Embryonic stem cells and cardiomyocyte differentiation: phenotypic and molecular analyses

Embryonic stem (ES) cell lines, derived from the inner cell mass (ICM) of blastocyst-stage embryos, are pluripotent and have a virtually unlimited capacity for self-renewal and differentiation into all cell types of an embryoproper. Both human and mouse ES cell lines are the subject of intensive investigation for potential applications in developmental biology and medicine. ES cells from both sources differentiate in vitro into cells of ecto-, endoand meso-dermal lineages, and robust cardiomyogenic differentiation is readily observed in spontaneously differentiating ES cells when cultured under appropriate conditions. Molecular, cellular and physiologic analyses demonstrate that ES cell-derived cardiomyocytes are functionally viable and that these cell derivatives exhibit characteristics typical of heart cells in early stages of cardiac development. Because terminal heart failure is characterized by a significant loss of cardiomyocytes, the use of human ES cell-derived progeny represents one possible source for cell transplantation therapies. With these issues in mind, this review will focus on the differentiation of pluripotent embryonic stem cells into cardiomyocytes as a developmental model, and the possible use of ES cell-derived cardiomyocytes as source of donor cells.     

The secretome from embryonic stem cell cardiomyogenesis: Same signals,different cellular feedbacks

Ischemic heart diseases are a global health problem that requires the search for alternative therapies to the current treatments. Thus, an understanding of how cardiomyogenic signals can affect cellular behavior would allow us to create strategies to improve the cell recovery in damaged tissues. In this study, we aimed to evaluate the effects of the conditioned medium (CM), collected at different time points during in vitro cardiomyogenesis of human embryonic stem cells (hESCs), to direct cell behavior. We assayed different cell types to demonstrate noncytotoxic effects from the collected CM and that the CM obtained at initial time points of cardiomyogenic differentiation could promote the cell proliferation. Otherwise, the secretome derived from cardiac committed cells during cardiomyogenesis was unable to improve angiogenesis or migration in endothelial cells, and ineffective to stimulate the differentiation of cardioblasts or increase the differentiation efficiency of hESC. Therefore, we demonstrated that the effectiveness of the CM response varies depending on the cell type and the differentiation step of hESC‐derived cardiomyocytes.     

Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells

Bone marrow mesenchymal stem cells have been shown to transdifferentiate into cardiomyocytes after 5-azacytidine treatment or co-culturing with rodent cardiomyocytes. We investigate if adult human bone marrow stem cells can be differentiated ex vivo into cardiomyocyte-like cells (CLCs) independent of cytotoxic agents or co-culturing technique. Sternal bone marrow was collected from 16 patients undergoing coronary artery bypass surgery. Mesenchymal stem cells were differentiated in a cardiomyogenic differentiation medium containing insulin, dexamethasone, and ascorbic acid. Differentiation towards CLCs was determined by induced expression of cardiomyocyte-specific proteins. Differentiated CLCs expressed multiple structural and contractile proteins that are associated with cardiomyocytes. Thin filament associated myofibrillar proteins were detected early in the cells, with cardiac troponin I, sarcomeric tropomyosin, and cardiac titin among the first expressed. Some CLCs were found to develop into a nascent cardiomyocyte phenotype with cross-striated myofibrils characterized by alpha-actinin-positive Z bands after 4-5 passages in differentiated culture. These lineage-defined CLCs may be potentially useful for repairing damaged myocardium.     

Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro

It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.     

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.     

胚胎干细胞向心肌细胞分化过程中能量代谢变化的研究

目的：定向诱导人胚胎干细胞分化为心肌细胞，对分化过程中胚胎干细胞、心肌祖细胞和心肌细胞糖酵解能力和线粒体氧化磷酸化能力进行实时定量检测，探索分化过程中细胞能量代谢表型的转换机制.方法：用GSK3抑制剂CHIR99021和Wnt信号通路小分子抑制剂IWP2的方法定向分化人胚胎干细胞为心肌祖细胞和心肌细胞；细胞免疫荧光检测人胚胎干细胞标志物，流式细胞术检测人心肌祖细胞和心肌细胞标志物；应用细胞外流量分析（Extracellular Flux Analysis）方法检测人胚胎干细胞、心肌祖细胞和心肌细胞能量代谢情况.结果：人胚胎干细胞干性保持稳定，均表达Nanog、OCT4、SOX2细胞标志物；在向心肌分化过程中，第7d心肌祖细胞标志物Isl1表达99%以上，分化第15d心肌细胞标志物cTnT表达83%以上；人胚胎干细胞糖酵解代谢能力最强，心肌细胞线粒体功能最强，心肌祖细胞处于两种代谢方式的过度阶段.结论：在人胚胎干细胞向心肌细胞分化的过程中，细胞糖酵解能力逐渐减弱，线粒体氧化磷酸化能力逐渐增强，细胞的能量代谢类型发生转变.     

