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。

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<ol><li>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-赖氨酸（PDL）50微克/毫升的PDL在黑暗中在室温（RT）。 1个半小时后，吸出PDL一次，去离子水冲洗锥形瓶。让烧瓶干播种前的细胞（ 表1）。 </li></ol> 2。开始分化的神经祖细胞源性祖细胞，少突胶质细胞（PDO） 该协议是酚盐人类中枢神经系统祖细胞在以前的出版物，它不属于本协议。人类中枢神经系统祖细胞中分离到前脑的一个为期8周的妊娠胎儿的大脑，按照与美国国立卫生研究院的指导方针。 <ol><li>成长PDL涂层烧瓶中neurobasal培养基补充有25 ng / ml的碱性成纤维细胞生长因子和20 ng / ml的表皮生长因子（EGF）（ 图1A）上的神经祖细胞的多能人口。不要使用他们，如果高于通路（11）（P11）。 </li><li>文化是约70％汇合时，开始分化的神经祖细胞。 </li><li>车型完整分化少突胶质细胞的介质，使用无血清的DMEM / HAMS F12 1:1培养基与牛血清白蛋白，L-谷氨酰胺，庆大霉素，N2成分，T3中，Shh，NT-3，碱性成纤维细胞生长因子，和PDGF-AA（ 表1） 。这种介质将被定义为与生长因子（低聚培养基+ GF）的寡核苷酸介质。 </li><li>删除PROGEN从烧瓶中的itor媒体，漂洗细胞一次磷酸盐缓冲盐水（PBS），然后添加低聚介质+ GF（2.3）中描述的开始少突胶质细胞的分化的详情。只有承诺的少突胶质谱系的细胞生长（ 图1B）。 3周在该培养基中培养细胞。 </li><li>每星期，当细胞是在90-95％汇合时，通过用胰蛋白酶（0.05％）-EDTA（0.1％）（4毫升的T75烧瓶中）。传送整个介质从入50毫升的锥形管中的细胞进行传代，该条件培养基将用于淬灭的胰蛋白酶。确保干出细胞。然后添加胰蛋白酶。 </li><li>培养细胞中的胰蛋白酶在室温下5分钟，轻轻拍打烧瓶数次，以帮助细胞脱落。 </li><li>淬灭的胰蛋白酶，通过与分离的细胞的条件培养基的烧瓶中加入4-5毫升。 </li><li>介质和细胞转移到相同的50毫升锥形管。的离心的培养基和细胞在1,200 rpm（〜300 xg离心）在RT下的5分钟。 </li><li>弃上清，重悬细胞沉淀，轻轻倾斜管，然后加入新鲜的寡核苷酸介质+ GF。轻轻地吸移管培养基和细胞向上和向下，使均匀的细胞悬浮液。 </li><li>计数到每一个新的PDL涂层的T75烧瓶中的细胞，并转移2.5e6。 </li></ol> 3。在少突胶质细胞分化的最后一步<ol><li>培养3周在低聚培养基+ GF，当细胞在70％至80％汇合后，启动的最终分化过程中（ 图1C）。 </li><li>准备培养基中以同样的方式作为寡核苷酸的培养基+ GF，但不添加四GF:Shh的，NT-3，碱性成纤维细胞生长因子，血小板衍生生长因子-AA。这四个GF未经将被定义为寡介质 - GF。 </li><li>吸液寡核苷酸的培养基+ GF从烧瓶中，一次，用PBS冲洗，然后添加介质的总体积为16毫升，¾（12毫升）的寡核苷酸的培养基 - GF和¼（4毫升）是PDN的条件培养基，为6日-10日）。从PDN是条件培养基的使用不是关键的分化过程中或细胞的生存。事实上，我们观察到PDO的唯一的直接接触与PDN细胞在共培养实验产生了影响对PDO生存的长度。生存时间寡媒介 - GF可以延长到两，三个星期，，如果PDO细胞共培养PDN细胞。区分人类神经祖细胞PDN以前的出版物中描述的协议，它不属于本协议。从培养基中的查询结果与多个进程（ 图1D）的细胞培养在逐步分化的生长因子撤出。 </li></ol> 4。流式细胞仪检测流式细胞仪检测比较少突胶质细胞在分化过程中的关系与他们的父母人口标记收购。 <ol><li>使用神经亲捐血者之血袋控制。种子PDO的涂有聚-D-赖氨酸在低聚培养基+ GF两个T75烧瓶中的2.5×10 6个细胞的细胞。 </li><li>一个星期后，更换介质，新鲜的寡核苷酸介质+ GF在烧瓶中控制细胞活力的文化，而其他瓶中寡媒介 - GF。 </li><li>在媒体使用PDO细胞中分离20 U /毫升，木瓜蛋白酶和胰蛋白酶。这是因为，木瓜蛋白酶具有平缓的过程中对细胞的影响保持细胞形态的解离。 </li><li>加入5毫升的Earle氏平衡盐溶液（EBSS）木瓜蛋白酶小瓶。将小瓶在37℃下10分钟或直到木瓜蛋白酶完全溶解，溶液出现清晰。