大鼠胎脑神经干细胞的培养分化及鉴定的实验研究

目的建立大鼠胎脑皮质神经干细胞的培养、扩增、诱导分化及鉴定的方法。方法选取孕14d胎鼠的大脑皮质作为细胞来源,在无血清培养基中添加B27、碱性成纤维细胞因子、表皮生长因子,建立胚胎神经干细胞的体外培养体系,用5%的胎牛血清诱导神经干细胞分化,用免疫荧光染色技术进行对神经干细胞及诱导分化细胞的鉴定。结果原代培养的神经干细胞折光性强,培养第2d开始形成细胞集落,第3d形成大的神经球。3～5代传代的细胞生长稳定。经胎牛血清诱导后第3d开始,神经球开始向周边发出突起,悬浮的细胞开始贴壁生长。原代及传代培养的神经球表达Nestin阳性,分化细胞分别表达NSE、GFAP和GALC阳性。结论应用无血清培养技术可在体外培养、扩增出神经干细胞。5%的胎牛血清可以诱导神经干细胞向神经元、星形胶质细胞和少突胶质细胞等成熟细胞分化。     

Folic acid deficiency inhibits neural rosette formation and neuronal differentiation from rhesus monkey embryonic stem cells

Evidence from epidemiological studies has proved that periconceptional use of folic acid (FA) can significantly reduce the risk of neural tube defects (NTDs). However, it is hard to explore when and how FA plays roles in neurogenesis and brain development in vivo, especially in human or other nonhuman primate systems. Primate embryonic stem cell (ESC) lines are ideal models for studying cell differentiation and organogenesis in vitro. In the present study, the roles of FA in neural differentiation were assessed in a rhesus monkey ESC system in vitro. The results showed no significant difference in the expression of neural precursor markers, such as nestin, Sox-1, or Pax-6, among neural progenitors obtained from different FA concentrations or with the FA antagonist methotrexate (MTX). However, FA depletion decreased cell proliferation and affected embryoid body (EB) and neural rosette formation, as well as neuronal but not neuroglia differentiation. Our data imply that the ESC system is a suitable model for further exploring the mechanism of how FA works in prevention of NTDs in primates.     

In vitro culture and differentiation of rat embryonic midbrain-derived neural stem cells

BACKGROUND: Midbrain-derived neural stem cells (mNSCs) can differentiate into functional mature dopamincrgic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson's disease. OBJECTIVE: To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING: An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital (China) from March to November 2007. MATERIALS: Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein (GFAP) antibody and cyclic nucleotide 3'-phosphohydrolase (CNPase) antibody were provided by Abeam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) were provided by R＆D Systems. METHODS: The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/Fl2, 1% N2 supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10 μg/mL polylysine and induced to differentiate in the DMEM/Fl2 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES: The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for βⅢ tubulin, GFAP and CNPase. RESULTS: Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tubulin. CONCLUSION: Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells following induction by EGF, FGF2 and N2 additive.     

In vitro culture and differentiation of rat embryonic midbrain-derived neural stem cells*☆

BACKGROUND： Midbrain-derived neural stem cells （mNSCs） can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson＇s disease. OBJECTIVE： To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING： An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital （China） from March to November 2007. MATERIALS： Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein （GFAP） antibody and cyclic nucleotide 3＇-phosphohydrolase （CNPase） antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor （EGF） and fibroblast growth factor-2 （FGF2） were provided by R＆D Systems. METHODS： The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/FI 2, 1% N： supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10ug/mL polylysine and induced to differentiate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES： The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for β Ⅲ tubulin, GFAP and CNPase. RESULTS： Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tu     

Reactive astrogliosis induces astrocytic differentiation of adult neural stem/progenitor cells in vitro

