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.     

Activation of Cardiac Stem Cells in Myocardial Infarction

The development of heart failure caused by acute myocardial infarction is accompanied by massive necrotic death of cardiomyocytes in lesion areas and subsequent pathological myocardial remodeling. Traditionally, the possibility of heart reparation has been considered to be severely limited or absent in the postnatal period. Endogenous cardiac stem cells with a regenerative potential have recently been described, but the mechanisms of activation of these cells remain poorly understood. The aim of our work was to obtain cardiac stem cells from the ischemic area of the myocardium and compare their functional properties with stem cells isolated from the healthy area of the myocardium. RT-PCR was used to quantify the gene expression in cardiac stem cells. In addition, differentiated cells were stained for specific markers using immunocytochemical method. Cardiac stem cells originating from the infarction area had a higher proliferative potential and a greater propensity to migrate in comparison to the cells originated from a healthy myocardial area. The expression level of several specific markers of cardiogenic, osteogenic and adipogenic differentiation upon induction of corresponding differentiation was higher in the cells from the infarction area than in cells from the healthy myocardium. We conclude that myocardial ischemia activates the internal regenerative potential of cardiac stem cells.     

Contemporary perspective on endogenous myocardial regeneration

Considering the complex nature of the adult heart, it is no wonder that innate regenerative processes, while maintaining adequate cardiac function, fall short in myocardial jeopardy. In spite of these enchaining limitations, cardiac rejuvenation occurs as well as restricted regeneration. In this review, the background as well as potential mechanisms of endogenous myocardial regeneration are summarized. We present and analyze the available evidence in three subsequent steps. First, we examine the experimental research data that provide insights into the mechanisms and origins of the replicating cardiac myocytes, including cell populations referred to as cardiac progenitor cells (i.e., c-kit+ cells). Second, we describe the role of clinical settings such as acute or chronic myocardial ischemia, as initiators of pathways of endogenous myocardial regeneration. Third, the hitherto conducted clinical studies that examined different approaches of initiating endogenous myocardial regeneration in failing human hearts are analyzed. In conclusion, we present the evidence in support of the notion that regaining cardiac function beyond cellular replacement of dysfunctional myocardium via initiation of innate regenerative pathways could create a new perspective and a paradigm change in heart failure therapeutics. Reinitiating cardiac morphogenesis by reintroducing developmental pathways in the adult failing heart might provide a feasible way of tissue regeneration. Based on our hypothesis “embryonic recall”, we present first supporting evidence on regenerative impulses in the myocardium, as induced by developmental processes.     

Stable benefit of embryonic stem cell therapy in myocardial infarction

Conventional therapies for myocardial infarction attenuate disease progression without contributing significantly to repair. Because of the capacity for de novo cardiogenesis, embryonic stem cells are considered a potential source for myocardial regeneration, yet limited information is available on their ultimate therapeutic value. We treated infarcted rat hearts with CGR8 embryonic stem cells preexamined for cardiogenicity, serially probed left ventricular function, and determined final pathological outcome. Stem cell delivery generated new cardiomyocytes of embryonic stem cell origin that integrated with host myocardium within infarct regions. This resulted in a functional benefit within 3 wk that remained sustained over 12 wk of continuous follow-up and included a vigorous inotropic response to beta-adrenergic challenge. Integration of stem cell-derived cardiomyocytes was associated with normalized ventricular architecture, little scar, and a decrease in signs of myocardial necrosis. In contrast, sham-treated infarcted hearts exhibited ventricular cavity dilation and aneurysm formation, poor ventricular function, and a lack of response to beta-adrenergic stimulation. No evidence of graft rejection, ectopy, sudden cardiac death, or tumor formation was observed after therapy. These findings indicate that embryonic stem cells, through differentiation within the host myocardium, can contribute to a stable beneficial outcome on contractile function and ventricular remodeling in the infarcted heart.     