这是一个用于解离细胞的木瓜蛋白酶溶液。 </li><li>添加0.5毫升EBSS一个脱氧核糖核酸（DNA酶）的小瓶（ 表1）。轻轻混匀。添加0.25毫升此溶液溶解的木瓜蛋白酶的小瓶。该制剂包含的最终浓度为约20 U / ml的木瓜蛋白酶和00.005％DNA酶。 </li><li>地点的木瓜蛋白酶溶液（如上面所述）上的细胞，并在37℃下孵育15-30分钟;监测使用显微镜的细胞脱离。用5毫升的介质带或不带GF和7分钟，在1,200 rpm的转速下离心，使反应停止。 </li><li>从两个烧瓶细胞沉淀转移到两个不同的5毫升的聚丙烯管中，并用它们与正常的生理介质（NPM:145毫摩尔NaCl，5 mM KCl中，1.8毫的CaCl 2，10mM的Hepes和10mM葡萄糖），补充有1毫克/毫升牛血清白蛋白（NPM + BSA）。 </li><li>执行表面免疫组化，在4℃下20分钟，使用具体和适当滴定的初级抗体A2B5，O4，GALC（ 表2）。 </li><li>与NPM + BSA洗涤细胞5分钟，并在4℃下，它们与特定的二级抗体，在4℃下孵育20分钟（ 表2）。 </li><li>染色细胞内的抗原，包括巢蛋白，胶质纤维酸性蛋白（GFAP），Ⅲ类た-微管蛋白，髓鞘碱性蛋白（MBP）（ 表2），固定细胞在2％多聚甲醛（PFA）的20分钟，在RT中，于-20℃下的15分钟用70％冷乙醇通透</li><li>用1×PBS洗涤细胞，和（ 表2）与特定的二级抗体孵育。 </li><li>区分的完整细胞和亚细胞碎片在流式细胞术分析，染色细胞与4,6 - 二脒基-2 - phenyllindole，二氢氯化物（DAPI）（Life Technologies公司，纽约州Grand Island）。 </li><li>在优化上述协议，我们进行了适当的控制实验，以确认各免疫试剂的特异性。简单地说，对于直接标记的抗体，适当的阴性对照相同的免疫球蛋白同种型类（或子类）和荧光染料共轭作为初级抗体（ 即 ，同种型对照）是一个直接的共轭的抗体。对于间接免疫的主要抗体，负相应的控制w作为二次抗体结合的荧光染料，专门针对的免疫球蛋白的类（或子类）的主机中，对一次抗体生成感兴趣的。当从多个主机的第一抗体（小鼠，兔，鸡）一起使用时，我们使用了针对每个主机和每个所用的第二抗体的一次抗体的二次抗体，典型的交叉吸附对其他主机的免疫球蛋白从尽量减少交叉主办免疫反应。阳性对照包括已知表达感兴趣的抗原，可以使用直接或间接的与主针对这些抗原的抗体的免疫反应的细胞制剂的直接或间接的免疫染色。上述的优化方法进行一次，为每种类型的免疫染色实验。控制免疫反应，没有发现显着的跨间小学和中学抗体的抗原决定簇的免疫反应。流式细胞仪进行均匀暂停荧光标记的细胞使用一个FACVantage SE细胞分选仪（BD Biosciences公司，圣何塞，加利福尼亚州）配备了3个激光器，它提供了激发波长调整到488纳米，647纳米，和广阔的紫外线（351-364纳米）（ 图2） 。 </li></ol> 5。免疫荧光法 注:对于免疫实验中，PDO镀寡中等+ GF或寡媒介- GF 2.5e5在6孔板涂有PDL。在停药后的生长因子的各时间点，将细胞固定在2％的PFA。抗体染色塑料6孔板，而不是盖玻片上进行。 <ol><li>取出介质，添加2％PFA。在室温下孵育10分钟。丢弃PFA。 ，每次5 min，用PBS洗涤细胞3次。要小心，不要干出细胞。 </li><li>细胞内的标记，用在PBS中的0.25％的仓鼠溶液中10分钟，在RT 3.Permeabilize细胞。 </li><li>为了防止非特异性结合，阻止细胞fHHG（1mM的HEPES缓冲液，2％马血清和10％山羊血清在Hanks&#39;平衡盐溶液）或10分钟。特定的初级抗体（ 表2）稀释到一个预先确定的浓度在HHG。将摊薄到细胞抗体。摇床或在4℃下过夜，在室温下孵育1小时。 </li><li>洗涤细胞3次，每次5分钟，用PBS。稀释适当的二级抗体与荧光染料到一个预先确定的浓度在HHG（ 表2），耦合。将稀释的第二抗体的混合物到细胞上。在室温孵育1小时在振荡器上在黑暗中。 </li><li>洗涤细胞3次，每次5分钟，用PBS。 </li><li>为了避免漂白加入10μl延长黄金DAPI染色的细胞，在它们上面放置盖玻片。可视化用Axiovert 200M的荧光显微镜（Zeiss，索恩伍德，NY），（ 图3）的标记的细胞。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tapping+into+the+glial+reservoir:+cells+committed+to+remaining+uncommitted.">Tapping into the glial reservoir: cells committed to remaining uncommitted.