Neural stem cells reside in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells (NSPCs) isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Recent studies have shown that endogenous or grafted NSPCs are activated after an injury and migrate toward lesioned areas. In these areas, reactive astrocytes are present and secrete numerous molecules and growth factors; however, it is not currently known whether reactive astrocytes can influence the lineage selection of NSPCs. We investigated whether reactive astrocytes could affect the differentiation, proliferation, and survival of adult NSPCs by modelling astrogliosis in vitro, using mechanical lesion of primary astrocytes. Initially, it was found that conditioned medium from lesioned astrocytes induced astrocytic differentiation of NSPCs without affecting neuronal or oligodendrocytic differentiation. In addition, NSPCs in coculture with lesioned astrocytes also displayed increased astrocytic differentiation and some of these NSPC-derived astrocytes participated in glial scar formation in vitro. When proliferation and survival of NSPCs were analyzed, no differential effects were observed between lesioned and nonlesioned astrocytes. To investigate the molecular mechanisms of the astrocyte-inducing activity, the expression of two potent inducers of astroglial differentiation, ciliary neurotrophic factor and leukemia inhibitory factor, was analyzed by Western blot and shown to be up-regulated in conditioned medium from lesioned astrocytes. These results demonstrate that lesioned astrocytes can induce astroglial differentiation of NSPCs and provide a mechanism for astroglial differentiation of these cells following brain injury.     

三种底物对体外培养的神经干细胞分化和迁移能力的影响

目的 :探讨不同底物对体外培养的神经干细胞分化和迁移能力的影响。 方法 :采用多聚鸟氨酸、层黏连蛋白、鼠尾胶原等作为底物 ,观察它们对大鼠神经干细胞分化的诱导和对细胞迁移能力的影响。 结果 :三种底物均能诱导神经干细胞的分化 ,诱导分化的能力为层黏连蛋白 >多聚鸟氨酸 >鼠尾胶原 ,多聚鸟氨酸与层黏连蛋白两者联用具有协同叠加效应 ,而且这些底物也具有介导分化后的神经细胞迁移的能力 ,各种底物对细胞迁移影响力的大小与其诱导分化的能力相似。 结论 :多聚鸟氨酸、层黏连蛋白和胶原不同程度上促进神经干细胞分化和迁移。     

Gastrodin blocks neural stem cell differentiation into glial cells mediated by kainic acid

Kainic acid can simulate excitatory amino acids in vitro.Neural stem cells,isolated from newborn Wistar rats,were cultured in vitro and exposed to 100-4 000 μM kainic acid for 7 days to induce neuronal cell differentiation,causing the number of astrocytes to be significantly increased.Treatment with a combination of 0.5 mg/L gastrodin and kainic acid also caused the number of differentiated neurons to be significantly increased compared with treatment with kainic acid alone.Experimental findings suggest that gastrodin reduces the excitability of kainic acid and induces neural stem cell differentiation into neurons.     

CNTF对神经干细胞体外增殖的影响

目的 探讨睫状神经营养因子(ciliary neurotrophic factor,CNTF)对神经干细胞体外增殖的影响.方法 原代培养新生SD大鼠的室下区神经干细胞并鉴定,经传代纯化后,用不同浓度CNTF (0.1、0.5、1、5、10、20、30 ng/ml)处理5～7 d,MTT实验检测神经干细胞增殖活性.不完全...     

In vitro differentiation of neural stem cells derived from human olfactory bulb into dopaminergic‐like neurons

