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.     

Progenitor cells isolated from the human heart: a potential cell source for regenerative therapy

Background. In recent years, resident cardiac progenitor cells have been identified in, and isolated from the rodent heart. These cells show the potential to form cardiomyocytes, smooth muscle cells, and endothelial cells in vitro and in vivo and could potentially be used as a source for cardiac repair. However, previously described cardiac progenitor cell populations show immature development and need co-culture with neonatal rat cardiomyocytes in order to differentiate in vitro. Here we describe the localisation, isolation, characterisation, and differentiation of cardiomyocyte progenitor cells (CMPCs) isolated from the human heart. Methods. hCMPCs were identified in human hearts based on Sca-1 expression. These cells were isolated, and FACS, RT-PCR and immunocytochemistry were used to determine their baseline characteristics. Cardiomyogenic differentiation was induced by stimulation with 5-azacytidine. Results. hCMPCs were localised within the atria, atrioventricular region, and epicardial layer of the foetal and adult human heart. In vitro, hCMPCs could be induced to differentiate into cardiomyocytes and formed spontaneously beating aggregates, without the need for co-culture with neonatal cardiomyocytes. Conclusion. The human heart harbours a pool of resident cardiomyocyte progenitor cells, which can be expanded and differentiated in vitro. These cells may provide a suitable source for cardiac regeneration cell therapy. (Neth Heart J 2008;16: 163-9.)     

Nkx2-5- and Isl1-expressing cardiac progenitors contribute to proepicardium

Correct delineation of the hierarchy of cardiac progenitors is a key step to understanding heart development, and will pave the way for future use of cardiac progenitors in the treatment of heart disease. Multipotent Nkx2-5 and Isl1 cardiac progenitors contribute to cardiomyocyte, smooth muscle, and endothelial lineages, which constitute the major lineages of the heart. Recently, progenitors located within the proepicardium and epicardium were reported to differentiate into cardiomyocytes, as well as smooth muscle and endothelial cells. However, the relationship of these proepicardial progenitors to the previously described Nkx2-5 and Isl1 cardiac progenitors is incompletely understood. To address this question, we performed in vivo Cre-loxP-based lineage tracing. Both Nkx2-5- and Isl1-expressing progenitors contributed to the proepicardium and expressed Wt1 and Tbx18, markers of proepicardial progenitor cells. Interestingly, Nkx2-5 knockout resulted in abnormal proepicardial development and decreased expression of Wt1, suggesting a functional role for Nkx2-5 in proepicardium formation. Taken together, these results suggest that Nkx2-5 and/or Isl1 cardiac progenitors contribute to proepicardium during heart development.     

Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages

Cell-tracing studies in the mouse indicate that the cardiac lineage arises from a population that expresses the vascular endothelial growth factor receptor 2 (VEGFR2, Flk-1), suggesting that it may develop from a progenitor with vascular potential. Using the embryonic stem (ES) cell differentiation model, we have identified a cardiovascular progenitor based on the temporal expression of the primitive streak (PS) marker brachyury and Flk-1. Comparable progenitors could also be isolated from head-fold stage embryos. When cultured with cytokines known to function during cardiogenesis, individual cardiovascular progenitors generated colonies that displayed cardiomyocyte, endothelial, and vascular smooth muscle (VSM) potential. Isolation and characterization of this previously unidentified population suggests that the mammalian cardiovascular system develops from multipotential progenitors.     

