Possible promotion of neuronal differentiation in fetal rat brain neural progenitor cells after sustained exposure to static magnetism

We have previously shown significant potentiation of Ca²⁺ influx mediated by N‐methyl‐D ‐aspartate receptors, along with decreased microtubules‐associated protein‐2 (MAP2) expression, in hippocampal neurons cultured under static magnetism without cell death. In this study, we investigated the effects of static magnetism on the functionality of neural progenitor cells endowed to proliferate for self‐replication and differentiate into neuronal, astroglial, and oligodendroglial lineages. Neural progenitor cells were isolated from embryonic rat neocortex and hippocampus, followed by culture under static magnetism at 100 mT and subsequent determination of the number of cells immunoreactive for a marker protein of particular progeny lineages. Static magnetism not only significantly decreased proliferation of neural progenitor cells without affecting cell viability, but also promoted differentiation into cells immunoreactive for MAP2 with a concomitant decrease in that for an astroglial marker, irrespective of the presence of differentiation inducers. In neural progenitors cultured under static magnetism, a significant increase was seen in mRNA expression of several activator‐type proneural genes, such as Mash1, Math1, and Math3, together with decreased mRNA expression of the repressor type Hes5. These results suggest that sustained static magnetism could suppress proliferation for self‐renewal and facilitate differentiation into neurons through promoted expression of activator‐type proneural genes by progenitor cells in fetal rat brain. © 2009 Wiley‐Liss, Inc.     

Subventricular zone neural progenitors from rapid brain autopsies of elderly subjects with and without neurodegenerative disease

In mice and in young adult humans, the subventricular zone (SVZ) contains multipotent, dividing astrocytes, some of which, when cultured, produce neurospheres that differentiate into neurons and glia. It is unknown whether the SVZ of very old humans has this capacity. Here, we report that neural stem/progenitor cells can also be cultured from rapid autopsy samples of SVZ from elderly human subjects, including patients with age‐related neurologic disorders. Histological sections of SVZ from these cases showed a glial fibrillary acidic protein (GFAP)‐positive ribbon of astrocytes similar to the astrocyte ribbon in human periventricular white matter biopsies that is reported to be a rich source of neural progenitors. Cultures of the SVZ contained 1) neurospheres with a core of Musashi‐1‐, nestin‐, and nucleostemin‐immunopositive cells as well as more differentiated GFAP‐positive astrocytes; 2) SMI‐311‐, MAP2a/b‐, and β‐tubulin(III)‐positive neurons; and 3) galactocerebroside‐positive oligodendrocytes. Neurospheres continued to generate differentiated progeny for months after primary culturing, in some cases nearly 2 years postinitial plating. Patch clamp studies of differentiated SVZ cells expressing neuron‐specific antigens revealed voltage‐dependent, tetrodotoxin‐sensitive, inward Na⁺ currents and voltage‐dependent, delayed, slowly inactivating K⁺ currents, electrophysiologic characteristics of neurons. A subpopulation of these cells also exhibited responses consistent with the kinetics and pharmacology of the h‐current. However, although these cells displayed some aspects of neuronal function, they remained immature, insofar as they did not fire action potentials. These studies suggest that human neural progenitor activity may remain viable throughout much of the life span, even in the face of severe neurodegenerative disease. J. Comp. Neurol. 515:269–294, 2009. © 2009 Wiley‐Liss, Inc.     

大鼠脂肪干细胞源性神经球分化为雪旺细胞样细胞

目的:体外诱导大鼠脂肪干细胞为雪旺细胞样细胞?方法:分离?培养和鉴定大鼠脂肪干细胞;表皮生长因子(epidermal growth factor, EGF)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)诱导大鼠脂肪干细胞为神经球,并对神经球进行鉴定;视黄酸?forskolin?血小板衍生生长因子(platelet-derived growth factor, PDGF)及heregulin等诱导神经球为雪旺细胞样细胞,并对雪旺细胞样细胞的形态和表面标志物进行鉴定?结果:大鼠脂肪干细胞为成纤维细胞样单层贴壁细胞,表达间质细胞标志物fibronectin,不表达神经干细胞标志物nestin,并能进行成脂和成骨分化?大鼠脂肪干细胞能被诱导为悬浮生长的神经球;神经球表达nestin,不表达fibronectin,并能进一步分化为雪旺细胞样细胞?雪旺细胞样细胞呈双极或三极,并表达雪旺细胞经典标志物胶质原纤维酸性蛋白(glial fibrillary acidic protein, GFAP)?S100和p75?结论:大鼠脂肪干细胞能在体外分化为雪旺细胞样细胞?这种雪旺细胞样细胞对于治疗神经系统疾病具有重要意义?     

