Adult neural stem cells, neurogenic niches, and cellular therapy

Niches are specialized microenvironments that regulate stem cells activity. In the nervous system, during development, niches control neural stem cells (NSCs) maturation and the formation of the neuronal network. In the adult, neurogenesis occurs in discrete areas of the brain, the subventricular zone and the hippocampus, where neurogenic niches have been identified and characterized. These niches, an angiogenic and an astroglial niche, control NSCs self-renewal and differentiation. Although the molecular and cellular mechanisms underlying the interactions between NSCs and their environment remain to be elucidated, neurogenic niches share similar developmentally conserved pathways with other niches. It is hypothesized that neurogenic niches underlie the properties and functions of NSCs in the adult central nervous system. Hence, neurogenic niches may not only hold the key to our understanding of neurogenesis in the adult brain, but also of the developmental potential of adult NSCs, and their potential for cellular therapy.     

Comparative analysis of neuroectodermal differentiation capacity of human bone marrow stromal cells using various conversion protocols

Human adult bone marrow-derived mesodermal stromal cells (hMSCs) are able to differentiate into multiple mesodermal tissues, including bone and cartilage. There is evidence that these cells are able to break germ layer commitment and differentiate into cells expressing neuroectodermal properties. There is still debate about whether this results from cell fusion, aberrant marker gene expression or real neuroectodermal differentiation. Here we extend our work on neuroectodermal conversion of adult hMSCs in vitro by evaluating various epigenetic conversion protocols using quantitative RT-PCR and immunocytochemistry. Undifferentiated hMSCs expressed high levels of fibronectin as well as several neuroectodermal genes commonly used to characterize neural cell types, such as nestin, beta-tubulin III, and GFAP, suggesting that hMSCs retain the ability to differentiate into neuroectodermal cell types. Protocols using a direct differentiation of hMSCs into a neural phenotype failed to induce significant changes in morphology and/or expression of markers of early and mature glial/neuronal cells types. In contrast, a multistep protocol with conversion of hMSCs into a neural stem cell-like population and subsequent terminal differentiation in mature glia and neurons generated relevant morphological changes as well as significant increase of expression levels of marker genes for early and late neural cell types, such as nestin, neurogenin2, MBP, and MAP2ab, accompanied by a loss of their mesenchymal properties. Our data provide an impetus for differentiating hMSCs in vitro into mature neuroectodermal cells. Neuroectodermally converted hMSCs may therefore ultimately help in treating acute and chronic neurodegenerative diseases. Analysis of marker gene expression for characterization of neural cells derived from MSCs has to take into account that several early and late neuroectodermal genes are already expressed in undifferentiated MSCs.     

Cannabinoid type 1 receptor blockade induces transdifferentiation towards a brown fat phenotype in white adipocytes

Aim: The endocannabinoid (EC) system is a major component in the control of energy homeostasis. It mediates a positive energy balance via central and peripheral pathways. Blockade of the cannabinoid type 1 receptor induces weight reduction and improves cardiovascular risk factors in overweight patients. Cannabinoid receptor type 1 (CB1R)‐deficient mice are resistant to diet‐induced obesity. The mechanisms responsible for these effects remain only partially elucidated. We hypothesized peripheral effects via direct modulation of adipocyte function to be an integral part of EC action on energy metabolism and insulin sensitivity. Methods: SV40 immortalized murine white and brown adipocytes were used for all experiments. We investigated the effect of CB1R blockade by stimulating the cells acutely and chronically with rimonabant, a selective antagonist for the CB1R, or by knocking down the receptor with small interfering RNA (siRNA). Changes in thermogenic mRNA and protein expression as well as mitochondrial biogenesis and function were assessed by real‐time RT‐PCR, immunoblotting, fluorescent staining techniques, electron microscopy and by measuring oxygen consumption. Results: Acute and chronic blockade of the CB1R with the selective antagonist rimonabant or by siRNA in murine white adipocytes strongly induced the thermogenic uncoupling protein‐1 (UCP‐1). UCP‐1 expression was increased in a time‐ and dose‐dependent manner both at the RNA and protein level. Furthermore, this effect was paralleled by enhanced peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α) expression. In accordance with these findings, AMP‐activated protein kinase (AMPK) phosphorylation was also increased after rimonabant treatment. Mitochondria‐specific fluorescent staining demonstrated an augmentation in the number of mitochondria. This was confirmed by electron microscopy images. Moreover, rimonabant treatment enhanced the cytochrome c oxidase activity and increased cellular oxygen consumption. Conclusions: Taken together, our data demonstrate that inhibition of peripheral CB1R action in adipocytes directly promotes transdifferentiation of white adipocytes into a mitochondria‐rich, thermogenic brown fat phenotype. Enhanced thermogenesis and insulin sensitivity may represent a peripheral mechanism contributing to weight loss and improved glucose homeostasis in rimonabant‐treated patients.     