This study describes a new accessible source of neuronal stem cells that can be used in Parkinson's disease cell transplant. The human olfactory bulb contains neural stem cells (NSCs) that are responsible for neurogenesis in the brain and the replacement of damaged cellular components throughout life. NSCs are capable of differentiating into neuronal and glial cells. We isolated NSCs from the olfactory bulb of brain‐death donors and differentiated them into dopaminergic neurons. The olfactory bulb tissues obtained were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F12, B27 supplemented with basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor. The NSCs and proliferation markers were assessed. The multipotentiality of olfactory bulb NSCs was demonstrated by their capacity to differentiate into neurons, oligodendrocytes and astrocytes. To generate dopaminergic neurons, olfactory bulb NSCs were differentiated in neurobasal medium, supplemented with B27, and treated with sonic hedgehog, fibroblast growth factor 8 and glial cell‐derived neurotrophic factor from the 7th to the 21st day, followed by detection of dopaminergic neuronal markers including tyrosine hydroxylase and aromatic l ‐amino acid decarboxylase. The cells were expanded, established in continuous cell lines and differentiated into the two classical neuronal phenotypes. The percentage of co‐positive cells (microtubule‐associated protein 2 and tyrosine hydroxylase; aromatic l‐amino acid decarboxylase and tyrosine hydroxylase) in the treated cells was significantly higher than in the untreated cells. These results illustrate the existence of multipotent NSCs in the adult human olfactory bulb that are capable of differentiating toward putative dopaminergic neurons in the presence of trophic factors. Taken together, our data encourage further investigations of the possible use of olfactory bulb NSCs as a promising cell‐based therapeutic strategy for Parkinson's disease.     

Time-sensitive effects of hypoxia on differentiation of neural stem cells derived from mouse embryonic stem cells in vitro

Abstract

Objectives:

Oxygen tension is an important component of microenvironment for the differentiation of embryonic stem cells including neural lineage. However, the comprehensive influence of hypoxia on neural differentiation during embryonic neural development has not yet been examined.

Methods:

In this study, we investigated the effect of low oxygen levels (5% O₂), or hypoxia, in two stages of neural differentiation in vitro: (1) inducing mouse embryonic stem cells into neural stem cells (NSCs); and then (2) inducing NSCs into neural progenitor cells in neurospheres.

Results:

In the first stage, NSCs generation was reduced under hypoxia. Less mature morphological changes (including neural marker) of NSCs were observed, suggesting the prevention of early differentiation under hypoxic conditions. Thus undifferentiated stem cells were maintained in this stage. However, in the second stage, hypoxia induced neural differentiation in neurospheres. Nevertheless, non-neural progenitor cell formation, such as mesoderm progenitor cell lines or epithelial cell lines, was restricted by low oxygen tension.

Discussions:

Our results demonstrate that hypoxia is essential for regulating neural differentiation and show the different effects on NSC differentiation dependent on the time-course of NSC development. In the early stage of NSCs induction, hypoxia inhibits neural differentiation and maintains the undifferentiated state; in the later stage of NSCs induction, hypoxia induces neural differentiation. Our study may contribute to the development of new insights for expansion and control of neural differentiation.     

大鼠神经干细胞体外培养

目的:介绍一种较成熟的大鼠神经干细胞体外培养方法。方法:取新生大鼠(1d之内)侧脑室前角周围的脑组织,用EDTA:胰蛋白酶(1:1)消化,制成单细胞悬液,以2-5×105个细胞/ml接种于无血清培养基中。观察其增殖、分化情况,并用nestin、GFAP、NSE免疫细胞化学对细胞进行定性。结果:培养细胞呈悬浮生长,具有增殖能力,倍增时间为2d,nestin阳性。诱导分化后细胞贴壁生长,体积增大,并发出形态各异的突起。可见GFAP和NSE阳性细胞。结论:培养细胞经鉴定为神经干细胞。本方法简单、易操作,可行性强。     

Effects of olfactory ensheathing cells on the proliferation and differentiation of neural stem cells

BACKGROUND:Olfactory ensheathing cells can promote oriented differentiation and proliferation of neural stem cells by cell-secreted neural factors. OBJECTIVE:To observe the effect of olfactory ensheathing cells on the differentiation and proliferation of neural stem cells. DESIGN,TIME AND SETTING:Cytology was performed at the Department of Neurology,Tongji Medical College,Huazhong University of Science and Technology,China,from September 2007 to October 2008. MATERIALS:Mouse anti-nestin polyclonal antibo...     