</a> J. Cell Biol. 188, 305-312 (2010).</li><li>Jakovcevski, I., Filipovic, R., Mo, Z., Rakic, S., Zecevic, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrocyte+development+and+the+onset+of+myelination+in+the+human+fetal+brain.">Oligodendrocyte development and the onset of myelination in the human fetal brain.</a> Front Neuroanat. 3, 5 (2009).</li><li>Rao, R. C., Boyd, J., Padmanabhan, R., Chenoweth, J. G., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+serum-free+derivation+of+oligodendrocyte+precursors+from+neural+stem+cell-enriched+cultures.">Efficient serum-free derivation of oligodendrocyte precursors from neural stem cell-enriched cultures.</a> Stem Cells. 27, 116-125 (2009).</li><li>D'Intino, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triiodothyronine+administration+ameliorates+the+demyelination/remyelination+ratio+in+a+non-human+primate+model+of+multiple+sclerosis+by+correcting+tissue+hypothyroidism.">Triiodothyronine administration ameliorates the demyelination/remyelination ratio in a non-human primate model of multiple sclerosis by correcting tissue hypothyroidism.</a> J. Neuroendocrinol. 23, 778-790 (2011).</li><li>Cui, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal+oligodendrocyte+progenitor+cells+from+different+gestational+stages+exhibit+substantially+different+potential+to+myelinate.">Human fetal oligodendrocyte progenitor cells from different gestational stages exhibit substantially different potential to myelinate.</a> Stem Cells Dev. , (2012).</li><li>Messam, C. A., Hou, J., Major, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexpression+of+nestin+in+neural+and+glial+cells+in+the+developing+human+CNS+defined+by+a+human-specific+anti-nestin+antibody.">Coexpression of nestin in neural and glial cells in the developing human CNS defined by a human-specific anti-nestin antibody.</a> Exp. Neurol. 161, 585-596 (2000).</li></ol>

