Cardiac regeneration: epicardial mediated repair

The hearts of lower vertebrates such as fish and salamanders display scarless regeneration following injury, although this feature is lost in adult mammals. The remarkable capacity of the neonatal mammalian heart to regenerate suggests that the underlying machinery required for the regenerative process is evolutionarily retained. Recent studies highlight the epicardial covering of the heart as an important source of the signalling factors required for the repair process. The developing epicardium is also a major source of cardiac fibroblasts, smooth muscle, endothelial cells and stem cells. Here, we examine animal models that are capable of scarless regeneration, the role of the epicardium as a source of cells, signalling mechanisms implicated in the regenerative process and how these mechanisms influence cardiomyocyte proliferation. We also discuss recent advances in cardiac stem cell research and potential therapeutic targets arising from these studies.     

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.     

Human cardiomyocyte progenitor cells: a short history of nearly everything

The high occurrence of cardiac disease in the Western world has driven clinicians and cardiovascular biologists to look for alternative strategies to treat patients. A challenging approach is the use of stem cells to repair the heart, in itself an inspiring thought. In the past 10 years, stem cells from different sources have been under intense investigation and, as a result, a multitude of studies have been published on the identification, isolation, and characterization, of cardiovascular progenitor cells and repair in different animal models. However, relatively few cardiovascular progenitor populations have been identified in human hearts, including, but not limited to, cardiosphere-derived cells, cKit+ human cardiac stem cells , Isl1+ cardiovascular progenitors, and, in our lab, cardiomyocyte progenitor cells (CMPCs). Here, we aim to provide a comprehensive summary of the past findings and present challenges for future therapeutic potential of CMPCs.     

Telocytes in human epicardium

The existence of the epicardial telocytes was previously documented by immunohistochemistry (IHC) or immunofluorescence. We have also demonstrated recently that telocytes are present in mice epicardium, within the cardiac stem‐cell niches, and, possibly, they are acting as nurse cells for the cardiomyocyte progenitors. The rationale of this study was to show that telocytes do exist in human (sub)epicardium, too. Human autopsy hearts from 10 adults and 15 foetuses were used for conventional IHC for c‐kit/CD117, CD34, vimentin, S‐100, τ, Neurokinin 1, as well as using laser confocal microscopy. Tissue samples obtained by surgical biopsies from 10 adults were studied by digital transmission electron microscopy (TEM). Double immunolabelling for c‐kit/CD34 and, for c‐kit/vimentin suggests that in human beings, epicardial telocytes share similar immunophenotype features with myocardial telocytes. The presence of the telocytes in human epicardium is shown by TEM. Epicardial telocytes, like any of the telocytes are defined by telopodes, their cell prolongations, which are very long (several tens of μm), very thin (0.1–0.2 μm, below the resolving power of light microscopy) and with moniliform configuration. The interconnected epicardial telocytes create a 3D cellular network, connected with the 3D network of myocardial telocytes. TEM documented that telocytes release shed microvesicles or exocytotic multivesicular bodies in the intercellular space. The human epicardial telocytes have similar phenotype (TEM and IHC) with telocytes located among human working cardiomyocyte. It remains to be established the role(s) of telocytes in cardiac renewing/repair/regeneration processes, and also the pathological aspects induced by their ‘functional inhibition’, or by their variation in number. We consider telocytes as a real candidate for future developments of autologous cell‐based therapy in heart 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.     

Limbal niche cells are a potent resource of adult mesenchymal progenitors

Limbal niche cells located in the limbal Palisades of Vogt are mesenchymal stem cells that reside next to limbal basal epithelial cells. Limbal niche cells are progenitors that express embryonic stem cell markers such as Nanog, Nestin, Oct4, Rex1, Sox2 and SSEA4, mesenchymal cell markers such as CD73, CD90 and CD105, and angiogenesis markers such as Flk‐1, CD31, CD34, VWF, PDGFRβ and α‐SMA, but negative for CD45. In addition, the stemness of limbal niche cells can be maintained during their cell culture in a three‐dimension environment. Furthermore, expanded limbal niche cells have the capability to undergo adipogenesis, chondrogenesis, osteogenesis and endogenesis in vitro, indicating that they are in fact a valuable resource of adult progenitors. Furthermore studies on how the limbal niche cells regulate the aforementioned stemness and corneal fate decision are warranted, as those investigations will shed new light on how mesenchymal progenitors reverse limbal stem cell deficiency and lead to new methods for limbal niche cell treatment.     

