Generation of regionally specified neurons in expanded glial cultures derived from the mouse and human lateral ganglionic eminence

The specific identity of neuronal precursors within the embryonic brain is, at present, not clear. Here we show that cultures with glial characteristics derived from the embryonic mouse or human lateral ganglionic eminence (LGE) can be expanded over many passages and maintain their glial identity. Interestingly, removal of serum and EGF from the culture medium results in the generation of large numbers of neurons. The neurons derived from these cultures display many characteristic features of striatal neurons, which normally derive from the LGE, even after extensive expansion in vitro. Furthermore, a portion of the neurons generated in these cultures were shown to arise from glial fibrillary acidic protein (GFAP)-expressing cells. These results demonstrate that at least a subpopulation of neurogenic LGE precursors exhibit glial characteristics.     

Neuronal differentiation following transplantation of expanded mouse neurosphere cultures derived from different embryonic forebrain regions

In vitro, expanded neurospheres exhibit multipotent properties and can differentiate into neurons, astrocytes and oligodendrocytes. In vivo, cells from neurospheres derived from mouse fetal forebrain have previously been reported to predominantly differentiate into glial cells, and not into neurons. Here we isolated stem/progenitor cells from E13.5 lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE) and cortical primordium, of a green fluorescent protein (GFP)-actin transgenic mouse. Free-floating neurospheres were expanded in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and implanted after five to six passages into the striatum, hippocampus and cortex of neonatal rats. Cell suspensions of primary LGE tissue were prepared and grafted in parallel. Grafted cells derived from the primary tissue displayed widespread incorporation into all regions, as visualized with the mouse-specific antibody M2, or mouse satellite DNA in situ hybridization, and differentiated into both neurons, astrocytes and oligodendrocytes. Grafts of neurosphere cells derived from the LGE, MGE and cortical primordium differentiated primarily into astrocytes, but contained low but significant numbers of GFP-immunoreactive neurons. Neurons derived from LGE neurospheres were of three types: cells with the morphology of medium-sized densely spiny projection neurons in the striatum; cells with interneuron-like morphologies in striatum, cortex and hippocampus; and cells integrating into SVZ and migrating along the RMS to the olfactory bulb. MGE- or cortical primordium-derived neurospheres differentiated into interneuron-like cells in both striatum and hippocampus. The results demonstrate the ability of in vitro expanded neural stem/progenitor cells to generate both neurons and glia after transplantation into neonatal recipients, and differentiate in a region-specific manner into mature neurons with morphological features characteristic for each target site.     

Excitatory amino acid receptors in primary cultures of glial cells from the retina.

Binding of 3H-L-aspartate to membranes from retinal glial cells in primary culture was characterized. Binding kinetics showed a saturable, reversible binding to three populations of sites with KB = 40, 200, and 1,300 nM. The first two were present at 1 day in vitro (DIV), whereas the latter two were observed at 12 DIV. The possibility of the 40 nM site being neuronal cannot be discarded, since some neurons are present at 1 DIV. In 12 DIV cultures, the presence or absence of sodium determined two different pharmacological patterns, comparable to those described for electrogenic glutamate transport in Müller cells, and QA metabotropic receptors in astrocytes, respectively. Results suggest that, as has been shown for some receptors in nerve tissue, the properties of glial cell receptors undergo age-dependent changes. In turn, this could be related to changes in the function of neurotransmitter substances during development.     

The interface between glial progenitors and gliomas

The mammalian brain and spinal cord contain heterogeneous populations of cycling, immature cells. These include cells with stem cell-like properties as well as progenitors in various stages of early glial differentiation. This latter population is distributed widely throughout gray and white matter and numerically represents an extremely large cell pool. In this review, we discuss the possibility that the glial progenitors that populate the adult CNS are one source of gliomas. Indeed, the marker phenotypes, morphologies, and migratory properties of cells in gliomas strongly resemble glial progenitors in many ways. We review briefly some salient features of normal glial development and then examine the similarities and differences between normal progenitors and cells in gliomas, focusing on the phenotypic plasticity of glial progenitors and the responses to growth factors in promoting proliferation and migration of normal and glioma cells, and discussing known mutational changes in gliomas in the context of how these might affect the proliferative and migratory behaviors of progenitors. Finally, we will discuss the “cancer stem cell” hypothesis in light of the possibility that glial progenitors can generate gliomas.     

