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.)     

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.     

骨髓干细胞的可塑性研究进展

成体干细胞在体内特定的微环境或体外人工培养条件下具有极强的可塑性分化潜能,其主要功能是负责组织细胞的生理性更新和病理性修复．骨髓组织中包括产生所有成熟血细胞系的造血干细胞(HSCs)、多潜能成体祖细胞和能分化为骨、软骨、脂肪的间充质干细胞(MSCs),这些细胞时还有向造血和骨髓以外的其他类型的成熟细胞分化如神经、肌肉、皮肤、心、肝、肾、肺等分化的能力．对最近几年国内外关于骨髓干细胞可塑性的实验研究进展作简要综述．     

Identification and biological characterization of chicken embryonic cardiac progenitor cells

Objectives: Many kinds of cardiac progenitor cell populations have been identified, including c‐kit⁺, Nkx2.5⁺s and GATA4⁺ cells. However, these progenitors have limited ability to differentiate into different cardiac cell types. Recently, a new kind of cardiac progenitor cell named the multipotent Isl1⁺ cardiovascular progenitor (MICPs) has been identified, which also expresses Nkx2.5, GATA4, CD34 and Flk1. Materials and methods: In this study, we have isolated and characterized MICPs from chicken embryonic heart tissues using immunofluorescence and PCR. Results: Results shown that they express markers of cardiac progenitor cells, with high clonality. They have the ability to self‐renew and can give rise to three types of heart cell in vitro. Conclusions: Myocytes, smooth muscle cells and endothelial cells. Our work provides evidence for a developmental paradigm of the heart, that endothelial and muscle lineage diversification arises from multipotent cardiac progenitor cells. Existence of these cells provides a new opportunity for myocardial injury repair.     

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.     

Therapeutic potential of stem/progenitor cells in human skeletal muscle for cardiovascular regeneration

Although myoblast transplantation in patients with ischemic heart failure results in a significant improvement of cardiac function, subsequent studies have consistently shown the myotubes formation in the absence of electromechanical coupling with the neighboring host myocardium, accompanied with the short-term release of paracrine effectors from implanted cells. One major pitfall of using myoblasts is that transplanted cells do not differentiate into cardiomyocytes, which may cause the inherent proarrhythmogenic events. Therefore, whether a discrete subpopulation in heterogeneous muscle-cell cultures is responsible for substantial cardiovascular regeneration has yet to be investigated. We describe here the isolation of progenitor cells from human skeletal muscle. These cells proliferated as non-adherent myospheres in suspension and displayed early embryonic factors and mesenchymal cell-like characteristics. Flow cytometric analyses demonstrated that CD56/N-CAM/Leu-19, a neural cell adhesion molecule abundantly present in myoblasts, was absent in myospheres but was expressed in an adherent cell population containing myogenic precursors. Myosphere-derived progenitor cells (MDPCs) differentiated in culture to produce cardiac, smooth muscle, and endothelial cells. Transplantation of MDPCs into ischemic hearts in NOD/scid mice promoted angiogenesis with substantial cardiovascular regeneration. Our results provide a foundation to further study the cell and biological function of human MDPCs which may have potential therapeutic implications.     

Stem cells, vascular smooth muscle cells and atherosclerosis

Stem cells have the ability to differentiate into a variety of cells to replace dead cells or to repair tissue. Recently, accumulating evidence indicates that mechanical forces, cytokines and other factors can influence stem cell differentiation into vascular smooth muscle cells (SMCs). In developmental process, SMCs originate from several sources, which show a great heterogenicity in different vessel walls. In adult vessels, SMCs display a less proliferative nature, but are altered in response to risk factors for atherosclerosis. Traditional view on SMC origins in atherosclerotic lesions is challenged by the recent findings that stem cells and smooth muscle progenitors contribute to the development of atherosclerotic lesions. Vascular progenitor cells circulating in human blood and the presence of adventitia in animals are recent discoveries, but the source of these cells is still unknown. The present review gives an update on the progress of stem cell and SMC research in atherosclerosis, and discusses possible mechanisms of stem/progenitor cell differentiation that contribute to the disease process.     