Comparison of various kinds of bone marrow stem cells for the repair of infarcted myocardium: single clonally purified non-hematopoietic mesenchymal stem cells serve as a superior source

A variety of adult stem cells have been used to transplant into the infarcted (MI) heart, however, comparative studies are lacking to show more suitable source of cells for transplantation. We have identified a single non-hematopoietic mesenchymal stem cell subpopulation (snMSCs) isolated from human bone marrow and clonally purified, that over 99% of them expressed MSC marker proteins and cardiomyocyte marker proteins when induction in vitro. We also compared the effects of the snMSCs with unpurified MSC (uMSCs), mononuclear cells (BMMNCs), or peripheral blood mononuclear cells (PBMNCs) on myocardial repair after induction of MI in rats. Ninety days later, we observed a better cardiac function assessed by ejection fraction, fraction of shortening and lung wet/dry weight ratios, less remodeling of left ventricle (LV), lower collagen density in the LV, and more vessels in the ischemic wall in the snMSCs transplantation group than in other cell-transplanted groups. Furthermore, the transplanted cells expressing cardiomyocyte specific proteins or vascular endothelial cell marker proteins were more in the snMSCs group than in other ones. We conclude that transplantation with single clonally purified MSCs seems to be more beneficial to the cardiac repair than with other stem cells after MI.     

Foetal and adult cardiomyocyte progenitor cells have different developmental potential

In the past years, cardiovascular progenitor cells have been isolated from the human heart and characterized. Up to date, no studies have been reported in which the developmental potential of foetal and adult cardiovascular progenitors was tested simultaneously. However, intrinsic differences will likely affect interpretations regarding progenitor cell potential and application for regenerative medicine. Here we report a direct comparison between human foetal and adult heart‐derived cardiomyocyte progenitor cells (CMPCs). We show that foetal and adult CMPCs have distinct preferences to differentiate into mesodermal lineages. Under pro‐angiogenic conditions, foetal CMPCs form more endothelial but less smooth muscle cells than adult CMPCs. Foetal CMPCs can also develop towards adipocytes, whereas neither foetal nor adult CMPCs show significant osteogenic differentiation. Interestingly, although both cell types differentiate into heart muscle cells, adult CMPCs give rise to electrophysiologically more mature cardiomyocytes than foetal CMPCs. Taken together, foetal CMPCs are suitable for molecular cell biology and developmental studies. The potential of adult CMPCs to form mature cardiomyocytes and smooth muscle cells may be essential for cardiac repair after transplantation into the injured heart.     

Regulation of cardiomyocyte proliferation during development and regeneration

The regulation of cardiomyocyte proliferation is important for heart development and regeneration. The proliferation patterns of cardiomyocytes are closely related to heart morphogenesis, size, and functions. The proliferation levels are high during early embryogenesis; however, mammalian cardiomyocytes exit the cell cycle irreversibly soon after birth. The cell cycle exit inhibits cardiac regeneration in mammals. On the other hand, cardiomyocytes of adult zebrafish and probably newts can proliferate after cardiac injury, and the hearts can be regenerated. Therefore, the ability to reproliferate determines regenerative ability. As in other cells, the relationship between proliferation and differentiation is very interesting, and is closely related to cardiac development, regeneration and homeostasis. In this review, these topics are discussed.     

Induced Pluripotent Stem Cell-Derived Cardiac Progenitors Differentiate to Cardiomyocytes and Form Biosynthetic Tissues

The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors’ ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and drug development studies.     