Differential responses of human lens epithelial cells to intraocular lenses in vitro: hydrophobic acrylic versus PMMA or silicone discs

Background The purpose of this study was to determine the influence of different materials of intraocular lenses (IOLs) on human lens epithelial cell behavior, including adhesion, migration, proliferation, apoptosis, and epithelial-mesenchymal transdifferentiation (EMT) in vitro. Methods Human lens epithelial cells (SRA 01/04) were grown on hydrophobic acrylic (Acrysof), polymethylmethacrylate (PMMA), and silicone IOLs. Cellular adhesion, migration, proliferation, and apoptotic assays were performed to assess cell behavior. The expression of EMT markers (fibronectin and type I collagen) produced by cells on IOLs was determined by immunoblotting and immunocytochemistry. Results Human lens epithelial cells exhibited preferred adhesion and reduced apoptosis when cultured on acrylic IOLs, in comparison to PMMA and silicone IOLs. Cells grown on acrylic lenses formed a confluent epithelial monolayer. Migration of lens epithelial cells under the acrylic lens was substantially blocked in an in vitro assay. In contrast, cells grown on PMMA and silicone lenses displayed a spindle-shaped, myofibroblast-like morphology, increased apoptosis, reduced adhesion, and enhanced production of EMT proteins such as fibronectin and type I collagen. The migration of lens epithelial cells under PMMA and silicone IOLs was substantial in the in vitro assay. Conclusion This report demonstrates that hydrophobic acrylic lenses are more capsular biocompatible than PMMA and silicone lenses. The in vitro assays are reliable measurements for evaluating the responses of human lens epithelial cells to different IOL materials, and could advance our understanding of the preferential capsular opacification conferred by different IOL materials.     

Hair cell regeneration in sensory epithelia from the inner ear of a urodele amphibian

The capacity of urodele amphibians to regenerate a variety of body parts is providing insight into mechanisms of tissue regeneration in vertebrates. In this study the ability of the newt, Notophthalmus viridescens, to regenerate inner ear hair cells in vitro was examined. Intact otic capsules were maintained in organotypic culture. Incubation in 2 mM gentamicin for 48 hours resulted in ablation of all hair cells from the saccular maculae. Thus, any hair cell recovery was not due to repair of damaged hair cells. Immature hair cells were subsequently observed at approximately 12 days posttreatment. Their number increased over the following 7-14 days to reach approximately 30% of the normal number. Following incubation of damaged tissue with bromodeoxyuridine (BrdU), labeled nuclei were confined strictly within regions of hair cell loss, indicating that supporting cells entered S-phase. Double labeling of tissue with two different hair cell markers and three different antibodies to BrdU in various combinations, however, all showed that the nuclei of cells that labeled with hair cell markers did not label for BrdU. This suggested that the new hair cells were not derived from those cells that had undergone mitosis. When mitosis was blocked with aphidicolin, new hair cells were still generated. The results suggest that direct phenotypic conversion of supporting cells into hair cells without an intervening mitotic event is a major mechanism of hair cell regeneration in the newt. A similar mechanism has been proposed for the hair cell recovery phenomenon observed in the vestibular organs of mammals.     

Temporal Expression of Notch in Preterm Rat Lungs Exposed to Hyperoxia

Notch signaling is essential in tissue and organdevelopment. Signals transmitted through theNotch receptor, in combination with other factorsinfluence cell differentiation, proliferation and ap optotic events at all stages of development and re habilitation or remolding of tissues[1]. Previous re searches revealed that Notch receptor/ligand per sistently exists over the whole period of fetal lungdevelopment[2], but it is still unknown how theywork during the chronic lun…     

Mutual paracrine effects of colorectal tumour cells and stromal cells: modulation of tumour and stromal cell differentiation and extracellular matrix component production in culture