没有利益冲突的声明。

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...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 终浓度 </td></tr><tr><td> DME / HAMS F12 1:1 </td><td>欧米茄科学家</td><td> DM-251 </td><td> 1X </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A9418 </td><td> 1％ </td></tr><tr><td>庆大霉素</td><td>质量生物</td><td> 120-098-031 </td><td> 50微克/毫升</td></tr><tr><td> L-谷氨酰胺</td><td>质量生物</td><td> 118-084-061 </td><td> 2毫米</td></tr><tr><td> T3 </td><td>西格玛</td><td> T2877 </td><td> 3纳米</td></tr><tr><td> N2组件</td><td> Gibco公司</td><td> 17502 </td><td> 1:100 </td></tr><tr><td> NT-3 </td><td> PeproTech公司公司</td><td> 450-03 </td><td> 2纳克/毫升</td></tr><tr><td>嘘</td><td> R＆D系统</td><td> 1314-SH/CF </td><td> 2纳克/毫升</td></tr><tr><td>碱性成纤维细胞生长因子</td><td> PeproTech公司公司</td><td> 100-18B </td><td> 20纳克/毫升</td></tr><tr><td> PDGF-AA </td><td> PeproTech公司公司</td><td> 100-13A </td><td> 10纳克/毫升</td></tr><tr><td> PDL </td><td>西格玛</td><td> P6407 </td><td> 50微克/立方米</td></tr><tr><td> PFA </td><td>电子显微镜科学</td><td> 15712 </td><td> 2％ </td></tr><tr><td>胰蛋白酶</td><td>质量生物</td><td> 118-087-721 </td><td></td></tr><tr><td>木瓜蛋白酶</td><td>沃辛顿</td><td> LK003178 </td><td> 20 U / ml的</td></tr><tr><td> DNA酶瓶</td><td>沃辛顿</td><td> LK003172 </td><td> 0.005％ </td></tr><tr><td> EBSS </td><td>沃辛顿</td><td> LK003188 </td><td></td></tr><tr><td>的ProLong黄金 同DAPI </td><td> Invitrogen公司</td><td> P36931 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1试剂 。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 初级抗体（均在1微克/毫升） </td><td></td><td> 种同型 </td><td> 源 </td><td> 二级抗体 </td></tr><tr><td colspan="5"> 流式细胞仪 </td></tr><tr><td> A2B5-生物素</td><td>鼠标</td><td> IgM抗体</td><td>礼品J.尼尔森</td><td>链霉亲和素PETR，Invitrogen公司，加利福尼亚州</td></tr><tr><td> O4FITC </td><td>鼠标</td><td> IgM抗体</td><td>从J.尼尔森的礼物</td><td></td></tr><tr><td> A2B5 </td><td>鼠标</td><td> IgM抗体</td><td>密理博，MA </td><td> gαmIgM抗体-FITC，Invitrogen公司，加利福尼亚州</td></tr><tr><td> O4 </td><td>鼠标</td><td> IgM抗体</td><td>密理博，MA </td><td> gαmIgM抗体-FITC，Invitrogen公司，加利福尼亚州</td></tr><tr><td> GALC </td><td>鼠标</td><td> IgG的3 </td><td>密理博，MA </td><td> 3，南方生物技术，AL-PEgαm抗体</td></tr><tr><td> MBP </td><td>鸡</td><td>卵黄抗体</td><td>密理博，MA </td><td> dαckIgY的AMCA，:杰克逊免疫。，PA </td></tr><tr><td>巢</td><td>鼠标</td><td>抗体1 </td><td> 2000 28 messam 等。 </td><td> gαm的IgG 1-PECy5，Invitrogen公司，加利福尼亚州</td></tr><tr><td> GFAP </td><td>兔</td><td>抗体</td><td>密理博，MA </td><td> gαrb的IgG-PE，PA，杰克逊免疫</td></tr><tr><td> βIII微管蛋白</td><td>鼠标</td><td>抗体2 </td><td>科文斯，CA </td><td> gαmIgG抗体2α-PETR，Invitrogen公司，CA </td></tr><tr><td colspan="6"> 免疫细胞化学 </td></tr><tr><td> βIII微管蛋白 （1:1,500） </td><td>鼠标</td><td>抗体2A </td><td>科文斯，CA </td><td> gαmIgG的图2a-FITC，Invitrogen公司，加利福尼亚州 （1:1,000） </td></tr><tr><td> GFAP （1:1,000） </td><td>兔</td><td>抗体</td><td>密理博，MA </td><td> ，gαrb，杰克逊的IgG-FITC免疫，PA （1:500） </td></tr><tr><td> MBP （1:50） </td><td>鸡</td><td>卵黄抗体</td><td>密理博，MA </td><td> ，dαck卵黄抗体-FITC，杰克逊免疫，PA （1:100） </td></tr><tr><td> O4 （1:100） </td><td>鼠标</td><td> IgM抗体</td><td>密理博，MA </td><td> gαmIgM型AF546，Invitrogen公司，加利福尼亚州 （1:100） </td></tr><tr><td> GALC （1:10） </td><td>兔</td><td>抗体</td><td>密理博，MA </td><td> gαmIgM型AF750，Invitrogen公司，加利福尼亚州 （1:100） </td></tr></tbody></table> 表2中。抗体（Abs）以下，用于流式细胞仪和免疫细胞化学检测抗体结合物:PE，藻红蛋白; PETR得克萨斯红，藻红蛋白; AMCA，氨基-甲基-香豆素-乙酸乙酯;循环，花青; FITC，异硫氰酸荧光素。 免疫球蛋白:免疫球蛋白AF:的Alexa Fluor;gαm:羊抗鼠;gαrb:羊抗兔;dαck:驴抗鸡。 </td></tr></tbody></table>