干细胞与心肌细胞替代治疗

胚胎干细胞及来源于骨髓、骨骼肌、血管、肝脏、皮肤、脂肪等组织器官的成体干细胞均有多向分化潜能。胚胎干细胞可分化为3个胚层的所有组织细胞。成体干细胞具有可塑性和转分化的潜能。在一定条件下,这些干细胞可被诱导分化为心肌细胞。成年心脏可能存在心肌干细胞,具有增殖和分化为包括跳动性心肌细胞的多种细胞的潜能。因此,干细胞可用于心肌细胞替代治疗,以替代死亡的心肌细胞,改善心脏功能,防治心肌梗塞后心衰、减少心肌重构等症状。本文对干细胞治疗心肌梗塞有关进展及问题作一综述。     

Heterogeneic nature of adult cardiac side population cells

Side population cells have been found in various types of adult tissue including heart and are presumed to be tissue-specific stem/progenitor cells. In the present study, we confirmed the presence of cardiac side population (cSP) cells, which showed both the Hoechst 33342 efflux ability and ABCG2 expression, in adult murine heart. Flow cytometric analysis showed that more than half of cSP cells expressed the endothelial marker VE-cadherin or the smooth muscle markers, α-smooth muscle actin and desmin. In addition, immunohistochemical analysis demonstrated that ABCG2⁺ cells were mainly localized within vascular walls. Quantitative RT-PCR analysis demonstrated that VE-cadherin⁻ cSP cells progressively expressed Nkx2.5 and cardiac troponin T with time in culture. VE-cadherin⁻ cSP cells also expressed mesodermal-mesenchymal-associated markers and differentiated into osteocytes and adipocytes. These results highlight the heterogeneic nature of cSP cells, consisting of vascular endothelial cells, smooth muscle cells, and mesenchymal stem/progenitor cells including potential cardiomyogenic cells.     

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.     

视黄酸通路的启示——从心脏发育到心肌谱系定向分化

多潜能干细胞具有无限增殖的能力,并能够分化为心肌细胞,因此在心脏再生方面拥有巨大潜力.胚胎发育过程为干细胞定向分化提供了重要线索,在过去的几年中,通过操控心脏发育关键通路,在心肌定向分化方面取得了重要进展,但是现有的分化方法仍不能稳定地诱导心肌细胞,表明现有的通路不能有效解决这些问题.视黄酸(RA)通路在心脏发育过程中发挥重要作用,RA缺失会导致心房变小、心室小梁减少、心肌壁增厚且细胞间连接松散.在体外心肌定向分化过程中,RA多用于促进多潜能干细胞向心房分化.但从RA通路基因敲除小鼠的表型来看,除了调控心肌亚型分化,RA在多个发育阶段发挥重要作用.深入解析RA在心肌分化各阶段的作用机制,将有助于获得高质量的心肌细胞.同时,研究RA在心内膜和心外膜分化中的作用机制也有助于解释RA通路敲除小鼠的心脏异常.总之,从RA在胚胎发育中的作用来看,需要更多的体外研究来揭示RA在心肌谱系分化中的作用.本文综述了RA通路在心脏发育的心肌分化过程中的作用,并探讨了需要解决的问题.     

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.     

'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells

The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.     

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.     

Graphene enhances the cardiomyogenic differentiation of human embryonic stem cells

Graphene has drawn attention as a substrate for stem cell culture and has been reported to stimulate the differentiation of multipotent adult stem cells. Here, we report that graphene enhances the cardiomyogenic differentiation of human embryonic stem cells (hESCs) at least in part, due to nanoroughness of graphene. Large-area graphene on glass coverslips was prepared via the chemical vapor deposition method. The coating of the graphene with vitronectin (VN) was required to ensure high viability of the hESCs cultured on the graphene. hESCs were cultured on either VN-coated glass (glass group) or VN-coated graphene (graphene group) for 21 days. The cells were also cultured on glass coated with Matrigel (Matrigel group), which is a substrate used in conventional, directed cardiomyogenic differentiation systems. The culture of hESCs on graphene promoted the expression of genes involved in the stepwise differentiation into mesodermal and endodermal lineage cells and subsequently cardiomyogenic differentiation compared with the culture on glass or Matrigel. In addition, the culture on graphene enhanced the gene expression of cardiac-specific extracellular matrices. Culture on graphene may provide a new platform for the development of stem cell therapies for ischemic heart diseases by enhancing the cardiomyogenic differentiation of hESCs.     

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.     

Expression of cardiac function genes in adult stem cells is increased by treatment with nitric oxide agents

Mesenchymal stem cells (MSCs) have received special attention for cardiomyoplasty because several studies have shown that they differentiate into cardiomyocytes both in vitro and in vivo. Nitric oxide (NO) is a free radical signaling molecule that regulates several differentiation processes including cardiomyogenesis. Here, we report an investigation of the effects of two NO agents (SNAP and DEA/NO), able to activate both cGMP-dependent and -independent pathways, on the cardiomyogenic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs). The cells were isolated, cultured and treated with NO agents. Cardiac- and muscle-specific gene expression was analyzed by indirect immunofluorescence, flow cytometry, RT-PCR and real-time PCR. We found that untreated (control) ADSCs and BM-MSCs expressed some muscle markers and NO-derived intermediates induce an increased expression of some cardiac function genes in BM-MSCs and ADSCs. Moreover, NO agents considerably increased the pro-angiogenic potential mostly of BM-MSCs as determined by VEGF mRNA levels.     

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy.