Quantitatively analyzing the protective effect of mesenchymal stem cells on cardiomyocytes in single-cell biochips

To understand how stem cells benefit native cardiomyocytes is crucial for cell-based therapies to rescue cardiomyocytes (CMCs) damaged during heart infarction and other cardiac diseases. However, the current conclusions on the protective effect of mesenchymal stem cells (MSCs) were obtained by analyzing the overall amount of protein and factor secretion in a conventional co-culture system. These results neglected the heterogeneity of MSC population and failed to determine the importance of cellular contact to the protective effects. To address these issues, we have constructed two biochips by microfabrication methods and laser-guided cell micropatterning technique. Using the biochips, the protective effect of MSCs on CMCs can be quantitatively analyzed at single-cell level with defined cellular contact. The role of cellular contact on protective effect can be clarified according to our statistical results.     

Network architecture of signaling from uncoupled helicase-polymerase to cell cycle checkpoints and trans-lesion DNA synthesis

Potentially, adult stem cell-based therapy provides a new therapeutic option for myocardial regeneration. However, to date, with regard to the benefits seen, the mechanisms involved in stem cell-based therapy are not well understood. Suggested pathways proposed so far include fusion of stem cells with cardiomyocytes, transdifferentiation into cardiac and vascular cells and secretion of paracrine factors. In a recent study, our group examined the fate of human adipose tissue-derived stem cells (hASCs) fused with rat cardiomyocytes after treatment with fusion-inducing hemagglutinating virus of Japan (HVJ). In this study, we demonstrated that cells of fused hASC cardiomyocytes display a cardiomyocyte phenotype and spontaneous rhythmic contraction and generate an action potential in vitro. As part of the work underlying this paper, we co-cultured rat neonatal cardiomyocytes with hASCs or pig bone marrow-derived mesenchymal stem cells (MSCs), where ASCs or MSCs had previously been transduced with a lentivirus encoding eGFP. Our data evidence early cardiac contractile proteins, such as Titin and MF20, identified in eGFP-positive cells, suggesting a cardiomyogenic phenotype. Recent work by others has shown that the myogenic conversion increased when BMSCs were cultured with apoptotic cells. In this Extra View article, we review the current understanding of stem cell-derived factors, fusion/partial fusion and the manner in which the exchange of cellular contents between stem cells and cardiomyocytes might contribute to the reprogramming of fully differentiated cardiomyocytes based on recently published literature.     

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.     

Cardiac differentiation of human pluripotent stem cells

Due to the extremely limited proliferative capacity of adult cardiomyocytes, human embryonic (pluripotent) stem cell derived cardiomyocytes (hESC-CMs) are currently almost the only reliable source of human heart cells which are suited to large-scale production. These cells have the potential for wide-scale application in drug discovery, heart disease research and cell-based heart repair. Embryonic atrial-, ventricular- and nodal-like cardiomyocytes can be obtained from differentiated human embryonic stem cells (hESCs). In recent years, several highly efficient cardiac differentiation protocols have been developed. Significant progress has also been made on understanding cardiac subtype specification, which is the key to reducing the heterogeneity of hESC-CMs, a major obstacle to the utilization of these cells in medical research and future cell-based replacement therapies. Herein we review recent progress in cardiac differentiation of hESCs and cardiac subtype specification, and discuss potential applications in drug screening and cell-based heart regeneration.     

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.     

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.     

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.     

Sublethal Caspase Activation Promotes Generation of Cardiomyocytes from Embryonic Stem Cells

Generation of new cardiomyocytes is critical for cardiac repair following myocardial injury, but which kind of stimuli is most important for cardiomyocyte regeneration is still unclear. Here we explore if apoptotic stimuli, manifested through caspase activation, influences cardiac progenitor up-regulation and cardiomyocyte differentiation. Using mouse embryonic stem cells as a cellular model, we show that sublethal activation of caspases increases the yield of cardiomyocytes while concurrently promoting the proliferation and differentiation of c-Kit⁺/α-actinin^low cardiac progenitor cells. A broad-spectrum caspase inhibitor blocked these effects. In addition, the caspase inhibitor reversed the mRNA expression of genes expressed in cardiomyocytes and their precursors. Our study demonstrates that sublethal caspase-activation has an important role in cardiomyocyte differentiation and may have significant implications for promoting cardiac regeneration after myocardial injury involving exogenous or endogenous cell sources.     

