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.)     

Mesodermal cell types induce neurogenesis from adult human hippocampal progenitor cells

Neurogenesis in the adult human brain occurs within two principle neurogenic regions, the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. Recent reports demonstrated the isolation of human neuroprogenitor cells (NPCs) from these regions, but due to limited tissue availability the knowledge of their phenotype and differentiation behavior is restricted. Here we characterize the phenotype and differentiation capacity of human adult hippocampal NPCs (hNPCs), derived from patients who underwent epilepsy surgery, on various feeder cells including fetal mixed cortical cultures, mouse embryonic fibroblasts (MEFs) and PA6 stromal cells. Isolated hNPCs were cultured in clonal density by transferring the cells to serum-free media supplemented with FGF-2 and EGF in 3% atmospheric oxygen. These hNPCs showed neurosphere formation, expressed high levels of early neuroectodermal markers, such as the proneural genes NeuroD1 and Olig2, the NSC markers Nestin and Musashi1, the proliferation marker Ki67 and significant activity of telomerase. The phenotype was CD15low/-, CD34-, CD45- and CD133-. After removal of mitogens and plating them on poly D-lysine, they spontaneously differentiated into a neuronal (MAP2ab+), astroglial (GFAP+), and oligodendroglial (GalC+) phenotype. Differentiated hNPCs showed functional properties of neurons, such as sodium channels, action potentials and production of the neurotransmitters glutamate and GABA. Co-culture of hNPCs with fetal cortical cultures, MEFs and PA6 cells increased neurogenesis of hNPCs in vitro, while only MEFs and PA6 cells also led to a morphological and functional neurogenic maturation. Together we provide a first detailed characterization of the phenotype and differentiation potential of human adult hNPCs in vitro. Our findings reinforce the emerging view that the differentiation capacity of adult hNPCs is critically influenced by non-neuronal mesodermal feeder cells.     

MicroRNA‐155 prevents necrotic cell death in human cardiomyocyte progenitor cells via targeting RIP1

To improve regeneration of the injured myocardium, cardiomyocyte progenitor cells (CMPCs) have been put forward as a potential cell source for transplantation therapy. Although cell transplantation therapy displayed promising results, many issues need to be addressed before fully appreciating their impact. One of the hurdles is poor graft‐cell survival upon injection, thereby limiting potential beneficial effects. Here, we attempt to improve CMPCs survival by increasing microRNA‐155 (miR‐155) levels, potentially to improve engraftment upon transplantation. Using quantitative PCR, we observed a 4‐fold increase of miR‐155 when CMPCs were exposed to hydrogen‐peroxide stimulation. Flow cytometric analysis of cell viability, apoptosis and necrosis showed that necrosis is the main cause of cell death. Overexpressing miR‐155 in CMPCs revealed that miR‐155 attenuated necrotic cell death by 40 ± 2.3%via targeting receptor interacting protein 1 (RIP1). In addition, inhibiting RIP1, either by pre‐incubating the cells with a RIP1 specific inhibitor, Necrostatin‐1 or siRNA mediated knockdown, reduced necrosis by 38 ± 2.5% and 33 ± 1.9%, respectively. Interestingly, analysing gene expression using a PCR‐array showed that increased miR‐155 levels did not change cell survival and apoptotic related gene expression. By targeting RIP1, miR‐155 repressed necrotic cell death of CMPCs, independent of activation of Akt pro‐survival pathway. MiR‐155 provides the opportunity to block necrosis, a conventionally thought non‐regulated process, and might be a potential novel approach to improve cell engraftment for cell therapy.     