Embryonic development of the proepicardium and coronary vessels

In the last few years, an increasing interest in progenitor cells has been noted. These cells are a source of undifferentiated elements from which cellular components of tissues and organs develop. Such progenitor tissue delivering stem cells for cardiac development is the proepicardium. The proepicardium is a transient organ which occurs near the venous pole of the embryonic heart and protrudes to the pericardial cavity. The proepicardium is a source of the epicardial epithelium delivering cellular components of vascular wall and interstitial tissue fibroblasts. It contributes partially to a fibrous tissue skeleton of the heart. Epicardial derived cells play also an inductive role in differentiation of cardiac myocytes into conductive tissue of the heart. Coronary vessel formation proceeds by vasculogenesis and angiogenesis. The first tubules are formed from blood islands which subsequently coalesce forming the primitive vascular plexus. Coronary arteries are formed by directional growth of vascular protrusions towards the aorta and establishing contact with the aortic wall. The coronary vascular wall matures by attaching smooth muscle cell precursors and fibroblast precursors to the endothelial cell wall. The cells of tunica media differentiate subsequently into vascular smooth muscle by acquiring specific contractile and cytoskeletal markers of smooth muscle cells in a proximal - distal direction. The coronary artery wall matures first before cardiac veins. Maturity of the vessel wall is demonstrated by the specific shape of the internal surface of the vascular wall.     

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1⁺ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1⁺ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1⁺ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.     

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.     

Wnt2 coordinates the commitment of mesoderm to hematopoietic, endothelial, and cardiac lineages in embryoid bodies

Our recent gene expression profiling analyses demonstrated that Wnt2 is highly expressed in Flk1(+) cells, which serve as common progenitors of endothelial cells, blood cells, and mural cells. In this report, we characterize the role of Wnt2 in mesoderm development during embryonic stem (ES) cell differentiation by creating ES cell lines in which Wnt2 was deleted. Wnt2(-/-) embryoid bodies (EBs) generated increased numbers of Flk1(+) cells and blast colony-forming cells compared with wild-type EBs, and had higher Flk1 expression at comparable stages of differentiation. Although Flk1(+) cells were increased, we found that endothelial cell and terminal cardiomyocyte differentiation was impaired, but hematopoietic cell differentiation was enhanced and smooth muscle cell differentiation was unchanged in Wnt2(-/-) EBs. Later stage Wnt2(-/-) EBs had either lower or undetectable expression of endothelial and cardiac genes compared with wild-type EBs. Consistently, vascular plexi were poorly formed and neither beating cardiomyocytes nor alpha-actinin-staining cells were detectable in later stage Wnt2(-/-) EBs. In contrast, hematopoietic cell gene expression was upregulated, and the number of hematopoietic progenitor colonies was significantly enhanced in Wnt2(-/-) EBs. Our data indicate that Wnt2 functions at multiple stages of development during ES cell differentiation and during the commitment and diversification of mesoderm: as a negative regulator for hemangioblast differentiation and hematopoiesis but alternatively as a positive regulator for endothelial and terminal cardiomyocyte differentiation.     

Cardiomyocyte precursors and telocytes in epicardial stem cell niche: electron microscope images

A highly heterogeneous population of stem and progenitor cells has been described by light immunohistochemistry in the mammalian adult heart, but the ultrastructural identity of cardiac stem cells remains unknown. Using electron microscopy, we demonstrate the presence of cells with stem features in the adult mouse heart. These putative cardiac stem cells are small (6–10 μm), round cells, with an irregular shaped nucleus, large nucleolus, few endoplasmic reticulum cisternae and mitochondria, but numerous ribosomes. Stem cells located in the epicardial stem cell niche undergo mitosis and apoptosis. Cells with intermediate features between stem cells and cardiomyocyte progenitors have also been seen. Moreover, electron microscopy showed that cardiomyocyte progenitors were added to the peripheral working cardiomyocytes. Telocytes make a supportive interstitial network for stem cells and progenitors in the stem cell niche. This study enhances the hypothesis of a unique type of cardiac stem cell and progenitors in different stages of differentiation. In our opinion, stem cells, cardiomyocyte progenitors and telocytes sustain a continuous cardiac renewal process in the adult mammalian heart.     

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.     

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.     

