来自祖，人脑少突胶质细胞培养系统
Summary
小学，人类胎儿脑源性，多能祖细胞增殖<em在体外</em同时保持分化成神经元和星形胶质细胞的能力。这项工作表明，通过调节选择生长因子，神经前体细胞可以被诱导分化阶段的少突胶质谱系。
Abstract
人类神经祖细胞分化成神经元和神经胶质细胞类型进行研究和比较分子调控神经细胞谱系的发展提供了一种模式。从胎儿中枢神经系统组织的神经前体细胞在体外扩增得到很好的特点。尽管成人皮层下白质和发展各种文化条件成髓磷脂生产的少突胶质细胞，少突胶质细胞在体外实验获得足够的人力胎儿神经祖细胞的定向分化的神经胶质祖细胞的识别和分离仍然存在困难。分化的半乳糖脑苷脂+（GALC）和O4 +少突胶质前体细胞或祖细胞神经前体细胞（OPC）从已报道使用第二孕期胎儿的大脑。然而，这些细胞不支持包括星形胶质细胞和神经元的细胞增殖的情况下，都将丢失迅速随着时间的推移在培养。仍需要文化系统的少突胶质细胞谱系适合在体外实验中产生。
文化的初级人少突胶质细胞，例如，可以是一种有用的模型来研究发病机制中的嗜神经感染剂，如人多瘤病毒，JC病毒，即在体 内感染这些细胞。这些培养细胞中也可以提供其他脱髓鞘疾病的中枢神经系统（CNS）的模型。小学，人类胎儿脑源性，多能神经祖细胞在体外增殖的能力，同时保持分化成神经元祖细胞源性神经元，（PDN）和星形胶质细胞（祖衍生的星形胶质细胞，PDA）的研究表明，神经祖细胞可诱导少突胶质谱系发展的阶段，许多（祖衍生的少突胶质细胞，PDO）区分。我们的文化神经祖细胞在DMEM-F12无血清培养基支持中执行与碱性成纤维细胞生长因子碱性成纤维细胞生长因子（bFGF），血小板衍生生长因子（PDGF-AA），刺猬（嘘），神经营养因子3（NT-3），N-2和三碘甲状腺原氨酸（T3）。 2.5e6细胞每75厘米瓶中大约每7天传代培养的细胞。使用这些条件，在培养的细胞的大部分保持形态，其特征在于由几道工序和明文预少突胶质细胞的标记物，如A2B5和O-4。当我们删除的四个生长因子（GF），碱性成纤维细胞生长因子（bFGF，PDGF-AA，嘘，NT-3）和空调媒体PDN，细胞开始获得更多的流程和快速的特定标志物，如GALC和髓鞘少突胶质细胞分化碱性蛋白（MBP）。我们进行了表型特征，使用多色流式细胞仪识别少突胶质细胞的特有标志。
Protocol
注：对于常规的培养的神经祖细胞和少突胶质谱系细胞，孵化完成，在37℃下，在湿润的5％CO 2气氛中。每2天，将培养基更换使用50〜100％的新鲜培养基，如果文化是40-70％汇合。在附近汇合的时间，培养传代在2-2.5e6/T75烧瓶通常在每周计划。 1。准备涂层瓶为了制备涂覆的烧瓶在100毫升去离子水（DI水），然后涂层的T75烧瓶中稀释5毫克的聚-D-赖氨?…
Representative Results
这是非常重要的，从70％-80％汇合的神经前体细胞培养物（ 图1A）启动的分化过程。许多细胞就会死亡后改变培养基从祖寡介质的，因为它包含了特殊的生长因子。这表明，没有承诺的少突胶质型神经祖细胞的增长将不支持新媒体（ 图1B）。孵育在一个星期的培养基+ GF低聚导致在中间文化表现出窄的，双极的形态（ 图1B）。将细胞保持在低聚培养基+ GF为3-4周…
Discussion
本协议描述了如何推导出胎儿主要的人类神经祖细胞和少突胶质细胞的表型的特征，同时使用流式细胞仪和免疫荧光染色。从胎儿中枢神经系统的神经祖细胞的扩张和增长已经非常好1-4。然而，获得足够的人力的少突胶质细胞， 在体外实验仍然很困难的，即使它是可能的识别和分离胶质前体从成人脑白质5-13。已经有不同的尝试各种文化条件下的发展产生少突胶质细胞14…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究是在美国国立卫生研究院，NINDS的院内研究计划。与显微镜的帮助和Pamela C.筛分帮助编辑，作者要感谢所有成员的实验室，分子医学，神经科学，里克·德赖弗斯。
Materials
| Name of the reagent | Company | Catalogue number | Final concentration | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| DME/HAMS F12 1:1 | Omega Scientist | DM-251 | 1X | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Bovine Albumin | Sigma | A9418 | 1% | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Gentamicin | Quality Biologicals | 120-098-031 | 50 μg/ml | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| L-Glutamine | Quality Biologicals | 118-084-061 | 2 mM | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| T3 | Sigma | T2877 | 3 nM | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| N2 Components | Gibco BRL | 17502 | 1:100 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| NT-3 | PeproTech Inc | 450-03 | 2 ng/ml | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Shh | R&D System | 1314-SH/CF | 2 ng/ml | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| bFGF | PeproTech Inc | 100-18B | 20 ng/ml | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| PDGF-AA | PeproTech Inc | 100-13A | 10 ng/ml | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| PDL | Sigma | P6407 | 50 μg/m | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| PFA | Electron Microscopy Sciences | 15712 | 2% | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Trypsin | Quality Biologicals | 118-087-721 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Papain | Worthington | LK003178 | 20 U/ml | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| DNase vials | Worthington | LK003172 | 0.005% | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| EBSS | Worthington | LK003188 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| ProLong Gold with DAPI |
Invitrogen | P36931 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Table 1. Reagents. |
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Table 2. Antibodies (Abs) used for flow cytometry and immunocytochemistry assays. Antibody conjugates: PE, phycoerythrin; PETR, phycoerythrin Texas Red; AMCA, amino-methyl-coumarin-acetate; Cy, cyanine; FITC, fluorescein isothiocyanate. Ig: immunoglobulin. AF: Alexa Fluor; gαm: goat anti-mouse; gαrb: goat anti-rabbit; dαck: donkey anti-chicken. |
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Tags
Progenitor-derived Oligodendrocyte Culture SystemHuman Fetal BrainDifferentiationNeural ProgenitorsNeuronal Cell TypesGlial Cell TypesMolecular RegulationNeural Cell Lineage DevelopmentIn Vitro ExpansionGlial ProgenitorsSub-cortical White MatterCulture ConditionsMyelin Producing OligodendrocytesGalactocerebroside+O4+ Oligodendrocyte Precursor Or Progenitor Cells (OPC)Neural Precursor CellsSupport Cells (astrocytes And Neurons)Culture SystemIn Vitro ExperimentationPrimary Human OligodendrocytesPathogenesisNeurotropic Infectious Agents (human PolyomavirusJCV)Demyelinating DiseasesCentral Nervous System (CNS)
Citar este artigo
Monaco, M. C. G., Maric, D., Bandeian, A., Leibovitch, E., Yang, W., Major, E. O. Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain. J. Vis. Exp. (70), e4274, doi:10.3791/4274 (2012).