Human endometrial stem cells confer enhanced myocardial salvage and regeneration by paracrine mechanisms

Human endometrial stem cells (EnSCs) have the potential to be ‘off the shelf’ clinical reagents for the treatment of heart failure. Here, using an immunocompetent rat model of myocardial infarction (MI), we provide evidence that the functional benefits of EnSC transplantation are principally and possibly exclusively through a paracrine effect. Human EnSCs were delivered by intramyocardial injection into rats 30 min. after coronary ligation. EnSC therapy significantly preserved viable myocardium in the infarct zone and improved cardiac function at 28 days. Despite increased viable myocardium and vascular density, there was scant evidence of differentiation of EnSCs into any cardiovascular cell type. Cultured human EnSCs expressed a distinctive profile of cytokines that enhanced the survival, proliferation and function of endothelial cells in vitro. When injected into the peri‐infarct zone, human EnSCs activated AKT, ERK1/2 and STAT3 and inhibited the p38 signalling pathway. EnSC therapy decreased apoptosis and promoted cell proliferation and c‐kit+ cell recruitment in vivo. Myocardial protection and enhanced post‐infarction regeneration by EnSCs is mediated primarily by paracrine effects conferred by secreted cytokines that activate survival pathways and recruit endogenous progenitor stem cells. Menstrual blood provides a potentially limitless source of biologically competent ‘off the shelf’ EnSCs for allogeneic myocardial regenerative medicine.     

Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance

Cellular therapy for myocardial injury has improved ventricular function in both animal and clinical studies, though the mechanism of benefit is unclear. This study was undertaken to examine the effects of cellular injection after infarction on myocardial elasticity. Coronary artery ligation of Lewis rats was followed by direct injection of human mesenchymal stem cells (MSCs) into the acutely ischemic myocardium. Two weeks postinfarct, myocardial elasticity was mapped by atomic force microscopy. MSC-injected hearts near the infarct region were twofold stiffer than myocardium from noninfarcted animals but softer than myocardium from vehicle-treated infarcted animals. After 8 wk, the following variables were evaluated: MSC engraftment and left ventricular geometry by histological methods, cardiac function with a pressure-volume conductance catheter, myocardial fibrosis by Masson Trichrome staining, vascularity by immunohistochemistry, and apoptosis by TdT-mediated dUTP nick-end labeling assay. The human cells engrafted and expressed a cardiomyocyte protein but stopped short of full differentiation and did not stimulate significant angiogenesis. MSC-injected hearts showed significantly less fibrosis than controls, as well as less left ventricular dilation, reduced apoptosis, increased myocardial thickness, and preservation of systolic and diastolic cardiac function. In summary, MSC injection after myocardial infarction did not regenerate contracting cardiomyocytes but reduced the stiffness of the subsequent scar and attenuated postinfarction remodeling, preserving some cardiac function. Improving scarred heart muscle compliance could be a functional benefit of cellular cardiomyoplasty.     

Intramyocardial transplantation of cardiac telocytes decreases myocardial infarction and improves post‐infarcted cardiac function in rats

The midterm effects of cardiac telocytes (CTs) transplantation on myocardial infarction (MI) and the cellular mechanisms involved in the beneficial effects of CTs transplantation are not understood. In the present study, we have revealed that transplantation of CTs was able to significantly decrease the infarct size and improved cardiac function 14 weeks after MI. It has established that CT transplantation exerted a protective effect on the myocardium and this was maintained for at least 14 weeks. The cellular mechanism behind this beneficial effect on MI was partially attributed to increased cardiac angiogenesis, improved reconstruction of the CT network and decreased myocardial fibrosis. These combined effects decreased the infarct size, improved the reconstruction of the LV and enhanced myocardial function in MI. Our findings suggest that CTs could be considered as a potential cell source for therapeutic use to improve cardiac repair and function following MI, used either alone or in tandem with stem cells.     

