Pluripotent stem cell‐based heart regeneration: From the developmental and immunological perspectives

Heart diseases such as myocardial infarction cause massive loss of cardiomyocytes, but the human heart lacks the innate ability to regenerate. In the adult mammalian heart, a resident progenitor cell population, termed epicardial progenitors, has been identified and reported to stay quiescent under uninjured conditions; however, myocardial infarction induces their proliferation and de novo differentiation into cardiac cells. It is conceivable to develop novel therapeutic approaches for myocardial repair by targeting such expandable sources of cardiac progenitors, thereby giving rise to new muscle and vasculatures. Human pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells can self‐renew and differentiate into the three major cell types of the heart, namely cardiomyocytes, smooth muscle, and endothelial cells. In this review, we describe our current knowledge of the therapeutic potential and challenges associated with the use of pluripotent stem cell and progenitor biology in cell therapy. An emphasis is placed on the contribution of paracrine factors in the growth of myocardium and neovascularization as well as the role of immunogenicity in cell survival and engraftment. (Part C) 96:98–107, 2012. © 2012 Wiley Periodicals, Inc.     

Reviewing the role of cardiac exosomes in myocardial repair at a glance

Exosome-based therapy is an emerging novel approach for myocardial infarction (MI) treatment. Exosomes are identified as extracellular vesicles that are produced within multivesicular bodies in the cells' cytosols and then are secreted from the cells. Exosomes are 30–100 nm in diameter that are released from viable cells and are different from other secreted vesicles such as apoptotic bodies and microvesicles in their origin and contents such as RNAs, proteins, and nucleic acid. The recent advances in exosome research have demonstrated the role of these bionanovesicles in the physiological, pathological, and molecular aspects of the heart. The results of in vitro and preclinical models have shown that exosomes from different cardiac cells can improve cardiac function following MI. For example, mesenchymal stem cells (MSCs) and cardiac progenitor cells (CPCs) containing exosomes can affect the proliferation, survival, and differentiation of cardiac fibroblasts and cardiomyocytes. Moreover, MSCs- and CPCs-derived exosomes can enhance the migration of endothelial cells. Exosome-based therapy approaches augment the cardiac function by multiple means, such as reducing fibrosis, stimulation of vascular angiogenesis, and proliferation of cardiomyocytes that result in replacing damaged heart tissue with newly generated functional myocytes. This review article aims to briefly discuss the recent advancements in the role of secreted exosomes in myocardial repair by focusing on cardiac cells-derived exosomes.     

Cell therapy for ischaemic heart disease: focus on the role of resident cardiac stem cells

Myocardial infarction results in loss of cardiomyocytes, scar formation, ventricular remodelling, and eventually heart failure. In recent years, cell therapy has emerged as a potential new strategy for patients with ischaemic heart disease. This includes embryonic and bone marrow derived stem cells. Recent clinical studies showed ostensibly conflicting results of intracoronary infusion of autologous bone marrow derived stem cells in patients with acute or chronic myocardial infarction. Anyway, these results have stimulated additional clinical and pre-clinical studies to further enhance the beneficial effects of stem cell therapy. Recently, the existence of cardiac stem cells that reside in the heart itself was demonstrated. Their discovery has sparked intense hope for myocardial regeneration with cells that are obtained from the heart itself and are thereby inherently programmed to reconstitute cardiac tissue. These cells can be detected by several surface markers (e.g. c-kit, Sca-1, MDR1, Isl-1). Both in vitro and in vivo differentiation into cardiomyocytes, endothelial cells and vascular smooth muscle cells has been demonstrated, and animal studies showed promising results on improvement of left ventricular function. This review will discuss current views regarding the feasibility of cardiac repair, and focus on the potential role of the resident cardiac stem and progenitor cells. (Neth Heart J 2009;17:199–207.)     