progenitor-derived oligodendrocyte culture system from human fetal brain

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。

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...

Watch this Scientific Journal Video about Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain at JoVE.com

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

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。

来自祖，人脑少突胶质细胞培养系统

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

progenitor-derived-oligodendrocyte-culture-system-from-human-fetal

Research

JoVE Journal

Neuroscience

14.8K 次观看.  National Institute of Neurological Disorders and Stroke, National Institutes of Health. 小学，人类胎儿脑源性，多能祖细胞增殖

视频: Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

从废弃的人类胎儿大脑皮质组织神经干细胞的生成

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

科研

神经科学

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

人类神经的聚集体cGMP的适用展开法干细胞和祖细胞来源从多能干细胞或胎儿脑组织

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

人类成人皮肤成纤维细胞诱导神经元高产培养的简单生成

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has provided important insights into fundamental processes of the brain like cognition, learning, and motor control. However, this approach cannot provide causal evidence for the involvement of brain areas of interest. Transcranial magnetic stimulation (TMS) is a powerful, noninvasive tool for studying the human brain that can overcome this limitation by transiently modifying brain activity. Here, we highlight recent advances using a paired-pulse, dual-site TMS method with two coils that causally probes cortico-cortical interactions in the human motor system during different task contexts. Additionally, we describe a dual-site TMS protocol based on cortical paired associative stimulation (cPAS) that transiently enhances synaptic efficiency in two interconnected brain areas by applying repeated pairs of cortical stimuli with two coils. These methods can provide a better understanding of the mechanisms underlying cognitive-motor function as well as a new perspective on manipulating specific neural pathways in a targeted fashion to modulate brain circuits and improve behavior. This approach may prove to be an effective tool to develop more sophisticated models of brain-behavior relations and improve diagnosis and treatment of many neurological and psychiatric disorders.

This article describes new approaches to measure and strengthen functionally specific neural pathways with transcranial magnetic stimulation. These advanced noninvasive brain stimulation methodologies can provide new opportunities for the understanding of brain-behavior relations and development of new therapies to treat brain disorders.

利用颅内磁刺激测量和操纵人体运动系统中功能特定的神经通路

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

共培养实验的奥里戈丁细胞和奥里戈丁酸基质的生成

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...