Progenitor cell therapy for heart disease

Many cell types are currently being studied as potential sources of cardiomyocytes for cell transplantation therapy to repair and regenerate damaged myocardium. The question remains as to which progenitor cell represents the best candidate. Bone marrow-derived cells and endothelial progenitor cells have been tested in clinical studies. These cells are safe, but their cardiogenic potential is controversial. The functional benefits observed are probably due to enhanced angiogenesis, reduced ventricular remodeling, or to cytokine-mediated effects that promote the survival of endogenous cells. Human embryonic stem cells represent an unlimited source of cardiomyocytes due to their great differentiation potential, but each step of differentiation must be tightly controlled due to the high risk of teratoma formation. These cells, however, confront ethical barriers and there is a risk of graft rejection. These last two problems can be avoided by using induced pluripotent stem cells (iPS), which can be autologously derived, but the high risk of teratoma formation remains. Cardiac progenitor cells have the advantage of being cardiac committed, but important questions remain unanswered, such as what is the best marker to identify and isolate these cells? To date the different markers used to identify adult cardiac progenitor cells also recognize progenitor cells that are outside the heart. Thus, it cannot be determined whether the cardiac progenitor cells identified in the adult heart represent resident cells present since fetal life or extracardiac cells that colonized the heart after cardiac injury. Developmental studies have identified markers of multipotent progenitors, but it is unknown whether these markers are specific for adult progenitors when expressed in the adult myocardium. Cardiac regeneration is dependent on the stability of the cells transplanted into the host myocardium and on the electromechanical coupling with the endogenous cells. Finally, the promotion of endogenous regenerative processes by mobilizing endogenous progenitors represents a complementary approach to cell transplantation therapy.     

Local activation of cardiac stem cells for post‐myocardial infarction cardiac repair

The prognosis of patients with myocardial infarction (MI) and resultant chronic heart failure remains extremely poor despite continuous advancements in optimal medical therapy and interventional procedures. Animal experiments and clinical trials using adult stem cell therapy following MI have shown a global improvement of myocardial function. The emergence of stem cell transplantation approaches has recently represented promising alternatives to stimulate myocardial regeneration. Regarding their tissue‐specific properties, cardiac stem cells (CSCs) residing within the heart have advantages over other stem cell types to be the best cell source for cell transplantation. However, time‐consuming and costly procedures to expanse cells prior to cell transplantation and the reliability of cell culture and expansion may both be major obstacles in the clinical application of CSC‐based transplantation therapy after MI. The recognition that the adult heart possesses endogenous CSCs that can regenerate cardiomyocytes and vascular cells has raised the unique therapeutic strategy to reconstitute dead myocardium via activating these cells post‐MI. Several strategies, such as growth factors, mircoRNAs and drugs, may be implemented to potentiate endogenous CSCs to repair infarcted heart without cell transplantation. Most molecular and cellular mechanism involved in the process of CSC‐based endogenous regeneration after MI is far from understanding. This article reviews current knowledge opening up the possibilities of cardiac repair through CSCs activation in situ in the setting of MI.     

Cardiac shock wave therapy: assessment of safety and new insights into mechanisms of tissue regeneration

Although low-energy extracorporeal cardiac shock wave (ECSW) therapy represents an attractive non-invasive treatment option for ischaemic heart disease, the precise mechanisms of its action and influence on the cardiac tissue remain obscure. The goal of this study was to evaluate the effects of SW application on cardiac function and structure. Four-month-old Fisher 344 rats were subjected to ECSW therapy. Echocardiographic measurements of cardiac function were performed at baseline and at 1 and 3 months after treatment. Signs of inflammation, apoptosis and fibrosis were evaluated by immunohistochemistry in the control and treated hearts. ECSW application did not provoke arrhythmia or increase the troponin-I level. At all time points, the left ventricular ejection fraction and fractional shortening remained stable. Histological analysis revealed neither differences in the extracellular matrix collagen content nor the presence of fibrosis; similarly, there were no signs of inflammation. Moreover, a population of cardiac cells that responded eagerly to ECSW application in the adult heart was identified; c-kit-positive, Ki67-positive, orthochromatic cells, corresponding to cardiac primitive cells, were 2.65-fold more numerous in the treated myocardium. In conclusion, non-invasive ECSW therapy is a safe and effective way of activating cardiac stem cells and myocardial regeneration. Because many factors influence cellular turnover in the ischaemic myocardium during the course of ischaemic heart disease, cardiac remodelling, and heart failure progression, studies to identify the optimal treatment time are warranted.     