Stem cell-derived exosomes as a therapeutic tool for cardiovascular disease

Mesenchymal stem cells(MSCs) have been used to treat patients suffering from acute myocardial infarction(AMI) and subsequent heart failure. Although it was originally assumed that MSCs differentiated into heart cells such as cardiomyocytes, recent evidence suggests that the differentiation capacity of MSCs is minimal and that injected MSCs restore cardiac function via the secretion of paracrine factors. MSCs secrete paracrine factors in not only naked forms but also membrane vesicles including exosomes containing bioactive substances such as proteins, messenger RNAs, and microR NAs. Although the details remain unclear, these bioactive molecules are selectively sorted in exosomes that are then released from donor cells in a regulated manner. Furthermore, exosomes are specifically internalized by recipient cells via ligand-receptor interactions. Thus, exosomes are promising natural vehicles that stably and specifically transport bioactive molecules to recipient cells. Indeed, stem cell-derived exosomes have been successfully used to treat cardiovascular disease(CVD), such as AMI, stroke, and pulmonary hypertension, in animal models, and their efficacy has been demonstrated. Therefore, exosome administration may be a promising strategy for the treatment of CVD. Furthermore, modifications of exosomal contents may enhance their therapeutic effects. Future clinical studies are required to confirm the efficacy of exosome treatment for CVD.     

Role of cardiac progenitor cell‐derived exosome‐mediated microRNA‐210 in cardiovascular disease

Cardiac progenitor cells are considered to be one of the most promising stem cells for heart regeneration and repair. The cardiac protective effect of CPCs is mainly achieved by reducing tissue damage and/or promoting tissue repair through a paracrine mechanism. Exosome is a factor that plays a major role in the paracrine effect of CPCs. By delivering microRNAs to target cells and regulating their functions, exosomes have shown significant beneficial effects in slowing down cardiac injury and promoting cardiac repair. Among them, miRNA‐210 is an important anoxic‐related miRNA derived from CPCs exosomes, which has great cardiac protective effect of inhibiting myocardial cell apoptosis, promoting angiogenesis and improving cardiac function. In addition, circulating miR‐210 may be a useful biomarker for the prediction or diagnosis of related cardiovascular diseases. In this review, we briefly reviewed the mechanism of miR‐210 derived from CPCs exosomes in cardiac protection in recent years.     

Transplantation of human villous trophoblasts preserves cardiac function in mice with acute myocardial infarction

Over the past decade, cell therapies have provided promising strategies for the treatment of ischaemic cardiomyopathy. Particularly, the beneficial effects of stem cells, including bone marrow stem cells (BMSCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), have been demonstrated by substantial preclinical and clinical studies. Nevertheless stem cell therapy is not always safe and effective. Hence, there is an urgent need for alternative sources of cells to promote cardiac regeneration. Human villous trophoblasts (HVTs) play key roles in embryonic implantation and placentation. In this study, we show that HVTs can promote tube formation of human umbilical vein endothelial cells (HUVECs) on Matrigel and enhance the resistance of neonatal rat cardiomyocytes (NRCMs) to oxidative stress in vitro. Delivery of HVTs to ischaemic area of heart preserved cardiac function and reduced fibrosis in a mouse model of acute myocardial infarction (AMI). Histological analysis revealed that transplantation of HVTs promoted angiogenesis in AMI mouse hearts. In addition, our data indicate that HVTs exert their therapeutic benefit through paracrine mechanisms. Meanwhile, injection of HVTs to mouse hearts did not elicit severe immune response. Taken together, our study demonstrates HVT may be used as a source for cell therapy or a tool to study cell‐derived soluble factors for AMI treatment.     

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.     

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.     

Exosomes from neuronal stem cells may protect the heart from ischaemia/reperfusion injury via JAK1/2 and gp130

Myocardial infarction requires urgent reperfusion to salvage viable heart tissue. However, reperfusion increases infarct size further by promoting mitochondrial damage in cardiomyocytes. Exosomes from a wide range of different cell sources have been shown to activate cardioprotective pathways in cardiomyocytes, thereby reducing infarct size. Yet, it is currently challenging to obtain highly pure exosomes in quantities enough for clinical studies. To overcome this problem, we used exosomes isolated from CTX0E03 neuronal stem cells, which are genetically stable, conditionally inducible and can be produced on an industrial scale. However, it is unknown whether exosomes from neuronal stem cells may reduce cardiac ischaemia/reperfusion injury. In this study, we demonstrate that exosomes from differentiating CTX0E03 cells can reduce infarct size in mice. In an in vitro assay, these exosomes delayed cardiomyocyte mitochondrial permeability transition pore opening, which is responsible for cardiomyocyte death after reperfusion. The mechanism of MPTP inhibition was via gp130 signalling and the downstream JAK/STAT pathway. Our results support previous findings that exosomes from non-cardiomyocyte-related cells produce exosomes capable of protecting cardiomyocytes from myocardial infarction. We anticipate our findings may encourage scientists to use exosomes obtained from reproducible clinical-grade stocks of cells for their ischaemia/reperfusion studies.     