GABAergic immunoreactivity is predominant in neurons derived from expanded human neural precursor cells in vitro

Neural precursor cells have been previously isolated from the developing human nervous system and their properties studied both in vitro and in transplantation paradigms in vivo. However, their ability to differentiate into neurons of different neurochemical phenotypes remains poorly defined. In this study, the default in vitro neuronal differentiation of hENPs derived from five different regions of the human embryonic brain (cerebral cortex, striatum, cerebellum, ventral mesencephalon, and spinal cord) was studied after varying periods of time in culture. The results were directly compared to those from similarly prepared murine ENPs. hENPs prepared from all five regions showed a significant reduction in the number of neurons generated at each passage, such that by passage 4 only between 5 and 10% of cells spontaneously adopted a neuronal phenotype after differentiation in vitro. A similar observation was obtained with murine ENPs. hENPs prepared from more caudal parts of the developing neuroaxis generated fewer neurons compared to the more rostral regions. The only neuronal phenotype identified in these cultures was GABA, with 15-60% of the neurons immunopositive for this neurotransmitter. Thus there appears to be important differences between hENPs dependent on region of origin and time in vitro under standard culture conditions, forming decreasing numbers of neurons with increasing time in culture and more caudal sites of harvest, and with the major identifiable neurotransmitter being GABA. Such characterisation is important in the process of learning how to manipulate the neuronal phenotype of these cells.     

Peripheral glial cell differentiation from neurospheres derived from adipose mesenchymal stem cells

Mesenchymal stem cells derived from bone marrow and adipose tissue are being considered for use in neural repair because they can differentiate after appropriate induction in culture into neurons and glia. The question we asked was if neurospheres could be harvested from adipose-derived stem cells and if they then could differentiate in culture to peripheral glial-like cells. Here, we demonstrate that adipose-derived mesenchymal stem cells can form nestin-positive non-adherent neurosphere cellular aggregates when cultured with basic fibroblast growth factor and epidermal growth factor. Dissociation of these neurospheres and removal of mitogens results in expression of the characteristic Schwann cell markers S100 and p75 nerve growth factor receptor and GFAP. The simultaneous expression of these glia markers are characteristic features of Schwann cells and olfactory ensheathing cells which have unique properties regarding remyelination and enhancement of axonal regeneration. When co-cultured with dorsal root ganglion neurons, the peripheral glial-like cells derived from adipose mesenchymal stem cells aligned with neuritis and stimulated neuritic outgrowth. These results indicate that neurospheres can be generated from adipose-derived mesenchymal stem cells, and upon mitogen withdrawal can differentiate into peripheral glial cells with neurotrophic effects.     

NSCs在OECs诱导下定向分化及神经元电生理特性研究

目的探索大鼠嗅鞘细胞（OECs）对神经干细胞（NSCs）分化的影响,并记录分化后神经元电生理特性。方法在无血清改良Eagle培养基（DMEM／F12）培养的NSCs中,加入OECs条件培养基,观察分化情况,巢蛋白（nestin）鉴定NSCs、神经丝蛋白200（NF200）鉴定神经元、膜片钳检测神经元电生理特性。结果OECs能促进NSCs分化为神经元,分化后的神经元记录到快速激活快速失活、能被河豚毒素（TTX）特异阻断的钠电流及慢激活慢失活、能被四乙基氯化铵（TEA）特异阻断的延迟整流性钾电流。结论OECs能诱导NSCs分化,分化后的神经元具有活跃的电生理特性,具有替代凋亡、坏死神经元的潜能。     

Different response of astrocytes and Bergmann glial cells to portacaval shunt: an immunohistochemical study in the rat cerebellum.

The present study was performed in order to follow the response of rat cerebellum astroglial cells (Bergmann glial cells and astrocytes) to long-term portacaval shunt (PCS), by means of glial fibrillary acidic protein (GFAP) and vimentin immunoreactivities. Bergmann glia accumulated GFAP in response to PCS, whereas astrocytes decreased GFAP immunoreactivity when compared to control rats. The increase of GFAP occurs in cells located in the cerebellar layer where glutamate is mainly released. Since the vimentin content remained unaltered in response to PCS, when compared to control rats, it can be concluded that only the GFAP filaments are affected by PCS. Nevertheless, GFAP immunoreactivity presents regional differences in the cerebellar astroglial population, and the factors responsible for these variations are still unknown.     

