我们感谢为C茹贝尔，V诺伊维尔，V Barroca酒店，J Tilliet和动物的设施之外，所有的工作人员;到J Baijer和N Dechamps的细胞分选; O.艾蒂安技术援助，并答Gouret为她提供行政协助。流式细胞仪和细胞分选进行了在红外对抗流式细胞仪共享资源，通过设备赠款DIM茎极，INSERM，ARC基金会，并建立了CEA。这项工作是由法国电力公司（EDF）的资助。 MD有来自La法甲癌症中心和LM的奖学金从区域法兰西岛（DIM生物疗法）。马克 - 安德烈·Mouthon和弗朗索瓦D. Boussin份额共同高级作者。

该协议被设计符合11月24日的欧盟理事会指令，1986（86/609 / EEC），并已通过了动物福利机构委员会（CETEA-CEA DSV IDF）。 1.基本设置之前，文化和电视显微镜<ol><li>使用玻璃底培养板或μ-板共聚焦显微镜录像。为1 - 5×10 3个细胞/孔，使用96孔平板和24孔平板超过5×10 3细胞/孔。 </li><li>在开始实验前至少一天，准备无菌聚-D-赖氨酸（PDL）涂层板用于粘附单层培养。补充足够的PDL（蒸馏水中的10微克/毫升），以每外套的底部，并培育O / N在37℃。除去PDL溶液，并允许所述板期间在层流的至少2小时，在干燥罩之前漂洗三次， 卫生署2 O操作。如果不立即使用，存储所述涂覆板在-20℃。 </li><li>通过混合NSC基础培养基和NSC扩散补编在9制备培养基:1的比例（见材料表），以及2微克/毫升肝素，20纳克/ ml纯化人重组表皮生长因子（EGF），和10ng / ml的人类重组成纤维细胞生长因子2（FGF-2）。预热培养基至37℃，在使用前的水浴。 </li><li>为SVZ解离，制备木瓜蛋白酶溶液:1mg / ml的木瓜蛋白酶（15 UI /毫升）在含有0.2毫克/毫升的L-半胱氨酸，0.2mg / ml的EDTA和0.01毫克/毫升DNA酶I厄尔的余额盐溶液（EBSS）于PBS中。通过经过0.2μm过滤器灭菌的溶液。平衡在使用前将溶液在37℃。 </li><li>以停止酶反应，制备蛋白酶抑制剂溶液（卵类粘蛋白）:含有0.7毫克/毫升的胰蛋白酶抑制剂II型F12培养基:DMEM。过滤使用0.2微米的过滤器的溶液。 </li><li>准备PBS 0.6％葡萄糖溶液，收集的大脑和PBS 0.15％BSA小号olution用于洗涤步骤和抗体染色。 </li><li>准备清扫工具:剪刀，解剖和搭售镊子，手术刀。浸泡其中在70％的乙醇。 </li></ol>收获成年小鼠的2大脑和SVZ Microdissections <ol><li>牺牲成年小鼠FUCCI 23（2至3个月大的和/或12个月大的老化研究）执行根据，以适当的制度准则颈椎脱位。 </li><li>喷用70％的乙醇的小鼠和用锋利的剪刀剪断头。 </li><li>做一个切口，沿头皮揭示的头骨。 </li><li>执行纵向中线切口处开始对用小剪刀的嗅球的颅底。一定要避免损坏底层的大脑与剪刀片。删除与弯钳，露出大脑在颅骨上开的部分。 </li><li>收集脑在含有0.6％葡萄糖的PBS 15毫米陪替氏培养皿。 </li><li> ðissect走嗅球。将脑在其背表面和使用解剖刀通过视交叉使冠部。 </li><li>在解剖显微镜下，定位朝上朝向实验者切割冠状面的脑切片的延髓一部分。 </li><li>解剖SVZ，取出用细弯钳隔膜然后插入的细钳子1尖端插入纹状体紧邻心室和从周围组织分离SVZ。有关详细信息，请参阅Azari 等人。24。将解剖SVZ到含有1ml的PBS-0.6％葡萄糖的培养皿。 </li></ol> 3. SVZ组织解离<ol><li>剁碎解剖SVZ在陪替氏培养皿，直到没有大块留。 </li><li>沿着用PBS-0.6％葡萄糖转移组织糜到15ml管中并离心，在200×g离心5分钟。 </li><li>弃去上清液，并添加1 ml的预热的木瓜蛋白酶（1毫克/毫升，在步骤1.4中制备），在37℃的水浴中补充有0.01毫克/毫升DNA酶I.孵育10分钟。使用1毫升每只小鼠木瓜蛋白酶。 </li><li>离心在200×g离心5分钟，丢弃上清液。 </li><li>添加1毫升预热的卵类粘蛋白（0.7毫克/毫升，在步骤1.5中制备）以停止木瓜蛋白酶活性。机械通过P1000的枪头轻轻吹打向上和向下20倍进一步分解组织糜成单细胞悬液。