Granulocyte colony-stimulating factor treatment enhances the efficacy of cellular cardiomyoplasty with transplantation of embryonic stem cell-derived cardiomyocytes in infarcted myocardium

We tested the hypothesis that granulocyte colony-stimulating factor (G-CSF) administration would enhance the efficacy of cellular cardiomyoplasty with embryonic stem (ES) cell-derived cardiomyocytes in infarcted myocardium. Three weeks after myocardial infarction by cryoinjury, Sprague-Dawley rats were randomized to receive either an injection of medium, ES cell-derived cardiomyocyte transplantation, G-CSF administration, or a combination of G-CSF administration and ES cell-derived cardiomyocyte transplantation. Eight weeks after treatment, the cardiac tissue formation, neovascularization, and apoptotic activity in the infarct regions were evaluated by histology and immunohistochemistry. The left ventricular (LV) dimensions and function of the treated heart were evaluated by echocardiography. Transplanted ES cell-derived cardiomyocytes survived and participated in the myocardial regeneration in the infarcted heart. A combination of G-CSF treatment and ES cell-derived cardiomyocyte transplantation significantly promoted angiogenesis and reduced the infarct area and cell apoptosis in the infarcted myocardium compared with ES cell-derived cardiomyocyte transplantation alone. The combination therapy also attenuated LV dilation, as compared with ES cell-derived cardiomyocyte transplantation alone. G-CSF treatment can enhance the efficacy of cellular cardiomyoplasty by ES cell-derived cardiomyocyte transplantation to treat myocardial infarction.     

Genistein Promotes Endothelial Colony-Forming Cell (ECFC) Bioactivities and Cardiac Regeneration in Myocardial Infarction

Although stem cell-mediated treatment of ischemic diseases offers significant therapeutic promise, the limitation in the therapeutic efficacy of transplanted stem cells in vivo because of poor engraftment remains a challenge. Several strategies aimed at improving survival and engraftment of stem cells in the ischemic myocardium have been developed, such as cell transplantation in combination with growth factor delivery, genetic modification of stem cells, and/or cell therapy using scaffolds. To improve therapeutic efficacy, we investigated the effects of genistein on the engraftment of transplanted ECFCs in an acute myocardial ischemia model. Results: We found that genistein treatment enhanced ECFCs'' migration and proliferation, which was accompanied by increases in the expression of ILK, α-parvin, F-actin, and phospholylation of ERK 1/2 signaling. Transplantation of genistein-stimulates ECFCs (GS-ECFCs) into myocardial ischemic sites in vivo induced cellular proliferation and secretion of angiogenic cytokines at the ischemic sites and thereby enhanced neovascularization and decreased myocardial fibrosis as well as improved cardiac function, as shown by echocardiography. Taken together, these data suggest that pretreatment of ECFCs with genistein prior to transplantation can improve the regenerative potential in ischemic tissues, providing a novel strategy in adult stem cell therapy for ischemic diseases.     

Human adult skeletal muscle stem cells differentiate into cardiomyocyte phenotype in vitro

