Cellular composition of long-term human spinal cord- and forebrain-derived neurosphere cultures

In vitro expanded neural precursor cells (NPCs) may provide a stable source for cell therapy. In search of the optimal cell source for spinal cord repair, we investigated influences of gestational age, regional heterogeneity, and long-term in vitro propagation. The cellular content of neurosphere cultures prior to and after in vitro differentiation was studied by immunocytochemistry and flow cytometry. Human forebrain and spinal cord NPCs deriving from first-trimester tissue were cultured as neurospheres in the presence of epidermal growth factor, basic fibroblast growth factor, and ciliary neurotrophic factor. Proteins characteristic for embryonic stem cells, i.e., Tra-1-60, Tra-1-81, and SSEA-4, were present in approximately 0.5% of the cells in donor tissues and neurospheres. The proportions of nestin- and proliferating cell nuclear antigen-immunoreactive (IR) cells were also maintained, whereas the CD133-IR population increased in vitro. Glial fibrillary acidic protein-IR cells increased in number, and in contrast the fraction of beta-tubulin III-IR cells decreased, at and beyond passage 5 in spinal cord but not forebrain cultures. However, dissociated and in vitro-differentiated forebrain- and spinal cord-derived neurospheres generated similar proportions of neurons, astrocytes, and oligodendrocytes. Gestational age of the donor tissue, which ranged from 4.5 to 12 weeks for forebrain and from 4.5 to 9.5 weeks for spinal cord, did not affect the proportion of cells with different phenotypes in culture. Thus, cellular composition of human neurosphere cultures differs as a result of long-term in vitro propagation and regional heterogeneity of source tissue, despite expansion under equal culture conditions. This could in turn imply that human spinal cord and forebrain NPCs present different repair potentials in in vivo settings.     

人类神经干细胞的长期培养和传代

目的 探讨人类神经干细胞的体外培养条件及其传代的方法。方法 采用机械方法从胎脑中分离神经细胞，应用N2培养基进行培养，bFGF和EGF刺激细胞扩增；传统方法和对神经球切割的方法进行传代培养；应用免疫组织化学染色对培养的细胞及其分化的细胞进行鉴定。结果 从胎脑当中成功培养出人类的神经干细胞，培养条件下呈悬浮状态生长，形成神经球，绝大多数的细胞表达波形蛋白和Musashil两种神经干细胞的标志物；这种细胞可分化为神经元和星型胶质细胞，早期的培养有少量的少突胶质细胞；在这种培养条件下，神经干细胞生长速度较慢，而采用切割神经球的方法保持了细胞间的，神经干细胞可获得较大的扩增速度。结论 在体外的培养条件下，可从胎脑组织中培养出神经干细胞，它可做为中枢神经系统疾病移植治疗的潜在细胞来源。     

Unique gene expression patterns indicate microglial contribution to neural stem cell recovery following irradiation

Ionizing radiation results in damage to neural stem cells and reduced neurogenesis. The aim of the present study was to determine intrinsic and extrinsic factors that influence neural stem cell survival following irradiation, using qPCR. Gene expression of hippocampal and SVZ neurospheres were analyzed following irradiation, and results demonstrated that irradiated hippocampal and SVZ stem cells displayed similar gene expression profiles for intrinsic genes. Irradiated microglia (extrinsic factor) isolated from the SVZ exhibited increased gene expression of growth factors involved in stem cell maintenance, proliferation, and survival. However, microglial genes in the irradiated hippocampus responded less favorably with respect to stem cell recovery. This might explain the superior recovery of SVZ compared to hippocampal stem cells following in vivo irradiation. In addition, our results show that a combination of growth factors, which were upregulated in SVZ microglia, increased the proliferation and decreased cell death of irradiated neurospheres in vitro.     