Interactions of tumour and stromal cells influence tumour cell proliferation and differentiation, stromal cell phenotypic transdifferentiation and secretion of extracellular matrix (ECM) components. In this study, we established a monolayer and a three-dimensional cell-to-cell interaction model between canine mammary stromal cells and human colonic carcinoma cell lines (Caco-2 and HT-29) to investigate mutual paracrine effects of tumour cells and stromal cells on (i) tumour cell differentiation, (ii) production of ECM components and (iii) phenotypic transdifferentiation of stromal cells. We showed that when Caco-2 or HT-29 cells are cultured in collagen gels, they form a few small solid cell clusters with no lumina, but when cocultured with stromal cells, the tumour cells formed glandular structures with central lumina. This fibroblast-induced organization and differentiation of Caco-2 cells (not HT-29 cells) appeared to be mediated by transforming growth factor-beta (TGF-beta). Culturing of stromal cells, Caco-2 cells or HT-29 cells alone in both monolayers and gels resulted in weak tenascin-C expression in stromal cells and HT-29 cells and no expression in the Caco-2 cells. Coculturing of stromal cells with tumour cells resulted in increased tenascin-C expression in the stromal cells and HT-29 cells and induced expression of tenascin-C in the Caco-2 cells. This induction and increased expression of tenascin-C appeared to be mediated by TGF-beta. Culturing of stromal cells, Caco-2 cells or HT-29 cells alone on monolayers and in gels resulted in a weak expression of chondroitin sulfate (CS), chondroitin-6-sulfate (C-6-S) and versican in stromal cells and no expression in Caco-2 and HT-29 cells. Coculturing of stromal cells with tumour cells on monolayers and in gels resulted in increased CS, C-6-S and versican expression in stromal cells. This tumour cell-induced expression of CS, C-6-S and versican appeared to be mediated by TGF-beta and platelet-derived growth factor (PDGF). Coculturing of Caco-2 and HT-29 and stromal cells promoted the transdifferentiation of stromal cells into myofibroblasts, and this appeared to be mediated by TGF-beta. These results suggest that TGF-beta and PDGF are part of a paracrine system involved in stromal-epithelial cell interaction important in stromal cell differentiation and ECM component production.     

Fibroblast Phenotype Plasticity: Relevance for Understanding Heterogeneity in "Fibroblastic" Tumors

Cellular transformations, reflecting phenotypic plasticity, characterize embryonic life, would-repair, physiological adaptation, and neoplasia. Fibroblastic tumors show a range of cellular differentiation, which can be rationalized in terms of phenotypic plasticity of the “normal” fibroblast. In this paper, the various kinds of fibroblast transformation are discussed, and some insights provided into the molecular mechanisms involved. Comparable molecular events may take place in neoplastic fibroblasts to produce the heterogeneous tumors nevertheless identified as fibroblastic. The following transformations are discussed: histiocytic, and fibrohistiocytic tumors; adipocytic, and lipogenic tumors; myofibroblastic, and myofibroblastic tumors. A definition of the fibroblast is required. This consists of spindle-cell morphology, vimentin-staining, and abundant rough endoplasmic reticulum. Transformation to histiocytic, lipogenic and myofibroblastic phenotypes requires the development of lysosomes, lipid droplets and lamina, and peripheral myofilaments and fibronexuses respectively. These occur in non-malignant transforming (transdifferentiating) fibroblasts, and also in tumors identified as fibrohistiocytic, lipogenic and myofibroblastic. The molecular basis of the myofibroblast transformation is probably the best studied. It is driven primarily by transforming growth factor β. Investigations into the mechanisms of differentiation in normal fibrobiasts could prove fertile ground for defining comparable differentiation in tumors. In this respect, there are very few publications on the presence of growth factors in tumors or tumor-like lesions. There is, however, increasing investigation into gene expression and gene products in tumors, which bear on the differentiation process. Ultimately, our understanding of the molecular events controlling differentiation in cancer will lead to control, cure and prevention.     

角膜缘基质诱导毛囊干细胞角膜上皮样转分化的研究

目的 体外诱导培养毛囊干细胞角膜上皮样分化.方法 体外分离培养毛囊干细胞,然后用角膜缘基质组织匀浆液对其进行诱导培养,并用免疫细胞化学(ICC)法检测诱导前后α6-integfin、CD34、K12的变化;RT-PCR方法检测细胞诱导前后K12的mRNA表达情况;流式细胞分析(FCM)检测毛囊干细胞角膜上皮样分化的阳性率.结果 体外培养的毛囊干细胞保持高增殖的状态,α6-integrin、CD34干细胞质表达.经过10 d的诱导培养,可见K12于细胞质阳性表达;RT-PCR结果显示毛囊干细胞经诱导后K12的mRNA明显表达;FCM结果亦显示毛囊干细胞经诱导培养后有较高比率的K12阳性细胞.结论 毛囊干细胞经角膜缘组织匀浆液体外诱导可成功地分化为角膜上皮样细胞.