Long-term, EGF-stimulated cultures of attached GFAP-positive cells derived from the embryonic mouse lateral ganglionic eminence: in vitro and transplantation studies

Long-term attached cultures, prepared from mouse embryonic days 15-17 lateral ganglionic eminence, were grown in a medium including epidermal growth factor and serum, and the survival, differentiation, and migration of cells from either early or late passages were analyzed following transplantation. The cultured cells had the morphology of type I astroglial cells, with the vast majority of the cells immunoreactive for glial fibrillary acidic protein (around 90%), the intermediate filament marker nestin, and also the mouse-specific neural markers M2 and M6. The cultures were kept over 25 passages (7 months). During the first 8 passages, the growth rate gradually declined, but it increased again after passage 9 and thereafter stabilized at values similar to those observed during the initial culture period. After passages 4-6 and 18, cell suspensions were implanted cross-species into the intact or lesioned striatum of adult (passages 4-5 only) or intact striatum of neonatal rats (passages 4-6 or 18). Both early and late passage cells formed M2 (and M6)-positive transplants. In the neonatal recipients, widespread migration was seen from the needle tract throughout most of the striatum, along the internal capsule, and into the globus pallidus. In the adult striatum, the cells remained mostly around the injection tract, or within 0.4-0.6 mm from the graft core. These long-term attached cultures are interesting to compare to nonattached neurosphere cultures, and might also offer a means of propagating relatively pure populations of astroglia-like cells for basic transplantation studies or for use in experimental trials with ex vivo gene transfer.     

Induction of dopaminergic neurons from growth factor expanded neural stem/progenitor cell cultures derived from human first trimester forebrain

Multipotent stem/progenitor cells derived from human first trimester forebrain can be expanded as free-floating aggregates, so called neurospheres. These cells can differentiate into neurons, astrocytes and oligodendrocytes. In vitro differentiation protocols normally yield γ-aminobutyric acid-immunoreactive neurons, whereas only few tyrosine hydroxylase (TH) expressing neurons are found. The present report describes conditions under which 4–10% of the cells in the culture become TH immunoreactive (ir) neurons within 24 h. Factors including acidic fibroblast growth factor (aFGF) in combination with agents that increase intracellular cyclic AMP and activate protein kinase C, in addition to a substrate that promotes neuronal differentiation appear critical for efficient TH induction. The cells remain THir after trypsinization and replating, even when their subsequent culturing takes place in the absence of inducing factors. Consistent with a dopaminergic phenotype, mRNAs encoding aromatic acid decarboxylase, but not dopamine-β-hydroxylase were detected by quantitative real time RT-PCR. Ten weeks after the cells had been grafted into the striatum of adult rats with unilateral nigrostriatal lesions, only very few of the surviving human neurons expressed TH. Our data suggest that a significant proportion of expandable human neural progenitors can differentiate into TH-expressing cells in vitro and that they could be useful for drug and gene discovery. Additional experiments, however, are required to improve the survival and phenotypic stability of these cells before they can be considered useful for cell replacement therapy in Parkinson's disease.     

成人脑神经干细胞体内外增殖、分化和迁移研究

目的探索从成人脑组织获取的神经干细胞（成人-hNSCs）在体外的增殖能力、分化特性、以及在裸鼠颅内的存活、迁移及分化情况。方法分别留取癫痫患者手术切除的颞叶脑组织和10W左右人类自然流产胎儿纹状体组织,体外分离成单细胞悬液,无血清培养基培养、传代并诱导分化。软琼脂糖集落形成实验检测NSCs的增殖能力。免疫荧光法检测NSCs标志物神经上皮巢蛋白（Nestin）和诱导分化后神经元标志物13．tubllin以及神经胶质细胞标志物胶质纤维酸性蛋白（GFAP）的表达;利用动物立体定向仪将体外悬浮培养2W的人NSCs移植入裸鼠颅内,检测NSCs在裸鼠脑组织局部的存活、迁移和分化状况。结果成人-hNSCs集落形成能力较胚胎脑组织来源的NSCs（胎儿-hNSCs）明显减弱,免疫荧光染色显示分离的NSCs呈Nestin阳性,诱导分化后可见（β-tubllin和GFAP阳性的神经细胞,其中80％的细胞为GFAP阳性的星形胶质细胞,20％左右为β-tubllin阳性细胞。分别将成人-hNSCs和胎儿．hNSCs移植入裸鼠纹状体,1个月后,冰冻切片,荧光显微镜观察到来源于成人脑组织来源的NSCs仅见沿针道的近距离迁移,免疫荧光染色在成人-hNSCs移植裸鼠颅内只检测到GFAP阳性的星形胶质细胞。而胎儿-hNSCs可穿过针道,沿大脑廉向脑实质广泛迁移,免疫荧光染色能检测到GFAP和少量（β-tubllin阳性细胞。结论成人脑组织和胚胎纹状体组织中均能成功分离到神经前体细胞,而与胎儿．hNSCs相比,成人-hNSCs体外增殖能力、多向分化潜能和体内迁移能力都明显减弱。     