胚胎大鼠脑纹状体和中脑神经干细胞的培养

目的研究大鼠脑不同部位胚胎神经干细胞的增殖分化特性。方法采用无血清培养基分离和培养大鼠脑的纹状体和中脑的神经干细胞,通过巢蛋白(nestin)表达和5-溴脱氧尿嘧啶(5-bromo-2'-deoxyuridine BrdU)染色,鉴定细胞的增殖能力;通过新生神经元、星形胶质细胞和少突胶质细胞的特异性免疫细胞化学染色,鉴定培养细胞的多潜能性。通过酪氨酸羟化酶的染色(tyrosine hydroxylase,TH)鉴定多巴胺神经元。结果二者在体外培养都可增殖成球,并能分化成神经系统3个谱系的细胞。纹状体增殖传代3个月,中脑培养细胞增殖维持3周。中脑干细胞分化TH阳性细胞比例高于纹状体。结论培养的胎鼠脑细胞是神经干细胞。纹状体干细胞增殖能力高于中脑干细胞,中脑干细胞更倾向于分化成TH阳性细胞。     

Effects of neurotrophin-3 on the differentiation of neural stem cells into neurons and oligodendrocytes

In this study,cells from the cerebral cortex of fetal rats at pregnant 16 days were harvested and cultured with 20 μg/L neurotrophin-3.After 7 days of culture,immunocytochemical staining showed that,22.4% of cells were positive for nestin,10.5% were positive for β-III tubulin(neuronal marker),and 60.6% were positive for glial fibrillary acidic protein,but no cells were positive for O4(oligodendrocytic marker).At 14 days,there were 5.6% nestin-,9.6% β-III tubulin-,81.1% glial fibrillary acidic protein-,and 2.2% O4-positive cells.In cells not treated with neurotrophin-3,some were nestin-positive,while the majority showed positive staining for glial fibrillary acidic protein.Our experimental findings indicate that neurotrophin-3 is a crucial factor for inducing neural stem cells differentiation into neurons and oligodendrocytes.     

Wnt3a影响神经干细胞分化的体外研究

目的探讨Wnt3a对胚胎大鼠海马神经干细胞（NSCs）体外分化的影响。方法采用机械分离、无血清传代培养法从胎鼠海马中获得NSCs,使用免疫荧光法对其干细胞特性及其受体Fzd3蛋白表达进行鉴定,观察Wnt3a对NSCs体外分化的影响。结果海马NSCs表达特异性标志物巢蛋白及胞膜蛋白Fzd3;体外诱导分化,Wnt3a处理组神经元及星形胶质细胞分化的比例分别为11．25％±0．62％和56．26％±4．82％,而对照组则为8．54％±0．48％和168．42％±5．54％;组间分化差异具有统计学意义（P〈0．05）。结论体外环境下Wnt3a能够促进NSCs向神经元分化,并抑制其向星形胶质细胞分化。     

Wnt-1对室管膜前下区神经干细胞体外分化的影响

目的 探讨室管膜前下区(anterior subventricular zone，SVZa)神经干细胞体外分化过程中，Nestin和Wnt-1表达的变化，以及Wnt-1对SVZa神经干细胞体外分化的影响。方法以免疫荧光染色方法观察在SVZa神经干细胞体外分化过程中，Nestin和Wnt-1在不同时间点的表达水平，同时以基因转染方法增强SVZa神经干细胞Wnt-1表达，观察Nestin、Wnt-1和Mash-1的表达以及神经元分化比例的改变。结果 (1)Nestin主要表达于SVZa神经干细胞分化的早期阶段，随着细胞的分化和成熟，Nestin的表达迅速减弱。(2)SVZa神经干细胞贴壁分化2h即有Wnt-1表达，其表达高峰在细胞分化开始后12h，以后随着细胞分化的进行而迅速降低。(3)SVZa神经干细胞转染Wnt-1基因后，Nestin的表达减弱，Mash-1的表达增强，神经元的分化比例增高，Mash-1的增强与神经元分化之间有很高的一致性。结论 (1)Wnt-1表达于SVZa神经干细胞分化过程之中，在细胞分化成熟后则不表达，Wnt-1可能参与调节SVZa神经干细胞的体外分化，并可作为神经干细胞处于分化状态的标志。(2)SVZa神经干细胞转染Wnt-1后表达Mash-1增强，并更多分化为神经元，Wnt-1可能通过Mash-1途径促使SVZa神经干细胞向神经元方向分化。     