Cell transplantation to repair or regenerate injured myocardium is a new frontier in the treatment of cardiovascular disease. Most studies on stem cell transplantation therapy in both experimental heart infarct and in phase-I human clinical trials have focused on the use of undifferentiated stem cells. Based on our previous observations demonstrating the presence of multipotent progenitor cells in human adult skeletal muscle, in this study we investigated the capacity of these progenitors to differentiate into cardiomyocytes. Here we show an efficient protocol for the cardiomyogenic differentiation of human adult skeletal muscle stem cells in vitro. We found that treatment with Retinoic Acid directed cardiomyogenic differentiation of skeletal muscle stem cells in vitro. After Retinoic Acid treatment, cells expressed cardiomyocyte markers and acquired spontaneous contraction. Functional assays exhibited cardiac-like response to increased extracellular calcium. When cocultured with mouse cardiomyocytes, Retinoic Acid-treated skeletal muscle stem cells expressed connexin43 and when transplanted into ischemic heart were detectable even 5 weeks after injection. Based on these results, we can conclude that human adult skeletal muscle stem cells, if opportunely treated, can transdifferentiate into cells of cardiac lineage and once injected into infarcted heart can integrate, survive in cardiac tissue and improve the cardiac function.     

Cardiomyocyte progenitor cell‐derived exosomes stimulate migration of endothelial cells

Patients suffering from heart failure as a result of myocardial infarction are in need of heart transplantation. Unfortunately the number of donor hearts is very low and therefore new therapies are subject of investigation. Cell transplantation therapy upon myocardial infarction is a very promising strategy to replace the dead myocardium with viable cardiomyocytes, smooth muscle cells and endothelial cells, thereby reducing scarring and improving cardiac performance. Despite promising results, resulting in reduced infarct size and improved cardiac function on short term, only a few cells survive the ischemic milieu and are retained in the heart, thereby minimizing long-term effects. Although new capillaries and cardiomyocytes are formed around the infarcted area, only a small percentage of the transplanted cells can be detected months after myocardial infarction. This suggests the stimulation of an endogenous regenerative capacity of the heart upon cell transplantation, resulting from release of growth factor, cytokine and other paracrine molecules by the progenitor cells – the so-called paracrine hypothesis. Here, we focus on a relative new component of paracrine signalling, i.e. exosomes. We are interested in the release and function of exosomes derived from cardiac progenitor cells and studied their effects on the migratory capacity of endothelial cells.     

Research of cardiomyocyte precursors in adult rat heart

Recent reports supported the existence of stem cells in adult hearts. However, phenotype and localization of these cells have not been completely described and it is unknown if cardiac regenerative potential differs from one subject to another. The aims of our work were to identify different populations of cardiac stem cells by the analysis of specific markers and to evaluate the expression variability of these markers in 12 adult rat hearts. The expression of CD9, taube nuss and nanog suggests the presence of stem cells from the earliest stages of embryogenesis in adult myocardium. Their different expression could be associated to the degree of stem cell differentiation. CD34 and c-Kit antibodies were used to detect stem cells committed to one or more specific tissue lineages and we found a strong immunoreactivity for CD34 exclusively in the endothelial cells and a low positivity for c-Kit in the interstitium and next to the vessels. Moreover, as c-Kit expression highly differed within all examined hearts, we suggest that cardiomyogenic potential is different among the various subjects. Undifferentiated cells with myogenic-committed phenotype expressing GATA-4 and nestin were found, respectively, in the interstitial and myocardial cells and in few interstitial cells. Therefore, the physiologic turn over of cardiomyocytes may occur in adult hearts as it has been shown in many others organs. The study of myogenic potential could be important to identify markers specific of stem cells in in vivo adult myocardium that may be used to purify these cells and evaluate their regenerative ability.     

Induced cardiac progenitor cells repopulate decellularized mouse heart scaffolds and differentiate to generate cardiac tissue

Native myocardium has limited regenerative potential post injury. Advances in lineage reprogramming have provided promising cellular sources for regenerative medicine in addition to research applications. Recently we have shown that adult mouse fibroblasts can be reprogrammed to expandable, multipotent, induced cardiac progenitor cells (iCPCs) by employing forced expression of five cardiac factors along with activation of canonical Wnt and JAK/STAT signaling. Here we aim to further characterize iCPCs by highlighting their safety, ease of attainability, and functionality within a three-dimensional cardiac extracellular matrix scaffold. Specifically, iCPCs did not form teratomas in contrast to embryonic stem cells when injected into immunodeficient mice. iCPC reprogramming was achieved in wild type mouse fibroblasts without requiring a cardiac-specific reporter, solely utilizing morphological changes to identify, clonally isolate, and expand iCPCs, thus increasing the versatility of this technology. iCPCs also show the ability to repopulate decellularized native heart scaffolds and differentiated into organized structures containing cardiomyocytes, smooth muscle, and endothelial cells. Optical mapping of recellularized scaffolds shows field-stimulated calcium transients that propagate across islands of reconstituted tissue and bipolar local stimulation demonstrates cell-cell coupling within scaffolds. Overall, iCPCs provide a readily attainable, scalable, safe, and functional cell source for a variety of application including drug discovery, disease modeling, and regenerative therapy.     