嗅鞘细胞对脂肪干细胞诱导分化的影响

目的 比较2种不同方法共培养脂肪干细胞后神经元特异性核蛋白(neuron specific nuclear protein,NeuN)的表达.方法 以transwell小室为共培养载体,实验组中将脂肪干细胞接种于上室,嗅鞘细胞接种于下室,对照组下室无嗅鞘细胞,只加入嗅鞘细胞条件培养液,共培养12 d后观察脂肪干细胞NeuN的表达.结果 共培养12 d后2组都有部分脂肪干细胞表达NeuN,实验组NeuN阳性率为303/345(87.8%),而对照组为120/320(37.5%),差异有统计学意义(χ~2=181.6,P＜0.05).结论 脂肪干细胞在与嗅鞘细胞共培养时更容易向神经元样细胞分化.     

Glial cell lineage in the cerebral cortex: a review and synthesis.

The present review is focused on the cell lineage relationships underlying gliogenesis in the cerebral cortex. Studies conducted both in vivo and in vitro suggest that the process of cortical gliogenesis involves a hierarchy of progressively restricted progenitor cell pools. In the cerebral cortex, as well as other areas of the central nervous system, glial cells differentiate from one another through a series of steps that can be defined at molecular, structural, and functional levels. Although the precise timing, sequence, and diversity of the steps involved in cortical gliogenesis are still not fully defined, the emerging picture suggests that both cell lineage and cell-cell interactions play a synergetic role in the determination and maintenance of the proper blend of glial cells in the cerebrum.     

Secretion of nerve growth factor in cultures of glial cells and neurons derived from different regions of the mouse brain

The regional ability of central neurons and glial cells to produce nerve growth factor (NGF) was studied in vitro. NGF secretion was compared in cultures of perinatal astrocytes or embryonic neurons that were derived from various mouse brain structures. No regional differences were detected among cultures of post-natal day 2 glial cells of hippocampal, cortical, striatal, or mesencephalic origin. In all cases, levels of NGF released by the cells were very similar. They were closely correlated to the growth rate as shown by the fact that exponentially growing cells produced relatively more factor than did confluent cells, a finding in agreement with previous observations. Unlike growth-phase cells, primary astrocytes immediately plated at high cell density did not secrete any assayable factor before the 7th day of culture. Levels of NGF found during the following days remained low. In contrast, striking differences were observed among cultures of embryonic neurons. NGF was found in relatively large amounts in cultures of embryonic day 17 or 19 striatal neurons, whereas media conditioned by neurons from the mesencephalon, cortex, or septum contained much less factor. Amounts of NGF assayed in cultures of hippocampal neurons varied with the time of sampling of this brain structure. Levels of factor were significantly higher in media conditioned by embryonic day 19 neurons than in media of embryonic day 17 neurons. However, amounts of NGF found in supernatants of hippocampal neurons remained smaller than those present in cultures of striatal nerve cells. Altogether, the results suggest that, in addition to astrocytes, central neurons may also synthesize and secrete NGF in vitro and that this phenomenum is dependent on both the origin and the developmental stage of the neuronal population.     