黄芪甲苷促进心肌干细胞分化的作用研究

目的 探讨黄芪甲苷对心肌干细胞分化的促进作用。方法 采用磁珠分选法,分离小鼠Sca-1＋心肌干细胞,通过免疫组化方法观察黄芪甲甙处理后心肌细胞表面标志蛋白desmin、α-sarcomeric？actin和C-TnT表达的变化,以判断是否对心肌干细胞分化有促进作用。结果 250 mg/L的黄芪甲甙诱导4周后免疫组化染色显示心肌干细胞明显表达desmin、α-sarcomeric actin和C-TnT。而未诱导的细胞desmin、α-sarcomeric actin、C-TnT 均为阴性。因此黄芪甲甙可以促进小鼠Sca-1＋心肌干细胞分化为心肌样细胞,这些细胞表达心肌特异性的蛋白。结论 黄芪甲苷对心肌干细胞分化的促进作用表明其在心肌损伤性疾病的康复中有潜在的治疗价值,值得进一步研究。     

Betacellulin regulates the proliferation and differentiation of retinal progenitor cells in vitro

Retinal progenitor cells (RPCs) hold great potential for the treatment of retinal degenerative diseases. However, their proliferation capacity and differentiation potential towards specific retinal neurons are limited, which limit their future clinical applications. Thus, it is important to improve the RPCs’ ability to proliferate and differentiate. Currently, epidermal growth factor (EGF) is commonly used to stimulate RPC growth in vitro. In this study, we find that betacellulin (BTC), a member of the EGF family, plays important roles in the proliferation and differentiation of RPCs. Our results showed that BTC can significantly promote the proliferation of RPCs more efficiently than EGF. EGF stimulated RPC proliferation through the EGFR/ErbB2‐Erk pathway, while BTC stimulated RPC proliferation more powerfully through the EGFR/ErbB2/ErbB4‐Akt/Erk pathway. Meanwhile, under differentiated conditions, the BTC‐pre‐treated RPCs were preferentially differentiated into retinal neurons, including photoreceptors, one of the most important types of cells for retinal cell replacement therapy, compared to the EGF‐pre‐treated RPCs. In addition, knockdown of endogenous BTC expression can also obviously promote RPC differentiation into retinal neuronal cells. This data demonstrate that BTC plays important roles in promoting RPC proliferation and differentiation into retinal neurons. This study may provide new insights into the study of RPC proliferation and differentiation and make a step towards the application of RPCs in the treatment of retinal degenerative diseases.     

成体干细胞的生物学特点及应用前景

成体干细胞存在于人和哺乳动物组织中,具有自我更新和一定的分化潜能,现已从骨髓,软骨,血液,神经,肌肉,脂肪,皮肤,角膜缘,肝脏,胰腺等许多组织中获得成体干细胞,发现部分组织成体干细胞具有多向分化潜能,成体干细胞的研究在再生医学中有十分广阔的应用前景。     

PI3K/Akt and CREB regulate adult neural hippocampal progenitor proliferation and differentiation

The phosphoinositide 3-OH kinase (PI3K)/Akt pathway has been implicated in regulating several important cellular processes, including apoptosis, survival, proliferation, and metabolism. Using both pharmacological and genetic means, we demonstrate here that PI3K/Akt plays a crucial role in the proliferation of adult hippocampal neural progenitor cells. PI3K/Akt transduces intracellular signals from multiple mitogens, including basic fibroblast growth factor (FGF-2), Sonic hedgehog (Shh), and insulin-like growth factor 1 (IGF-1). In addition, retroviral vector-mediated over-expression of wild type Akt increased cell proliferation, while a dominant negative Akt inhibited proliferation. Furthermore, wild type Akt over-expression reduced glial (GFAP) and neuronal (beta-tubulin III) marker expression during differentiation, indicating that it inhibits cell differentiation. We also show that activation of the cAMP response element binding protein (CREB), which occurs in cells stimulated by FGF-2, is limited when Akt signaling is inhibited, demonstrating a link between Akt and CREB. Over-expression of wild type CREB increases progenitor proliferation, whereas dominant negative CREB only slightly decreases proliferation. These results indicate that PI3K/Akt signaling integrates extracellular signaling information to promote cellular proliferation and inhibit differentiation in adult neural progenitors.     