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.     

Adult stem cell homing and differentiation in vitro on composite fibrin matrix

Strategies to generate differentiated cells from haematopoetic progenitor cells will enhance potential use of adult stem cells for therapeutic transplantation or tissue engineering. Transplantation of undifferentiated stem cells into recipient tissue hinges on the hypothesis of a milieu dependent differentiation and it has been suggested that a clot-equivalent scaffold is crucial for these circulating cells to anchor and multiply. Here a natural scaffold, fibrin along with fibronectin, gelatin and growth factors has been used to induce endothelial progenitor cells and smooth muscle progenitor cells to differentiate into endothelial cells and smooth muscle cells, respectively, from peripheral blood mononuclear cells. Characteristics of endothelial cells have been verified by the detection of mRNA for and immunostaining the cells for von Willebrand factor, uptake of acetylated low-density lipoproteins and measurement of released nitric oxide in the culture medium, as nitrite. The specific molecules that characterized smooth muscle cells were alpha smooth muscle actin and calponin, besides deposition of collagen type I and elastin, onto the culture matrix. The adhesive proteins used for the fabrication of endothelial progenitor cells matrix and smooth muscle progenitor cells matrix were the same, but specific differentiation was brought about by modulating the growth factor composition in the matrix and in the culture medium. Both endothelial and smooth muscle cells were consistently developed from 20 ml of human blood.     

hDlk-1: a cell surface marker common to normal hepatic stem/progenitor cells and carcinomas

Advances in stem cell biology have clarified that a tumour is a collection of heterogeneous cell populations, and that only a small fraction of tumour cells possesses the potential to self-renew. Delta-like 1 protein (Dlk-1) is a surface antigen present on foetal hepatic stem/progenitor cells but absent from mature hepatocytes in neonatal and adult rodent liver. Using a monoclonal antibody (mAb) against hDlk-1, Yanai et al. (Dlk-1, a cell surface antigen on foetal hepatic stem/progenitor cells, is expressed in hepatocellular, colon, pancreas and breast carcinomas at a high frequency. J. Biochem. 2010;148:85-92) have shown that human (h) Dlk-1 is expressed in human foetal, but not adult, liver and that 20% of all hepatocellular carcinomas (HCCs) are hDlk-1(+). Importantly, an even higher percentage of HCCs in younger patients are hDLK-1(+). These authors also found that hDlk-1 is present at high frequency in colon adenocarcinomas, pancreatic islet carcinomas and small cell lung carcinomas. Here, I discuss the implications of the expression of foetal hepatic stem/progenitor cell antigens on carcinoma cells.     

The adult human brain harbors multipotent perivascular mesenchymal stem cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain.     

BMP4 regulates vascular progenitor development in human embryonic stem cells through a smad‐dependent pathway

The signals that direct pluripotent stem cell differentiation into lineage‐specific cells remain largely unknown. Here, we investigated the roles of BMP on vascular progenitor development from human embryonic stem cells (hESCs). In a serum‐free condition, hESCs sequentially differentiated into CD34+CD31?, CD34+CD31+, and then CD34?CD31+ cells during vascular cell development. CD34+CD31+ cells contained vascular progenitor population that gives rise to endothelial cells and smooth muscle cells. BMP4 promoted hESC differentiation into CD34+CD31+ cells at an early stage. In contrast, TGFβ suppressed BMP4‐induced CD34+CD31+ cell development, and promoted CD34+CD31? cells that failed to give rise to either endothelial or smooth muscle cells. The BMP‐Smad inhibitor, dorsomorphin, inhibited phosphorylation of Smad1/5/8, and blocked hESC differentiation to CD34+CD31+ progenitor cells, suggesting that BMP Smad‐dependent signaling is critical for CD34+CD31+ vascular progenitor development. Our findings provide new insight into how pluripotent hESCs differentiate into vascular cells. J. Cell. Biochem. 109: 363–374, 2010. © 2009 Wiley‐Liss, Inc.     