Expression of the Huntington’s disease transgene in neural stem cell cultures from R6/2 transgenic mice

Huntington’s disease (HD) is an inherited neurodegenerative disorder resulting in neuronal cell death in discrete brain regions due to an expanded CAG repeat of the huntingtin gene. The transgenic mouse model R6/2 expresses exon 1 of the human huntingtin gene with >150 CAG repeats, which produces mutant HD protein with an expanded poly-glutamine tract. We have established a neuronal stem cell system deriving from transgenic HD R6/2 neonatal brains as a renewable source for neurons and glia to facilitate studies of HD neuropathology and therapies. These R6/2 stem cell cultures can be cryopreserved and revived. Thawed neural progenitors can be expanded, established as continuous cell lines, and induced to differentiate into glia and neurons. Using standard culture conditions, there was no detectable morphological difference between wild type and HDR6/2 cells. Western analysis reveals that R6/2, but not wild type neurospheres, express the expanded repeat transgenic protein. Immunocytochemistry reveals that at a higher antibody concentration, huntingtin can be localized in the nucleus and the cytoplasm of wild type and R6/2 cells. We conclude that the R6/2 neuronal stem cell culture is a valuable tool for investigating HD pathogenesis and potential genetic or pharmacological interventions.     

Effects of lead exposure on proliferation and differentiation of neural stem cells derived from different regions of embryonic rat brain

Lead is a potent neurotoxin, causing brain damage and cognitive deficits in children even at low exposure levels. Although lead neurotoxicity can occur after prenatal or postnatal exposure, little is known of the effects of lead on embryonic neural stem cells (NSCs) or the extent to which NSCs originating in different brain regions may be differentially sensitive to the effects of lead exposure. The present study examined the effects of lead on proliferation and differentiation of neural stem cells (NSCs) originating from E15 rat cortex (CX), striatum (ST) or ventral mesencephalon (VM). Free-floating neurospheres were grown under standard conditions or in lead (0.01-100 microM)-containing conditioned media for 5 days and proliferation assessed by 3H-thymidine uptake. In other studies, control and lead-exposed neurospheres were collected, dissociated and re-plated in control or lead-containing differentiation media for 7 days. Cells were immunostained for visualization of mature neural and glial markers and counted. Lead exposure (0.01-10 microM) had no effect on neurosphere viability but caused a significant dose-dependent inhibition of proliferation in VM and ST but not CX neurospheres. The number of MAP2 positive neurons differentiated from lead-exposed neurospheres of VM and ST origin (but not CX) was significantly decreased from control as were the number of oligodendrocytes obtained, regardless of their region of origin. In contrast, lead exposure significantly increased the number of astrocytes obtained regardless of site of origin. These data suggest that even low levels of lead can differentially affect proliferation and differentiation of embryonic NSCs originating from different brain regions and supports the need for prevention of prenatal lead exposure.     

脂肪干细胞向神经细胞分化的研究现状

源于中胚层的脂肪与骨髓组织中的干细胞,均具有多向分化潜能,在特殊的环境条件诱导下,可向神经细胞分化.多种物质如氢化考地松、丙戊酸、丁羟茴醚、弗司扣林、表皮生长因子和成纤维细胞生长因子、β-巯基乙醇等可对脂肪干细胞向神经细胞的分化进行调控.脂肪来源的干细胞向神经细胞分化应用于中枢神经损伤功能改变有一定的效果,是值得深入研究的课题之一.     

Endogenous factors derived from embryonic cortex regulate proliferation and neuronal differentiation of postnatal subventricular zone cell cultures

In rodents, the subventricular zone (SVZ) harbours neural stem cells that proliferate and produce neurons throughout life. Previous studies showed that factors released by the developing cortex promote neurogenesis in the embryonic ventricular zone. In the present report, we studied in the rat the possible involvement of endogenous factors derived from the embryonic cortex in the regulation of the development of postnatal SVZ cells. To this end, SVZ neurospheres were maintained with explants or conditioned media (CM) prepared from embryonic day (E) 13, E16 or early postnatal cortex. We demonstrate that early postnatal cortex-derived factors have no significant effect on SVZ cell proliferation or differentiation. In contrast, E13 and E16 cortex release diffusible, heat-labile factors that promote SVZ cell expansion through increased proliferation and reduced cell death. In addition, E16 cortex-derived factors stimulate neuronal differentiation in both early postnatal and adult SVZ cultures. Fibroblast growth factor (FGF)-2- but not epidermal growth factor (EGF)-immunodepletion drastically reduces the mitogenic effect of E16 cortex CM, hence suggesting a major role of endogenous FGF-2 released by E16 cortex in the stimulation of SVZ cell proliferation. The evidence we provide here for the regulation of SVZ cell proliferation and neuronal differentiation by endogenous factors released from embryonic cortex may be of major importance for brain repair research.