Derivation of primitive neural stem cells from human-induced pluripotent stem cells

Human-induced pluripotent stem cells (hiPSCs) have facilitated studies on organ development and differentiation into specific lineages in in vitro systems. Although numerous studies have focused on cellular differentiation into neural lineage using hPSCs, most studies have initially evaluated embryoid body (EB) formation, eventually yielding terminally differentiated neurons with limited proliferation potential. This study aimed to establish human primitive neural stem cells (pNSCs) from exogene-free hiPSCs without EB formation. To derive pNSCs, we optimized N2B27 neural differentiation medium through supplementation of two inhibitors, CHIR99021 (GSK-3 inhibitor) and PD0325901 (MEK inhibitor), and growth factors including basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (hLIF). Consequently, pNSCs were efficiently derived and cultured over a long term. pNSCs displayed differentiation potential into neurons, astrocytes, and oligodendrocytes. These early NSC types potentially promote the clinical application of hiPSCs to cure human neurological disorders.     

经脑室植入神经干细胞在脑室周围白质软化新生大鼠脑内的迁移分化

背景：对新生动物进行脑内神经干细胞移植,所植入神经干细胞的增殖、迁移、分化、轴突形成以及髓鞘化等能力,均远远高于成年动物。 目的：对经脑室植入神经干细胞在脑室周围白质软化新生大鼠脑内的迁移分化功能进行观察,探讨神经干细胞移植对治疗早产儿脑室周围白质软化的可行性。 方法：2日龄脑室周围白质软化新生大鼠在建模后72 h进行经脑室神经干细胞移植。外源性神经干细胞用PKH26荧光素标记。脑立体定位仪下经脑室穿刺部位：前-后=1.5 mm,中线－外侧=-2 mm,深度=1.5 mm;移植细胞浓度为5×1010 L-1;移植总量为2 μL;移植速度0.5 μL/min。应用激光共聚焦显微镜分别对植入神经干细胞于植入后1,2,3,7,14,21 d进行动态观察。并分别进行各分化细胞的免疫荧光分析。 结果与结论：应用激光共聚焦显微镜对植入神经干细胞进行动态观察,证实经脑室植入的外源性神经干细胞在脑内具有良好的迁移能力,3 d内大部分移行至脑室周围区域,并分布在损伤严重部位。2周左右,神经干细胞在脑室周围区域主要分化为OL前体,部分分化为神经元及星形胶质细胞。提示经脑室神经干细胞移植对早产儿脑室周围白质软化具有很大的治疗潜力。     

慢病毒介导CD-TK融合基因转染对神经干细胞体外增殖的影响

目的探讨慢病毒介导胞嘧啶脱氨酶（cytosine deaminase,CD）及胸腺嘧啶激酶（thymidine kinase,TK）融合基因转染对神经干细胞体外增殖的影响。方法原代培养新生Wistar大鼠室下区神经干细胞并鉴定,经传代纯化后,用CD-TK基因包装的慢病毒处理3～5 d。MTT实验检测神经干细胞增殖活性,免疫荧光技术检测转染后神经干细胞GFAP、NSE表达。结果 MTT结果显示CD-TK融合基因转染对神经干细胞增殖无影响（P〉0.05）。免疫荧光结果显示：神经干细胞Nestin标记阳性;CD-TK融合基因转染后,神经干细胞NSE及GFAP均表达阳性。结论 CD-TK融合基因转染对神经干细胞增殖无影响。