Tracking adult stem cells

The maintenance of stem-cell-driven tissue homeostasis requires a balance between the generation and loss of cell mass. Adult stem cells have a close relationship with the surrounding tissue--known as their niche--and thus, stem-cell studies should preferably be performed in a physiological context, rather than outside their natural environment. The mouse is an attractive model in which to study adult mammalian stem cells, as numerous experimental systems and genetic tools are available. In this review, we describe strategies commonly used to identify and functionally characterize adult stem cells in mice and discuss their potential, limitations and interpretations, as well as how they have informed our understanding of adult stem-cell biology. An accurate interpretation of physiologically relevant stem-cell assays is crucial to identify adult stem cells and elucidate how they self-renew and give rise to differentiated progeny.     

Morphological and phenotypical characterization of human endothelial progenitor cells in an early stage of differentiation

The exact phenotype and lineage of endothelial progenitor cells (EPCs) are still a matter of debate and different expansion protocols are used to obtain them. In this study, EPC expansion from peripheral blood mononuclear cells was analyzed within the first week of culture. Both the adherent and suspended cells, of which the latter usually discarded, were considered. We provide, for the first time, a systematic study of EPC phenotype and functional features within the first 3 days of culture. Moreover, within the 2nd day, both cellular fractions displayed a significant increase in endothelial marker expression which correlated with EPC properties.     

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.     

Toll-like receptors in the functional orientation of cardiac progenitor cells

Cardiac progenitor cells (CPCs) have the potential to differentiate into several cell lineages with the ability to restore in cardiac tissue. Multipotency and self-renewal activity are the crucial characteristics of CPCs. Also, CPCs have promising therapeutic roles in cardiac diseases such as valvular disease, thrombosis, atherosclerosis, congestive heart failure, and cardiac remodeling. Toll-like receptors (TLRs), as the main part of the innate immunity, have a key role in the development and differentiation of immune cells. Some reports are found regarding the effect of TLRs in the maturation of stem cells. This article tried to find the potential role of TLRs in the dynamics of CPCs. By showing possible crosstalk between the TLR signaling pathways and CPCs dynamics, we could achieve a better conception related to TLRs in the regeneration of cardiac tissue.     

Continuous clonal labeling reveals uniform progenitor potential in the adult exocrine pancreas

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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.     

MiR-155 inhibits cell migration of human cardiomyocyte progenitor cells (hCMPCs) via targeting of MMP-16

Undesired cell migration after targeted cell transplantation potentially limits beneficial effects for cardiac regeneration. MicroRNAs are known to be involved in several cellular processes, including cell migration. Here, we attempt to reduce human cardiomyocyte progenitor cell (hCMPC) migration via increasing microRNA‐155 (miR‐155) levels, and investigate the underlying mechanism. Human cardiomyocyte progenitor cells (hCMPCs) were transfected with pre‐miR‐155, anti‐miR‐155 or control‐miR (ctrl‐miR), followed by scratch‐ and transwell‐ assays. These functional assays displayed that miR‐155 over‐expression efficiently inhibited cell migration by 38 ± 3.6% and 59 ± 3.7% respectively. Conditioned medium from miR‐155 transfected cells was collected and zymography analysis showed a significant decrease in MMP‐2 and MMP‐9 activities. The predicted 3′‐UTR of MMP‐16, an activator of MMP‐2 and ‐9, was cloned into the pMIR‐REPORT vector and luciferase assays were performed. Introduction of miR‐155 significantly reduced luciferase activity which could be abolished by cotransfection with anti‐miR‐155 or target site mutagenesis. By using MMP‐16 siRNA to reduce MMP‐16 levels or by using an MMP‐16 blocking antibody, hCMPC migration could be blocked as well. By directly targeting MMP‐16, miR‐155 efficiently inhibits cell migration via a reduction in MMP‐2 and ‐9 activities. Our study shows that miR‐155 might be used to improve local retention of hCMPCs after intramyocardial delivery.     

人胚胎干细胞向内皮祖细胞诱导分化及其应用研究进展

近年来,内皮细胞的应用价值不断提高,应用领域不断拓宽,但其来源有限,成为研究应用的主要障碍.胚胎干细胞在体外可分化为多种组织细胞系,有可能成为获取内皮细胞的另一来源.就人胚胎干细胞向内皮祖细胞分化、分离方法、相关分子机制及内皮祖细胞应用价值等进行阐述,以期能够引起更多的关注,推动其研究的进展.