恶性胶质瘤细胞株U251中肿瘤干细胞的分离、培养及鉴定

目的:从恶性胶质瘤细胞株U251中分离、培养并鉴定肿瘤干细胞。方法:将U251细胞置于含EGF、bFGF、LIF及B27的无血清培养基中培养,形成悬浮生长的细胞球后经免疫磁珠分离获取CD133阳性细胞,采用单细胞克隆法继续在上述培养液中培养。应用细胞免疫荧光染色对肿瘤干细胞及其分化细胞进行鉴定。结果:在恶性胶质瘤细胞株U251中成功分离出肿瘤干细胞,在上述无血清培养液中呈悬浮生长,具有很强的自我更新与繁殖能力,免疫荧光染色显示该细胞表达CD133,诱导分化后可分化成为神经元与星形胶质细胞。结论:体外培养的恶性胶质瘤细胞株U251中存在脑肿瘤干细胞,并能将其分离、培养及诱导分化。     

Microglia instruct subventricular zone neurogenesis

Microglia are increasingly implicated as a source of non-neural regulation of postnatal neurogenesis and neuronal development. To evaluate better the contributions of microglia to neural stem cells (NSCs) of the subventricular neuraxis, we employed an adherent culture system that models the continuing proliferation and differentiation of the dissociated neuropoietic subventricular tissues. In this model, neuropoietic cells retain the ability to self-renew and form multipotent neurospheres, but progressively lose the ability to generate committed neuroblasts with continued culture. Neurogenesis in highly expanded NSCs can be rescued by coculture with microglial cells or microglia-conditioned medium, indicating that microglia provide secreted factor(s) essential for neurogenesis, but not NSC maintenance, self-renewal, or propagation. Our findings suggest an instructive role for microglial cells in contributing to postnatal neurogenesis in the largest neurogenic niche of the mammalian brain.     

体外培养人类神经干细胞神经球中髓鞘的形成

目的：探索人类神经干细胞的体外培养条件和其形成的神经球的超微结构。方法：从胎脑皮质中机械分离细胞，N2培养基培养和扩增人类神经干细胞，应用免疫细胞化学方法对培养的细胞进行鉴定。对形成的神经球应用透射电镜进行超微结构研究。结果：从胎儿的脑皮质中成功分离培养出神经干细胞，形成典型的神经球，大部分细胞表达神经干细胞的标志物波形蛋白，细胞贴壁后可诱导分化 为神经元和胶质。早期培养的神经球中有典型的髓鞘形成。结论：从胎儿的脑皮质中成功地分离、培养出人类的神经干细胞，细胞呈悬浮状态生长，形成神经球。这种早期培养的神经干细胞有形成髓鞘的能力，可作为治疗人类脱髓鞘病变的潜在细胞来源。     

Behavior of hippocampal stem/progenitor cells following grafting into the injured aged hippocampus

Multipotent neural stem/progenitor cells (NSCs) from the embryonic hippocampus are potentially useful as donor cells to repopulate the degenerated regions of the aged hippocampus after stroke, epilepsy, or Alzheimer's disease. However, the efficacy of the NSC grafting strategy for repairing the injured aged hippocampus is unknown. To address this issue, we expanded FGF-2-responsive NSCs from the hippocampus of embryonic day 14 green fluorescent protein-expressing transgenic mice as neurospheres in vitro and grafted them into the hippocampus of 24-month-old F344 rats 4 days after CA3 region injury. Engraftment, migration, and neuronal/glial differentiation of cells derived from NSCs were analyzed 1 month after grafting. Differentiation of neurospheres in culture dishes or after placement on organotypic hippocampal slice cultures demonstrated that these cells had the ability to generate considerable numbers of neurons, astrocytes, and oligodendrocytes. Following grafting into the injured aged hippocampus, cells derived from neurospheres survived and dispersed, but exhibited no directed migration into degenerated or intact hippocampal cell layers. Phenotypic analyses of graft-derived cells revealed neuronal differentiation in 3%-5% of cells, astrocytic differentiation in 28% of cells, and oligodendrocytic differentiation in 6%-10% cells. The results demonstrate for the first time that NSCs derived from the fetal hippocampus survive and give rise to all three CNS phenotypes following transplantation into the injured aged hippocampus. However, grafted NSCs do not exhibit directed migration into lesioned areas or widespread neuronal differentiation, suggesting that direct grafting of primitive NSCs is not adequate for repair of the injured aged brain without priming the microenvironment.     