Host tissue response in stem cell therapy

Preclinical and clinical trials of stem cell therapy have been carried out for treating a broad spectrum of diseases using several types of adult stem cells. While encouraging therapeutic results have been obtained, much remains to be investigated regarding the best cell type to use, cell dosage, delivery route, long-term safety, clinical feasibility, and ultimately treatment cost. Logistic aspects of stem cell therapeutics remain an area that requires urgent attention from the medical community. Recent cardiovascular trial studies have demonstrated that growth factors and cytokines derived from the injected stem cells and host tissue appear to contribute largely to the observed therapeutic benefits, indicating that trophic actions rather than the multilineage potential (or stemness) of the administered stem cells may provide the underlying tissue healing power. However, the capacity for trophic factor production can be aberrantly downregulated as seen in human heart disease. Skeletal muscle is a dynamic tissue with an impressive ability to continuously respond to environmental stimuli. Indeed, a relation exists between active skeletal muscle and low cardiovascular risk, highlighting the critical link between the skeletal muscle and cardiovascular systems. Adding to this notion are recent studies showing that stem cells injected into skeletal muscle can rescue the failing rodent heart through activation of the muscle trophic factor network and mobilization of bone marrow multilineage progenitor cells. However, aging and disease can adversely affect the host tissue into which stem cells are injected. A better understanding of the host tissue response in stem cell therapy is necessary to advance the field and bridge the gap between preclinical and clinical findings.     

A common progenitor at the heart of development

Formation of the heart requires the coordinated functions of cardiac myocytes, smooth muscle cells, endothelial cells, and connective tissue elements. Several recent studies now reveal that these different cell types arise from a common progenitor (). These findings raise interesting questions about the lineage relationships of cardiovascular progenitor cell populations and suggest possibilities for cardiac repair in both congenital and acquired heart disease.     

In vivo cardiovasculogenesis by direct injection of isolated adult mesenchymal stem cells

The characterization of mesenchymal stem cells (MSCs) is of biological and clinical interest. We demonstrate that isolated MSCs, defined by CD34(low) c-kit(+) CD140a(+) Sca-1(high), are able to differentiate into cardiomyocytes, endothelial cells, and pericytes or smooth muscle cells by direct injection into adult heart. In skeletal muscle and lung, they also contributed to formation of the vasculature. MSCs did not transform into malignant cells or form excess extracellular matrix. This study suggests that MSCs may supply an ideal donor source of cardiovascular cells in patients with cardiopulmonary diseases.     

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.     

Vascular wall resident progenitor cells: a source for postnatal vasculogenesis

Here, we report the existence of endothelial precursor (EPC) and stem cells in a distinct zone of the vascular wall that are capable to differentiate into mature endothelial cells, hematopoietic and local immune cells, such as macrophages. This zone has been identified to be localized between smooth muscle and adventitial layer of human adult vascular wall. It predominantly contains CD34-positive (+) but CD31-negative (-) cells, which also express VEGFR2 and TIE2. Only few cells in this zone of the vascular wall are positive for CD45. In a ring assay using the fragments of human internal thoracic artery (HITA), we show here that the CD34+ cells of the HITA-wall form capillary sprouts ex vivo and are apparently recruited for capillary formation by tumor cells. New vessels formed by these vascular wall resident EPCs express markers for angiogenically activated endothelial cells, such as CEACAM1, and also for mature endothelial cells, such as VE-cadherin or occludin. Vascular wall areas containing EPCs are found in large and middle sized arteries and veins of all organs studied here. These data suggest the existence of a ;vasculogenic zone' in the wall of adult human blood vessels, which may serve as a source for progenitor cells for postnatal vasculogenesis, contributing to tumor vascularization and local immune response.