胎鼠脊髓源性神经干细胞分离培养与鉴定

目的:研究胎鼠的脊髓源性神经干细胞的分离培养方法并观察其增殖和分化能力。方法:利用显微操作技术分离获得胎鼠脊髓组织、无血清培养技术和酶消化法结合机械法传代培养神经干细胞、免疫细胞化学方法鉴定神经干细胞和分化情况。结果:建立了胎鼠脊髓源性神经干细胞的分离、培养和鉴定的方法,观察到了脊髓源性神经干细胞具有较强的增殖能力,在添加有5ng／mlEGF和5ng／mlbFGF的无血清培养液中可贴壁分化为神经元、少突细胞和星形胶质细胞。结论:在体外培养条件下分离培养的胎鼠脊髓源性神经干细胞具有干细胞的特性即较强的增殖能力和多向分化潜能。     

从人胚胎干细胞畸胎瘤中有效获取间充质干细胞和神经前体细胞

通过人胚胎干细胞(human embryonic stem cells,hESC)体外分化方法和畸胎瘤形成可以分化获得多种成体细胞．但目前尚不清楚是否可以从hESCs畸胎瘤中分离某些特异性细胞．通过体外筛选方法,有效地从hESCs畸胎瘤中分离出神经前体细胞(neural progenitor cells,NPCs)和间充质干细胞(mesenchymal stem cells,MSCs)．这种hESCs畸胎瘤来源的NPCs和MSCs与体内神经前体细胞和间充质干细胞有着相似的分子标记和特性,并具有进一步的分化潜能——分别可以诱导成为神经元、神经胶质细胞、脂肪细胞和骨骼细胞等．根据人胚胎干细胞畸胎瘤中含有不同分化阶段的外胚层、中胚层和内胚层的组织或细胞,认为人胚胎干细胞畸胎瘤可以作为另一个细胞来源以获取多种(包括人胚胎干细胞体外分化难以得到的)各种前体/干细胞和终末分化细胞．     

兔胚胎神经干细胞的分离、培养和鉴别

目的：研究兔胎脑神经干细胞体外生长特性,为探讨神经干细胞的临床应用及神经系统的发育奠定基础。方法：采用含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的N2无血清培养技术,取18天龄兔胚胎脑组织,分离神经干细胞,并观察分离的细胞体外培养、增殖、分化潜能,免疫组化鉴定。结果：从18天龄兔胎脑皮质和纹状体中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈半贴壁状态生长,形成神经球,可传代。细胞呈Nestin免疫反应阳性;在含血清培养基中培养时则分化,分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原。结论：来自兔胎脑神经干细胞能在体外培养、增殖并保持传代能力。无血清N2EGF、bFGF培养基有利于兔胎脑神经干细胞的存活和增殖,含血清培养基能诱导兔胎脑神经干细胞分化。     

科学出版社新书

干细胞及其分化细胞彩色图谱郑月茂,张翊华,张雅蓉著主要展示了编著者多年来科研积累的252幅干细胞及其分化细胞彩色图片,同时展示了其他科研人员有关胚胎干细胞、诱导型多能干细胞、造血干细胞和神经干细胞及其分化细胞的图片6幅。本书介绍了各类干细胞的特性、分离培养和诱     

神经干细胞分离培养方法的介绍

在成体的许多组织中发现了多能干细胞,这些干细胞可以进行自我复制,参与组织的正常修复。神经干细胞在体外能分化为神经元、星形胶质细胞和少突胶质细胞,并具有多向分化潜能。成体神经干细胞和胚胎干细胞都能分化成成体神经系统中的各种神经细胞。神经干细胞具有自我更新能力,因此神经干细胞可以应用于神经损伤或者神经疾病的修复。本文概述了神经干细胞体外分离培养的方法及其生长影响因子。     

Notch信号通路对神经干细胞增殖分化的作用

神经干细胞作为一种具有自我更新能力和多向分化潜能的细胞,它的增殖和分化受到多种源于自身或外在、邻近或远程细胞信号通路的调控,各种细胞因子及胞间通讯在神经干细胞的增殖和分化中发挥着重要的作用。近年来的多种研究表明,Notch信号通路正是这样一种可以通过相邻细胞的配体与受体相互作用,从而传递信号,进一步发挥其生物学功能的重要信号通路。该通路参与了神经干细胞维持自我形态及向多种具有不同功能的神经细胞分化的过程．对于研究神经干细胞的增殖和分化具有巨大的意义。该文将就当前Notch信号通路对神经干细胞增殖分化影响的相关研究进行简要综述。     