Stem cell engineering for treatment of heart diseases: Potentials and challenges

Heart disorders are a major health concern worldwide responsible for millions of deaths every year. Among the many disorders of the heart, myocardial infarction, which can lead to the development of congestive heart failure, arrhythmias, or even death, has the most severe social and economic ramifications. Lack of sufficient available donor hearts for heart transplantation, the only currently viable treatment for heart failure other than medical management options (ACE inhibition, beta blockade, use of AICDs, etc.) that improve the survival of patients with heart failure emphasises the need for alternative therapies. One promising alternative replaces cardiac muscle damaged by myocardial infarction with new contractile cardiomyocytes and vessels obtained through stem cell-based regeneration.We report on the state of the art of recovery of cardiac functions by using stem cell engineering. Current research focuses on (a) inducing stem cells into becoming cardiac cells before or after injection into a host, (b) growing replacement heart tissue in vitro, and (c) stimulating the proliferation of the post-mitotic cardiomyocytes in situ. The most promising treatment option for patients is the engineering of new heart tissue that can be implanted into damaged areas. Engineering of cardiac tissue currently employs the use of co-culture of stem cells with scaffold microenvironments engineered to improve tissue survival and enhance differentiation. Growth of heart tissue in vitro using scaffolds, soluble collagen, and cell sheets has unique advantages. To compensate for the loss of ventricular mass and contractility of the injured cardiomyocytes, different stem cell populations have been extensively studied as potential sources of new cells to ameliorate the injured myocardium and eventually restore cardiac function. Unresolved issues including insufficient cell generation survival, growth, and differentiation have led to mixed results in preclinical and clinical studies. Addressing these limitations should ensure the successful production of replacement heart tissue to benefit cardiac patients.     

Mesenchymal stem cell therapy: A promising cell‐based therapy for treatment of myocardial infarction

For decades, mesenchymal stem (MSCs) cells have been used for cardiovascular diseases as regenerative therapy. This review is an attempt to summarize the types of MSCs involved in myocardial infarction (MI) therapy, as well as its possible mechanisms effects, especially the paracrine one in MI focusing on the studies (human and animal) conducted within the last 10 years. Recently, reports showed that MSC therapy could have infarct‐limiting effects after MI in both experimental and clinical trials. In this context, various types of MSCs can help cardiac regeneration by either revitalizing the cardiac stem cells or revascularizing the arteries and veins of the heart. Furthermore, MSCs could produce paracrine growth factors that increase the survival of nearby cardiomyocytes, as well as increase angiogenesis through recruitment of stem cell from bone marrow or inducing vessel growth from existing capillaries. Recent research suggests that the paracrine effects of MSCs could be mediated by extracellular vesicles including exosomes. Exosomal microRNAs (miRNAs) released by MSCs are promising therapeutic hotspot target for MI. This could be attributed to the role of miRNA in cardiac biology, including cardiac regeneration, stem cell differentiation, apoptosis, neovascularization, cardiac contractility and cardiac remodeling. Furthermore, gene‐modified MSCs could be a recent promising therapy for MI to enhance the paracrine effects of MSCs, including better homing and effective cell targeted tissue regeneration. Although MSC therapy has achieved considerable attention and progress, there are critical challenges that remains to be overcome to achieve the most effective successful cell‐based therapy in MI.     

Repair of Ischemic Heart Disease with Novel Bone Marrow-Derived Multipotent Stem Cells

Congestive heart failure is a growing, worldwide epidemic. The major causes of heart failure are related to irreversible damage resulting from myocardial infarction (heart attack). The long-standing axiom has been that the myocardium has a limited capacity for self-repair or regeneration; and the irreversible loss of cardiac muscle and accompanying contraction and fibrosis of myocardial scar tissue, sets into play a series of events, namely, progressive ventricular remodeling of nonischemic myocardium that ultimately leads to progressive heart failure. The loss of cardiomyocyte survival cues is associated with diverse pathways for heart failure, underscoring the importance of maintaining the number of viable cardiomyocytes during heart failure progression. Currently, no medication or procedure used clinically has shown efficacy in replacing the myocardial scar with functioning contractile tissue. Therefore, given the major morbidity and mortality associated with myocardial infarction and heart failure, new approaches have been sought to address the principal pathophysiologic deficits responsible for these conditions, resulting from the loss of cardiomyocytes and viable blood vessels. Recently, the identification of stem cells from bone marrow capable of contributing to tissue regeneration has ignited significant interest in the possibility that cell therapy could be employed therapeutically for the repair of damaged myocardium. In this review, we will discuss the currently available bone marrow-derived stem progenitor cells for myocardial repair and focus on the advantages of using recently identified novel bone marrow-derived multipotent stem cells (BMSC)     