神经球石蜡包埋和巢蛋白免疫组化染色

目的应用细胞团石蜡包埋技术，检测神经球内部的神经干细胞标记物巢蛋白(nestin)表达。方法从孕17d的SD大鼠胚胎大脑海马部位分离，培养神经干细胞，石蜡包埋神经球，通过免疫组化方法检测神经球内部的nestin表达。结果培养中的神经球均显示nestin阳性表达，但阳性细胞比例和细胞团中阳性细胞所处部位有较大差异。结论细胞团石蜡包埋技术检测神经球内部的nestin表达，能全面反映神经球中神经干细胞存在的比例，可作为优化培养技术的实验基础。     

Analysis of differentiation of stem/progenitor cells in tissue culture of neocortical primordium of mouse brain

Differentiation of neural stem/progenitor cells from neocortical primordium of the brain from 14-day mouse embryos was studied by immunohistochemical methods during their culturing. Non-differentiated cells expressing nestin and vimentin persisted in freely floating neurospheres throughout the experiment. Glioblasts, neuroblasts, and differentiated neurons were found in neurospheres cultured in differentiating medium. However, neurons disappeared with increasing the number of passages, the formation of neuroblasts was terminated, and only astrocytes and nestin-positive cells were seen in the culture. It was found that cells of mouse embryonic neocortex lose the capacity for spontaneous multipotent differentiation during culturing. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 4, pp. 189–195, October, 2007     

大鼠受损视神经中神经前体细胞的变化及预变性腓总神经的调节

为了研究大鼠受损视神经内神经前体细胞的变化及调节,本研究建立了成年雄性大鼠视神经损伤及自体腓总神经移植模型,分正常组、损伤组和移植组,体外培养视神经的神经前体细胞,在相差显微镜下观测各组神经前体细胞神经球的形态和数目,以免疫荧光细胞化学方法对神经球细胞进行鉴定。结果显示:正常组神经球较小、较少,多数细胞表达nestin、GFAP或半乳糖脑苷脂(GC);视神经损伤后神经球的总数有所增加,但大神经球数明显减少,nestin、GFAP或GC阳性细胞也明显减少;神经移植后增加了各类神经球数,nestin、GFAP或GC阳性细胞也明显增加。本研究提示成年大鼠视神经内神经前体细胞较少,增殖能力较弱;视神经损伤也伤及其神经前体细胞并抑制其增殖;自体神经移植能保护神经前体细胞并促进其增殖。     

Stem cells as source for retinal pigment epithelium transplantation

Inherited maculopathies, age related macular degeneration and some forms of retinitis pigmentosa are associated with impaired function or loss of the retinal pigment epithelium (RPE). Among potential treatments, transplantation approaches are particularly promising. The arrangement of RPE cells in a well-defined tissue layer makes the RPE amenable to cell or tissue sheet transplantation. Different cell sources have been suggested for RPE transplantation but the development of a clinical protocol faces several obstacles. The source should provide a sufficient number of cells to at least recover the macula area. Secondly, cells should be plastic enough to be able to integrate in the host tissue. Tissue sheets should be considered as well, but the substrate on which RPE cells are cultured needs to be carefully evaluated. Immunogenicity can also be an obstacle for effective transplantation as well as tumorigenicity of not fully differentiated cells. Finally, ethical concerns may represent drawbacks when embryo-derived cells are proposed for RPE transplantation. Here we discuss different cell sources that became available in recent years and their different properties. We also present data on a new source of human RPE. We provide a protocol for RPE differentiation of retinal stem cells derived from adult ciliary bodies of post-mortem donors. We show molecular characterization of the in vitro differentiated RPE tissue and demonstrate its functionality based on a phagocytosis assay. This new source may provide tissue for allogenic transplantation based on best matches through histocompatibility testing.     

Spontaneous neural differentiation of stem cells in culture of human olfactory epithelium

We studied differentiation of stem cells in dissociated cultures of olfactory epithelium. Staining with anti-nestin antibodies revealed stem cells in the primary monolayer culture of the olfactory epithelium from adult human. Proliferation of these cells during culturing in serum-containing medium in the presence of nerve growth factors FGF2 and NGF led to the formation of neurospheres freely floating in the medium or attached to the substrate. Further long-term culturing and cloning of dissociated cells from these neurospheres in media not containing nerve growth factors led to spontaneous neural differentiation of the olfactory epithelium stem cells. The cells with phenotypic signs of differentiated neurons were stained with antibodies against β-tubulin and neurospecific enolase. Differentiated neurons formed diffuse and spatially organized neuronal networks. We hypothesized that factors triggering neural differentiation of olfactory epithelium stem cells are produced by astrocytes present in these cultures. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 4, pp. 183–188, October, 2007