成年鼠脑中神经干细胞的纯化

神经干细胞具有自我复制和多向分化的潜能 ,它可分化为成熟神经元和神经胶质细胞 ,人们希望利用神经干细胞的分化潜能治疗帕金森病等神经系统疾病 ,但首先必须分离纯化这些细胞。以往 ,人们从发育外周神经系统中纯化神经干细胞 ,而脑中干细胞纯化率从未超过 5 %。最近 ,ＲｏｄｎｅｙＬ .Ｒｉｅｔｚｅ等以 80 %的纯化率分离成年鼠脑干细胞并检测这些细胞的特性。他们发现 ,其中一种可在室管膜和脑室下腔区域找到的干细胞活性很强 ,在 ｑｕｅｒｋｏｐｆ变异鼠 (一种嗅觉神经元缺陷的鼠 )中 ,这种细胞选择性缺失 ,提示它可能是离体主要的功能性…     

成体干细胞可塑性的事实、质疑和展望

成体干细胞的可塑性是指存在于成年组织或器官中的不成熟细胞跨胚层分化的一种能力。近年来相关研究很多,有人认为成体干细胞具有可塑性,如造血干细胞可以分化为神经外胚层细胞和内胚层细胞：有人对其持怀疑态度,认为成年造血干细胞发育可塑性证据不足,成体干细胞不能跨胚层分化。由于分离纯化、检测手段等的局限,大多数研究均存在这样或那样的不足和误区,彻底研究清楚还有很长的路要走。     

RCMV感染星形胶质细胞对神经干细胞分化的影响

探讨大鼠巨细胞病毒（rat cytomegalovirus,RCMV）感染大鼠星形胶质细胞后,对神经干细胞分化的影响。原代分离培养新生大鼠星形胶质细胞和胚胎海马神经干细胞,将星形胶质细胞感染RCMV后和神经干细胞在Transwell24孔共培养体系下进行共培养,同时设对照组;用免疫荧光染色等方法检测神经干细胞与感染RCMV的星形胶质细胞共培养后,其分化细胞中神经元微管相关蛋白（microtubule-associated protein 2,MAP2）和星形胶质细胞胶质纤维酸性蛋白（glial fibril—lary acidic protein,GFAP）的表达。结果发现,感染RCMV的星形胶质细胞与神经干细胞共培养时,神经干细胞分化减慢,分化成的神经元和星形胶质细胞比率低于对照组,提示星形胶质细胞感染RCMV后可抑制神经干细胞的分化,可能与RCMV影响星形胶质细胞合成和分泌各种营养因子,干扰了神经干细胞的分化进程有关。     

脐血干细胞向不同体细胞分化的研究进展

脐血干细胞是一种具有多分化潜能的原始细胞,具备自我更新和增殖的能力,并能在特定因素的影响或诱导下,向多种细胞或组织分化。脐血来源的间充质干细胞不但可以分化为骨、脂肪和软骨,还可以转变成带有神经、肝脏及骨骼肌特异标记的细胞,并且具有应用到组织损伤修复、基因治疗载体和造血干细胞共移植等方面的潜力。旨在对于脐血干细胞在一定条件下分化为多种细胞研究进展进行综述。     

Establishment and characterization of a pluripotent stem cell line derived from human amniotic membranes and initiation of germ layers in vitro

OBJECTIVES: Pluripotent stem cells are proposed to be used in regenerative therapy and may exist in the human amniotic membrane. The present article is aimed at establishing a pluripotent stem cell line from human placenta. METHODS: HAM-1 (stem cell line derived from human amniotic membranes) was established by the colonial cloning technique using aMEM culture medium containing 10 ng/ml of EGF, 10 ng/ml of hLIF and 10% fetal bovine serum. RESULTS: HAM-1 cells appeared to maintain a normal karyotype indefinitely in vitro and expressed markers characteristic of stem cells from mice and human, namely alkaline phosphatase. Also, these cells contributed to the formation of chimeric mouse embryoid bodies and gave rise to cells of all germ layers in vitro. CONCLUSIONS: This study demonstrates that human amniotic membranes derived stem cells have a wide developmental capability and might be utilized to regenerate different types of cells or tissues for transplantation therapy.     

Plant-derived human recombinant growth factors and serum albumin maintain stemness of human-induced pluripotent stem cells

Stem cells are an important therapeutic source for recovery and regeneration, as their ability of self-renewal and differentiation offers an unlimited supply of highly specialized cells for therapeutic transplantation. Growth factors and serum are essential for maintaining the characteristics of stem cells in culture and for inducing differentiation. Because growth factors are produced mainly in bacterial (Escherichia coli) or animal cells, the use of such growth factors raises safety concerns that need to be resolved for the commercialization of stem cell therapeutics. To overcome this problem, studies on proteins produced in plants have been conducted. Here, we describe the functions of plant-derived fibroblast growth factor 2 (FGF2) and human serum albumin in the maintenance and differentiation of human-induced pluripotent stem cells (hiPSCs). Plant-derived FGF2 and human epidermal growth factor EGF were able to differentiate hiPSCs into neural stem cells (NSCs). These NSCs could differentiate into neuronal and glial cells. Our results imply that culturing stem cells in animal-free culture medium, which is composed of plant-derived proteins, would facilitate stem cell application research, for example, for cell therapy, by reducing contamination risk.     