Cardiac regenerative potential of cardiosphere‐derived cells from adult dog hearts

The regenerative potential of cardiosphere‐derived cells (CDCs) for ischaemic heart disease has been demonstrated in mice, rats, pigs and a recently completed clinical trial. The regenerative potential of CDCs from dog hearts has yet to be tested. Here, we show that canine CDCs can be produced from adult dog hearts. These cells display similar phenotypes in comparison to previously studied CDCs derived from rodents and human beings. Canine CDCs can differentiate into cardiomyocytes, smooth muscle cells and endothelial cells in vitro. In addition, conditioned media from canine CDCs promote angiogenesis but inhibit cardiomyocyte death. In a doxorubicin‐induced mouse model of dilated cardiomyopathy (DCM), intravenous infusion of canine CDCs improves cardiac function and decreases cardiac fibrosis. Histology revealed that injected canine CDCs engraft in the mouse heart and increase capillary density. Out study demonstrates the regenerative potential of canine CDCs in a mouse model of DCM.     

Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.     

Endothelial cells regulate cardiomyocyte development from embryonic stem cells

The molecules and environment that direct pluripotent stem cell differentiation into cardiomyocytes are largely unknown. Here, we determined a critical role of receptor tyrosine kinase, EphB4, in regulating cardiomyocyte generation from embryonic stem (ES) cells through endothelial cells. The number of spontaneous contracting cardiomyocytes, and the expression of cardiac‐specific genes, including α‐MHC and MLC‐2V, was significantly decreased in EphB4‐null ES cells. EphB4 was expressed in endothelial cells underneath contracting cardiomyocytes, but not in cardiomyocytes. Angiogenic inhibitors, including endostatin and angiostatin, inhibited endothelial cell differentiation and diminished cardiomyogenesis in ES cells. Generation of functional cardiomyocytes and the expression of cardiac‐specific genes were significantly enhanced by co‐culture of ES cells with human endothelial cells. Furthermore, the defects of cardiomyocyte differentiation in EphB4‐deficient ES cells were rescued by human endothelial cells. For the first time, our study demonstrated that endothelial cells play an essential role in facilitating cardiomyocyte differentiation from pluripotent stem cells. EphB4 signaling is a critical component of the endothelial niche to regulate regeneration of cardiomyocytes. J. Cell. Biochem. 111: 29–39, 2010. © 2010 Wiley‐Liss, Inc.     

Apelin/APJ 与胚胎心肌分化

胚胎发育中心脏祖细胞迁移至生心区并分化为心肌细胞是心脏形成的基础。研究心肌分化对了解心脏发育异常以及应用干细胞治疗缺血性心脏病具有重要意义。最近研究发现apelin/APJ信号通路与祖细胞的迁移及心肌分化有关。本文就apelin/APJ与胚胎心肌分化的关系做一综述。     

Cardiac pathological changes of Atlantic salmon (Salmo salar L.) affected with heart and skeletal muscle inflammation (HSMI)

Heart and skeletal muscle inflammation (HSMI) is a disease of marine farmed Atlantic salmon where the pathological changes associated with the disease involve necrosis and an infiltration of inflammatory cells into different regions of the heart and skeletal muscle. The aim of this work was to characterize cardiac changes and inflammatory cell types associated with a clinical HSMI outbreak in Atlantic salmon using immunohistochemistry. Different immune cells and cardiac tissue responses associated with the disease were identified using different markers. The spectrum of inflammatory cells associated with the cardiac pathology consisted of mainly CD3(+) T lymphocytes, moderate numbers of macrophages and eosinophilic granulocytes. Proliferative cell nuclear antigen (PCNA) immuno-reaction identified significantly increased nuclear and cytoplasmic staining as well as identifying hypertrophic nuclei. Strong immunostaining was observed for major histocompatibility complex (MHC) class II in HSMI hearts. Although low in number, a few positive cells in diseased hearts were detected using the mature myeloid cell line granulocytes/monocytes antibody indicating more positive cells in diseased than non-diseased hearts. The recombinant tumor necrosis factor-α (TNFα) antibody identified stained macrophage-like cells and endothelial cells around lesions in addition to eosinophilic granular cells (EGCs). These findings suggested that the inflammatory response in diseased hearts comprised of mostly CD3(+) T lymphocytes and eosinophilic granular cells and hearts exhibited high cell turnover where DNA damage/repair might be the case (as identified by PCNA, caspase 3 and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) reactivity).