Critical role of Lin28‐TNFR2 signalling in cardiac stem cell activation and differentiation

Tumour necrotic factor receptor‐2 (TNFR2) has been to be cardiac‐protective and is expressed in cardiac progenitor cells. Our goal is to define the mechanism for TNFR2‐mediated cardiac stem cell activation and differentiation. By employing a protocol of in vitro cardiac stem cell (CSC) differentiation from human inducible pluripotent stem cell (hiPSC), we show that expression of TNFR2 precedes expression of CSC markers followed by expression of mature cardiomyocyte proteins. Activation of TNFR2 by a specific agonist promotes whereas inhibition of TNFR2 by neutralizing antibody diminishes hiPSC‐based CSC differentiation. Interestingly, pluripotent cell factor RNA‐binding protein Lin28 enhances TNFR2 protein expression in early CSC activation by directly binding to a conserved Lin28‐motif within the 3'UTR of Tnfr2 mRNA. Furthermore, inhibition of Lin28 blunts TNFR2 expression and TNFR2‐dependent CSC activation and differentiation. Our study demonstrates a critical role of Lin28‐TNFR2 axis in CSC activation and survival, providing a novel strategy to enhance stem cell‐based therapy for the ischaemic heart diseases.     

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.     

基因谱系示踪技术揭示小鼠Tbx18+心脏祖细胞向心系细胞分化的潜能

心脏祖细胞(cardiac progenitor cells,CPCs)的研究对阐明先天性心脏病的机制及治疗心血管疾病具有重要意义.哺乳动物的心脏组织由多种不同CPCs分化形成.转录因子Tbx18在发育中的心外膜中表达,对心脏的发育形成起重要的调节作用.为了在组织及活体细胞水平检测和阐明Tbx18+CPC的分化潜能,应用Cre-LoxP系统建立Tbx18+CPCs基因命运谱系示踪模型:Tbx18-Cre/Rosa26R-EYFP和Tbx18-Cre/Rosa26R-LacZ双杂合基因敲入小鼠.该双杂合基因敲入小鼠通过Cre的表达能有效地示踪Tbx18+细胞在胚胎和成年小鼠中的分化命运.Tbx18-Cre/Rosa26R-EYFP双杂合小鼠心脏能非常容易地利用流式细胞分选系统(FACS)分离出YFP+细胞,也可在倒置共聚焦显微镜下观察.应用X-gal染色分析其表达模式,揭示Tbx18命运谱系参与心房肌、室间隔、心室肌、冠状动脉、瓣膜等的形成.应用免疫荧光技术初步揭示Tbx18+CPCs向心脏肌钙蛋白T(cTNT)阳性心肌细胞和平滑肌肌球蛋白重链11(MYH11)阳性血管平滑肌细胞分化的潜能.心脏是一个由多种肌肉和非肌肉组织细胞构成的复杂器官.推测Tbx18可能在心脏祖细胞向肌源性细胞分化的信号通路中起重要调节作用.在上述研究中应用基因谱系示踪技术,验证Tbx18可作为一类CPCs的标志,为更深入揭示心脏祖细胞向心系细胞的分化潜能打下基础.     

Reaching out: junctions between cardiac telocytes and cardiac stem cells in culture

Telocytes (TCs) were previously shown by our group to form a tandem with stem/progenitor cells in cardiac stem cell (CSC) niches, fulfilling various roles in cardiac renewal. Among these, the ability to ‘nurse’ CSCs in situ, both through direct physical contact (junctions) as well as at a distance, by paracrine signalling or through extracellular vesicles containing mRNA. We employed electron microscopy to identify junctions (such as gap or adherens junctions) in a co‐culture of cardiac TCs and CSCs. Gap junctions were observed between TCs, which formed networks, however, not between TCs and CSCs. Instead, we show that TCs and CSCs interact in culture forming heterocellular adherens junctions, as well as non‐classical junctions such as puncta adherentia and stromal synapses. The stromal synapse formed between TCs and CSCs (both stromal cells) was frequently associated with the presence of electron‐dense nanostructures (on average about 15 nm in length) connecting the two opposing membranes. The average width of the synaptic cleft was 30 nm, whereas the average length of the intercellular contact was 5 μm. Recent studies have shown that stem cells fail to adequately engraft and survive in the hostile environment of the injured myocardium, possibly as a result of the absence of the pro‐regenerative components of the secretome (paracrine factors) and/or of neighbouring support cells. Herein, we emphasize the similarities between the junctions described in co‐culture and the junctions identified between TCs and CSCs in situ. Reproducing a CSC niche in culture may represent a viable alternative to mono‐cellular therapies.     

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.