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.To further characterize each tumor''s BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 °C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.     

Generation of human cortical neurons from a new immortal fetal neural stem cell line

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.     

光遗传技术通过Wnt/β-Catenin通路对新生神经元的影响*

目的:探讨Wnt/β-catenin信号通路光遗传技术在促进新生神经元成熟中的作用。方法:从胎鼠大脑皮层中提取神经干细胞,用携带DCX-ChR2-EGFP基因的慢病毒感染神经干细胞,观察神经干细胞分化为新生神经元后DCX的表达。实验细胞分为3组(n=9):对照组、NSCs+EGFP和NSCs+ChR2组。其中对照组为正常培养的NSCs(NSCs组);NSCs+EGFP组为携带DCX-EGFP基因慢病毒感染神经干细胞组;NSCs+ChR2组为携带DCX-ChR2-EGFP基因慢病毒感染神经干细胞组。病毒感染后48 h后连续3 d行470 nm蓝激光照射,然后检测各组NeuN⁺阳性细胞(成熟神经元标志物)的密度和NeuN⁺/Hoechst比值情况;Western blot检测各组成熟神经元相关蛋白MAP2、NeuN、Neurog2、NeuroD1和GluR2蛋白表达水平和Wnt/β-catenin通道相关蛋白TCF4和β-catenin蛋白的表达水平。用L-型钙通道阻断剂100 μmol/L维拉帕米或50 μg/ml的β-catenin抑制剂Dkk1处理NSCs+ChR2组细胞,然后行Western blot检测各组MAP2、NeuN、Neurog2、NeuroD1和GluR2蛋白表达水平。结果:连续3 d 470 nm蓝激光照射后,NSCs+ChR2组中NeuN⁺阳性细胞密度(成熟细胞)和NeuN⁺/Hoechst明显高于NSCs组和NSCs+EGFP组(P均＜0.05);Western blot检测的MAP2、NeuN、Neurog2、NeuroD1、GluR2蛋白及Wnt/β-catenin通路相关蛋白β-catenin、TCF4表达水平均明显高于NSCs组和NSCs+EGFP组(P均＜0.01);L-型钙通道阻断剂维拉帕米或β-catenin抑制剂Dkk1处理NSCs+ChR2组细胞后MAP2、Neurog2、NeuroD1和GluR2蛋白表达水平明显下降(P均＜ 0.01),NeuN表达水平也下降(P＜0.05)。证明ChR2通道蛋白开放产生阳离子内流促进新生神经元成熟,是通过Wnt/β-catenin信号通路实现的。结论:光遗传学方法通过Wnt/β-catenin信号通路促进新生神经元成熟。     

Inhibition of neuronal nitric oxide synthase activity promotes migration of human‐induced pluripotent stem cell‐derived neural stem cells toward cancer cells

The breakthrough in derivation of human‐induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells, including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy, we investigated in this study the tumor tropism of hiPSC‐derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC‐NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non‐migratory hiPSC‐NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down‐regulated in migratory hiPSC‐NSCs. Using nNOS inhibitors and nNOS siRNAs, we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC‐NSCs toward cancer cells, and that inhibition of its activity or down‐regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell‐mediated cancer therapy.     

Effects of Homocysteine on ERK Signaling and Cell Proliferation in Fetal Neural Stem Cells In Vitro

The aim of the present study was to determine if the excitatory amino acid homocysteine (Hcy) alters ERK signaling and cell proliferation in fetal neural stem cells (NSCs) in vitro. NSCs were isolated from fetal rats and grown in serum-free suspension medium. The cells were identified as NSCs by their expression of immunoreactive Sox2. NSCs were assigned to one of four treatment groups: vehicle control, low-dose Hcy group (Hcy-L, medium contained 30 μmol/L Hcy), middle-dose Hcy group (Hcy-M, 100 μmol/L Hcy) and high-dose Hcy group (Hcy-H, 300 μmol/L Hcy). Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Protein expression levels of ERK1/2 and phosphorylated ERK1/2 were detected by Western blot. The effects of Hcy on NSC death, including apoptosis, were assessed by using flow cytometry and trypan blue exclusion. The results showed that NSCs grew as neurospheres in the serum-free medium. Hcy decreased ERK1/2 protein phosphorylation and NSC proliferation, but it did not induce cell death or apoptosis within the concentration from 30 to 300 μmol/L. The above results are consistent with the hypothesis that Hcy decreases fetal NSC proliferation by inhibiting ERK signaling.