心肌球源性细胞在心力衰竭中的应用

冠状动脉或其分支阻塞而引起心肌缺血可导致急性心肌梗死,梗死区域周围形成瘢痕组织后心脏收缩功能下降,心室发生病理性重塑,最终出现充血性心力衰竭。心肌球源性细胞（CDC）是来自心肌的干细胞,在体外可以分化为心肌细胞和血管内皮细胞。在体内可以触发自身心肌细胞增殖和通过旁分泌募集祖细胞。旁分泌介质不但拥有干细胞的作用,且没有细胞移植相关的并发症。临床实验证明了CDC可以促进心肌梗死后的心脏功能的恢复。长期疗效还有待大规模临床试验的验证。     

The roles of mesenchymal stem cells (MSCs) therapy in ischemic heart diseases

Growing cell-based myocardial therapies which could lead to successful myocardial repair attracts medical interest. Even more intriguing is the observation that MSCs appears to be a more potent material among kinds of stem cells for the transplantation, the mechanism for this benefit remains unclear. However, the therapeutic contribution of MSCs to myocardial repair can be caused by multiple factors including: direct differentiation into cardiac tissue including cardiomyocytes, smooth muscle cell, and vascular endothelial cells; secreting a variety of cytokines and growth factors that have paracrine activities; spontaneous cell fusion; and stimulating endogenous repair. In addition, MSCs possess local immunosuppressive properties, and MSCs mobilization is widely used clinically for transplantation. We will discusses the potential mechanisms of MSCs repair for ischemic heart diseases.     

Prometheus's heart: what lies beneath

A heart attack kills off many cells in the heart. Parts of the heart become thin and fail to contract properly following the replacement of lost cells by scar tissue. However, the notion that the same adult cardiomyocytes beat throughout the lifespan of the organ and organism, without the need for a minimum turnover, gives way to a fascinating investigations. Since the late 1800s, scientists and cardiologists wanted to demonstrate that the cardiomyocytes cannot be generated after the perinatal period in human beings. This curiosity has been passed down in subsequent years and has motivated more and more accurate studies in an attempt to exclude the presence of renewed cardiomyocytes in the tissue bordering the ischaemic area, and then to confirm the dogma of the heart as terminally differentiated organ. Conversely, peri-lesional mitosis of cardiomyocytes were discovered initially by light microscopy and subsequently confirmed by more sophisticated technologies. Controversial evidence of mechanisms underlying myocardial regeneration has shown that adult cardiomyocytes are renewed through a slow turnover, even in the absence of damage. This turnover is ensured by the activation of rare clusters of progenitor cells interspersed among the cardiac cells functionally mature. Cardiac progenitor cells continuously interact with each other, with the cells circulating in the vessels of the coronary microcirculation and myocardial cells in auto-/paracrine manner. Much remains to be understood; however, the limited functional recovery in human beings after myocardial injury clearly demonstrates weak regenerative potential of cardiomyocytes and encourages the development of new approaches to stimulate this process.     

Is adult cardiac myocyte still able to proliferate?

Adult cardiac myocytes do not divide anymore. Mechanically overloaded hearts undergo hypertrophy and then fail. Cardiac hypertrophy is mainly caused by myocyte hypertrophy without myocyte proliferation, except during end-stage heart failure. By contrast, non muscular myocardial cells, such as the endothelial cells of the vessels, not only hypertrophy but are also able to proliferate. Recent works have suggested that these new cells are likely to be progenitor cells originating from bone marrow or vascular endothelium. These cells may form chimeras in the donor heart following heart transplantation. It is possible to mimic such an adaptative process by injecting progenitor cells either within the myocardium, or through the coronary circulation. Two type of cells have been utilised so far, namely bone marrow cells and myoblasts (or satellite cells) from skeletal muscles. The first clinical applications after myocardial infarction have been recently reported and showed the safety of the procedure and the possibility of improving myocardial function.     

Direct delivery of syngeneic and allogeneic large-scale expanded multipotent adult progenitor cells improves cardiac function after myocardial infarct

BACKGROUND: Multipotent adult progenitor cells (MAPC) comprise interesting candidates for myocardial regeneration because of a broad differentiation ability and immune privilege. We aimed to compare the improvement of cardiac function by syngeneic and allogeneic MAPC produced on a large scale using a platform optimized from MAPC research protocols. METHODS: Myocardial infarction was induced in Lewis rats by direct left anterior descending ligation followed immediately by direct injection into the infarct border zone of either Sprague-Dawley or Lewis MAPC from large-scale expansions. Echocardiography was performed to evaluate improvement in cardiac function, and immunohistochemistry was performed to identify MAPC within the infarct zone. RESULTS: Significant increases were observed in functional performance in animals transplanted with expanded MAPC compared with saline controls, with no significant differences between the syngeneic and allogeneic groups. Immunostaining demonstrated significant engraftment of expanded MAPC at 1 day after acute myocardial infarction, with <10% of either syngeneic or allogeneic cells remaining at 6 weeks. At this point there was no evidence of myocardial regeneration. However, a significant increase in vascular density within the infarct zone in MAPC-transplanted animals was observed, and MAPC were found to produce high levels of VEGF in culture. DISCUSSION: These findings support a model in which delivery of expanded MAPC following acute myocardial infarction results in improvement in cardiac function because of paracrine effects resulting in vascular density increases, as well as potentially other trophic effects, supporting newly injured cardiac myocytes. Thus transplantation with MAPC may represent a promising therapeutic strategy with application in the stimulation of neovascularization in ischemic heart 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.     

Stem cell-derived exosomes-an emerging tool for myocardial regeneration

Cardiovascular diseases(CVDs) continue to represent the number one cause of death and disability in industrialized countries. The most severe form of CVD is acute myocardial infarction(AMI), a devastating disease associated with high mortality and disability. In a substantial proportion of patients who survive AMI, loss of functional cardiomyocytes as a result of ischaemic injury leads to ventricular failure, resulting in significant alteration to quality of life and increased mortality. Therefore, many attempts have been made in recent years to identify new tools for the regeneration of functional cardiomyocytes. Regenerative therapy currently represents the ultimate goal for restoring the function of damaged myocardium by stimulating the regeneration of the infarcted tissue or by providing cellsthat can generate new myocardial tissue to replace the damaged tissue. Stem cells(SCs) have been proposed as a viable therapy option in these cases. However, despite the great enthusiasm at the beginning of the SC era, justified by promising initial results, this therapy has failed to demonstrate a significant benefit in large clinical trials. One interesting finding of SC studies is that exosomes released by mesenchymal SCs(MSCs) are able to enhance the viability of cardiomyocytes after ischaemia/reperfusion injury, suggesting that the beneficial effects of MSCs in the recovery of functional myocardium could be related to their capacity to secrete exosomes. Ten years ago, it was discovered that exosomes have the unique property of transferring miRNA between cells, acting as miRNA nanocarriers. Therefore, exosomebased therapy has recently been proposed as an emerging tool for cardiac regeneration as an alternative to SC therapy in the post-infarction period. This review aims to discuss the emerging role of exosomes in developing innovative therapies for cardiac regeneration as well as their potential role as candidate biomarkers or for developing new diagnostic tools.     

Cyclosporin-A potently induces highly cardiogenic progenitors from embryonic stem cells

Though cardiac progenitor cells should be a suitable material for cardiac regeneration, efficient ways to induce cardiac progenitors from embryonic stem (ES) cells have not been established. Extending our systematic cardiovascular differentiation method of ES cells, here we show efficient and specific expansion of cardiomyocytes and highly cardiogenic progenitors from ES cells. An immunosuppressant, cyclosporin-A (CSA), showed a novel effect specifically acting on mesoderm cells to drastically increase cardiac progenitors as well as cardiomyocytes by 10-20 times. Approximately 200 cardiomyocytes could be induced from one mouse ES cell using this method. Expanded progenitors successfully integrated into scar tissue of infracted heart as cardiomyocytes after cell transplantation to rat myocardial infarction model. CSA elicited specific induction of cardiac lineage from mesoderm in a novel mesoderm-specific, NFAT independent fashion. This simple but efficient differentiation technology would be extended to induce pluripotent stem (iPS) cells and broadly contribute to cardiac regeneration.