidates were further analyzed using knowledge-based platforms, paring the list to 20 candidates, which were then validated by real-time polymerase chain reaction (RTPCR). Next, we tested whether the time-of-day gene expression profiles could be indicative of disease progression by comparing the 1- vs. 8-wk TAC. Lastly, since protein expression is functionally relevant, we monitored time-of-day cycling for the analogous cardiac proteins. This approach is generally applicable and can lead to new understanding of disease.     

The role of stem cells in cardiac regeneration

After myocardial infarction, injured cardiomyocytes are replaced by fibrotic tissue promoting the development of heart failure. Cell transplantation has emerged as a potential therapy and stem cells may be an important and powerful cellular source. Embryonic stem cells can differentiate into true cardiomyocytes, making them in principle an unlimited source of transplantable cells for cardiac repair, although immunological and ethical constraints exist. Somatic stem cells are an attractive option to explore for transplantation as they are autologous, but their differentiation potential is more restricted than embryonic stem cells. Currently, the major sources of somatic cells used for basic research and in clinical trials originate from the bone marrow. The differentiation capacity of different populations of bone marrow-derived stem cells into cardiomyocytes has been studied intensively. The results are rather confusing and difficult to compare, since different isolation and identification methods have been used to determine the cell population studied. To date, only mesenchymal stem cells seem to form cardiomyocytes, and only a small percentage of this population will do so in vitro or in vivo. A newly identified cell population isolated from cardiac tissue, called cardiac progenitor cells, holds great potential for cardiac regeneration. Here we discuss the potential of the different cell populations and their usefulness in stem cell based therapy to repair the damaged heart.     

Ventricular arrhythmias.

Sudden cardiac death claims thousands of Canadians annually. Ventricular tachycardia and fibrillation account for up to 85% of these deaths. Identifying the patients at risk remains a major challenge. Those who have recurrent ventricular tachycardia or have been resuscitated from ventricular fibrillation are generally considered to be at highest risk. Although ventricular premature beats in the absence of previous ventricular tachycardia or fibrillation are not helpful in identifying such patients in most cases, they can indicate increased risk for sudden cardiac death in the presence of a structural cardiac abnormality, particularly recent myocardial infarction; however, the need for treatment in such cases is speculative and is being investigated. Treatment is mandatory for survivors of an episode of ventricular fibrillation and those with recurrent sustained ventricular tachycardia or torsade de pointes ventricular tachycardia. The approach to management is either invasive or noninvasive. Selection of an antiarrhythmic agent is facilitated by knowledge of some basic electrophysiologic features of the heart and of the classification of antiarrhythmic drugs. However, drug therapy has to be individualized on the basis of efficacy, left ventricular function and adverse effects or potential adverse effects of the drug. Amiodarone therapy or nonpharmacologic therapy should be considered if a suitable antiarrhythmic agent cannot be found.