Myocardial regeneration with bone-marrow-derived stem cells

Despite significant therapeutic advances, heart failure remains the predominant cause of mortality in the Western world. Ischaemic cardiomyopathy and myocardial infarction are typified by the irreversible loss of cardiac muscle (cardiomyocytes) and vasculature composed of endothelial cells and smooth muscle cells, which are essential for maintaining cardiac integrity and function. The recent identification of adult and embryonic stem cells has triggered attempts to directly repopulate these tissues by stem cell transplantation as a novel therapeutic option. Reports describing provocative and hopeful examples of myocardial regeneration with adult bone-marrow-derived stem and progenitor cells have increased the enthusiasm for the use of these cells, yet many questions remain regarding their therapeutic potential and the mechanisms responsible for the observed therapeutic effects. In this review article we discuss the current preclinical and clinical advances in bone-marrow-derived stem or progenitor cell therapies for regeneration or repair of the ischaemic myocardium and their multiple related mechanisms involved in myocardial repair and regeneration.     

Elastin overexpression by cell-based gene therapy preserves matrix and prevents cardiac dilation

After a myocardial infarction, thinning and expansion of the fibrotic scar contribute to progressive heart failure. The loss of elastin is a major contributor to adverse extracellular matrix remodelling of the infarcted heart, and restoration of the elastic properties of the infarct region can prevent ventricular dysfunction. We implanted cells genetically modified to overexpress elastin to re‐establish the elastic properties of the infarcted myocardium and prevent cardiac failure. A full‐length human elastin cDNA was cloned, subcloned into an adenoviral vector and then transduced into rat bone marrow stromal cells (BMSCs). In vitro studies showed that BMSCs expressed the elastin protein, which was deposited into the extracellular matrix. Transduced BMSCs were injected into the infarcted myocardium of adult rats. Control groups received either BMSCs transduced with the green fluorescent protein gene or medium alone. Elastin deposition in the infarcted myocardium was associated with preservation of myocardial tissue structural integrity (by birefringence of polarized light; P < 0.05 versus controls). As a result, infarct scar thickness and diastolic compliance were maintained and infarct expansion was prevented (P < 0.05 versus controls). Over a 9‐week period, rats implanted with BMSCs demonstrated better cardiac function than medium controls; however, rats receiving BMSCs overexpressing elastin showed the greatest functional improvement (P < 0.01). Overexpression of elastin in the infarcted heart preserved the elastic structure of the extracellular matrix, which, in turn, preserved diastolic function, prevented ventricular dilation and preserved cardiac function. This cell‐based gene therapy provides a new approach to cardiac regeneration.     

Molecular mechanisms of cardiomyocyte regeneration and therapeutic outlook

Differently from some lower vertebrates, which can completely regenerate their heart, in higher vertebrates cardiac injury generally leads to progressive failure. Induction of cycle re-entry in terminally differentiated cardiomyocytes and stem-cell transplantation are strategies to increase the regenerative potential of the heart. As experimental and clinical studies progress, demonstrating that adult stem-cell administration has a favorable impact on myocardial function, the identification of cardiac stem cells suggests that some endogenous repair mechanisms actually exist in the mammalian heart. However, a deeper understanding of the mechanism that drives cardiomyocyte proliferation and stem-cell-mediated cardiac repair is required to translate such strategies into effective therapies.