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.     

Cardiac potential of stem cells from whole human umbilical cord tissue

We investigated the role of stem cells from human umbilical cord tissue in cardiomyocyte regeneration. The umbilical cord stem cells were initially characterized and differentiated in a myocardial differentiation medium containing 5‐azacytidine for 24 h. Differentiation into cardiomyocytes was determined by expression of cardiac specific markers, like cardiac α‐actin, connexin43, myosin, Troponin T, and ultrastructural analysis. In vivo, the transplanted umbilical cord stem cells were sprouting from local injection and differentiated into cardiomyocyte‐like cells in a rat myocardial infarction model. Echocardiography revealed increasing left ventricular function after umbilical cord stem cell transplantation. These results demonstrate that umbilical cord stem cells can differentiate into cardiomyocyte‐like cells both in vitro and in vivo. Therefore, human umbilical cord might represent a source of stem cells useful for cellular therapy and myocardial tissue engineering. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon. J. Cell. Biochem. 107: 926–932, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

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.     

Telocytes in exercise‐induced cardiac growth

Exercise can induce physiological cardiac growth, which is featured by enlarged cardiomyocyte cell size and formation of new cardiomyocytes. Telocytes (TCs) are a recently identified distinct interstitial cell type, existing in many tissues and organs including heart. TCs have been shown to form a tandem with cardiac stem/progenitor cells in cardiac stem cell niches, participating in cardiac regeneration and repair. Although exercise‐induced cardiac growth has been confirmed as an important way to promote cardiac regeneration and repair, the response of cardiac TCs to exercise is still unclear. In this study, 4 weeks of swimming training was used to induce robust healthy cardiac growth. Exercise can induce an increase in cardiomyocyte cell size and formation of new cardiomyocytes as determined by Wheat Germ Lectin and EdU staining respectively. TCs were identified by three immunofluorescence stainings including double labelling for CD34/vimentin, CD34/platelet‐derived growth factor (PDGF) receptor‐α and CD34/PDGF receptor‐β. We found that cardiac TCs were significantly increased in exercised heart, suggesting that TCs might help control the activity of cardiac stem/progenitor cells, cardiomyocytes or endothelial cells. Adding cardiac TCs might help promote cardiac regeneration and renewal.     

Myocardial regeneration potential of adipose tissue-derived stem cells

Various tissue resident stem cells are receiving attention from basic scientists and clinicians as they hold promise for myocardial regeneration. For practical reasons, adipose tissue-derived stem cells (ASCs) are attractive cells for clinical application in repairing damaged myocardium based on the following advantages: abundant adipose tissue in most patients and easy accessibility with minimally invasive lipoaspiration procedure. Several recent studies have demonstrated that both cultured and freshly isolated ASCs could improve cardiac function in animal model of myocardial infarction. The mechanisms underlying the beneficial effect of ASCs on myocardial regeneration are not fully understood. Growing evidence indicates that transplantation of ASCs improve cardiac function via the differentiation into cardiomyocytes and vascular cells, and through paracrine pathways. Paracrine factors secreted by injected ASCs enhance angiogenesis, reduce cell apoptosis rates, and promote neuron sprouts in damaged myocardium. In addition, Injection of ASCs increases electrical stability of the injured heart. Furthermore, there are no reported cases of arrhythmia or tumorigenesis in any studies regarding myocardial regeneration with ASCs. This review summarizes the characteristics of both cultured and freshly isolated stem cells obtained from adipose tissue, their myocardial regeneration potential, and the underlying mechanisms for beneficial effect on cardiac function, and safety issues.     

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.     

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.     

Cardiac stem/progenitor cells, secreted proteins, and proteomics

Stem cell-based therapy is emerging as a novel approach for myocardial repair over conventional cardiovascular therapies. In addition to embryonic stem cells and adult stem cells from noncardiac sources, there is a small population of resident stem cells in the heart from which new cardiac cells (myocytes, vascular endothelial cells and smooth muscle cells) can be derived and used for cardiac repair in case of heart injury. It has been proposed that the clinical benefit of stem cells may arise from secreted proteins that mediate regeneration in a paracrine/autocrine manner. To be able to track the regulatory pathway on a molecular basis, utilization of proteomics in stem cell research is essential. Proteomics offers a tool that can address questions regarding stem cell response to disease/injury.     

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.