Preparation of a tissue-like cortical primary culture from embryonic rats using Matrigel and serum free Start V Medium

To address the scientific quest for unravelling signalling pathways crucial in CNS development and function, cell culture systems have to be developed that are mimicking the physiological state of brain cells more efficiently. Here, we describe a method for cultivation of a virtual three-dimensional structure consisting of neural stem cell-derived cell types by using Matrigel as surface substrate and Start V as a serum free medium. We demonstrate that free floating dissociated cells form attached neurospheres from which cells start migration to surrounding areas and develop a virtual three-dimensional cell structure composed of neurons, glia and neural stem cells. Neuronal precursor cells differentiate into cholinergic and GABAergic cells and express vesicle proteins. Further, neuronal cells are interwoven with Nestin positive stem cells and GFAP positive astrocytes. Additionally, oligodendrocytes and microglia can also be detected in this neural tissue-like structure. As an example for studying cell migration we added externally microglial cells (BV2) and performed a confocal time lapse study. It revealed, that co-cultivated microglial cells migrated towards neurospheres within 14 h. Thus, the described method provides a serum free, tissue-like primary cell culture system of neural cells useful for the investigations of basic cell-cell interactions under in vitro conditions.     

Dynamic behavior of cells within neurospheres in expanding populations of neural precursors

Large-scale expansion of neural stem and progenitor cells will be essential for clinically treating the large number of patients suffering from neurodegenerative disorders such as Parkinson's disease. Other applications of neural stem cell technology include further research in areas such as neural development or drug testing. Neural stem cells can be grown in vitro as tissue aggregates known as neurospheres, and in the current study, experiments were performed to determine the spatial arrangement and behavior of the cells within the neurosphere structure. A protocol utilizing sulfonated lipophilic fluorescent dyes was developed to effectively label populations of neural stem and progenitor cells without compromising cell density during culture. Cells retained the labels for at least 7 days. Using the labeling protocol, we discovered that the cells within the neurospheres were mobile and, moreover, the cells on the periphery of the neurospheres could migrate into the center of the neurospheres. Most important, the mixing time of two merging neurospheres was observed to be the same order of magnitude as the neural stem cell doubling time (approximately 20 h). This study is the first to show that the neurosphere system is dynamic, and these results will serve as a stepping stone to more in-depth studies of the neurosphere microenvironment.     

人胚海马神经干细胞体外培养及分化研究

目的 研究人胚胎海马神经干细胞体外长期培养的条件和其在自主分化条件下的分化能力和分化特点。方法 从人胚胎海马分离神经干细胞。采用无血清培养法，进行体外培养、扩增，形成神经球。使神经球贴壁分化，分化培养基不含有任何细胞有丝分裂促进剂。使用5-溴脱氧尿嘧啶核苷(BrdU)标记分裂增生的细胞，观察细胞的分裂增殖情况。使用免疫细胞化学法鉴定神经干细胞及其在不加诱导剂下的自主分化能力。结果 从人胚胎海马分离的神经干细胞具有增殖能力，细胞倍增时间为3．2d。BrdU检测有正在分裂、增殖的细胞。细胞贴壁分化后可以出现Nestin、GFAP、Tuj-1表达阳性的细胞。神经干细胞共培养6个月，传代14代。结论 分离培养的海马神经干细胞具有自我更新和增殖能力，可以长期培养。在不加任何诱导剂的自主分化条件下可以向神经元、胶质细胞分化。少突胶质细胞的培养需要不同的培养条件。分离培养的干细胞具有神经干细胞的特征。可用于基础和临床的相关研究。