避免气泡。 </li><li>通过无菌20微米的过滤器在一个新的15ml试管中通过细胞悬浮液。请一定要洗的细胞过滤器，用PBS 0.15％BSA以避免丢失的细胞。 </li><li>离心在200×g离心10分钟，丢弃上清液。重悬的细胞在含0.15％BSA的100μl的PBS中。 </li></ol> 4.免疫荧光染色的细胞分选对于电池使用FUCCI红小鼠（ 图2A），请使用以下排序抗体:CD24藻红蛋白cyanine7结合物的PC7] CD15 / LEX异硫氰酸荧光素[FITC]偶联和Ax647结合EGF配体。 注:LEX + EGFR +细胞和表皮生长因子受体阳性细胞并不丰富的成年SVZ:≈600和1500个细胞/小鼠分别为9。我们建议的2至3只小鼠汇集SVZ细胞有足够的材料。不超过12只小鼠在同一天的工作，以使细胞分选持续时间不超过3小时。请记住，太多的小鼠在同一天的工作会导致增加的细胞分选的持续时间可能导致增加的细胞死亡和/或细胞分化。 <ol><li>执行FACS染色在100μlPBS中0.15％BSA的每只小鼠（或在200μl的一组2至3只小鼠为最佳染色）的。 </li><li>准备对照管。使用补偿珠粒根据制造商以制备单一颜色控制管9; S协议。选择细胞的级分（从一只小鼠是足够提取出的细胞的1/10），并分离其在4个试管以制备1阴性对照管（未标记细胞）和3荧光减一（FMO）对照管。重悬在200μl每管的PBS 0.15％BSA的细胞。 提示:对于LEX-FITC FMO控制，标记细胞CD24-PC7（1:50）和Ax647结合的表皮生长因子配体（1:200）;为CD24-PC7 FMO控制，标记细胞CD15 / LEX-FITC（1:50）和Ax647共轭的EGF配体（1:200）和用于Ax647共轭EGF配体FMO控制，与CD15 / LeX-标记细胞FITC（1:50）和CD24-PC7（1:50）。 </li><li>对于用于细胞分选的管，使用下面的抗体在所指示的稀释在PBS中0.15％的BSA:CD24-PC7（1:50），CD15 / LEX-FITC（1:50）和Ax647共轭的EGF配体（1: 200）。 </li><li>在黑暗中孵育20分钟，在4℃。用1毫升的PBS 0.15％BSA和离心机在200×g离心10分钟洗涤。悬浮细胞在20081，L每脑PBS 0.15％BSA的。保持细胞分选管在冰上并立即进行细胞分选。 </li><li>如果使用FUCCI绿小鼠，使用分离柱之前的细胞分选作为LEX FITC抗体股比FUCCI绿色荧光相同的发射波长分开LEX阳性和LEX阴性级分（图3A，B）。 <ol><li>首先，在100μlPBS中的0.15％的BSA的标记，在4℃在黑暗中的细胞与小鼠抗人LEX抗体（1:50）15分钟。 </li><li>用1毫升的PBS 0.15％BSA和离心机在200×g离心洗细胞10分钟，然后在黑暗中标记细胞与抗小鼠I​​gM微珠（1:10）15分钟，在4℃。 </li><li>用1毫升的PBS 0.15％BSA和离心机在200×g离心洗细胞10分钟。悬浮细胞在500微升PBS 0.15％BSA和倒通过磁场分离柱的细胞图式，如图3℃。用1毫升的PBS 0.15％B洗柱SA获得莱克斯阴性部分。 </li><li>以获得LEX阳性分数，取出来自磁场分离柱和洗脱细胞用2ml PBS中0.15％的BSA。 </li><li>继续CD24-PC7和Ax647结合的EGF配体染色在4.2所示。 </li></ol> </li></ol> 5.细胞分选注:细胞分选在FACS分拣机在40psi用86微米noozle。 520/35纳米（FITC），575 / 26nm（PE），670 / 20nm的（Ax647）和740LP（PC7）:使用下面的过滤器设置荧光收集。补偿是必要的，以防止假阳性信号作为重叠的FUCCI红色荧光的发射光谱和PC7染料之间找到。 <ol><li>以细胞分选前夕，增加一个重要的染料从死细胞活歧视。我们使用HO（见材料表）在2微克/毫升终浓度。通过流式细胞分选机，并选择日运行阴性对照管（未标记的细胞）使用侧向角散射（SSC）和前向散射（FSC）参数（图1A）E细胞。 注:死细胞排除通过选通只的HO-阴性级分（ 图1A&#39;），然后双峰被排除通过选择脉冲宽度阴性部分（图1A&#39;&#39;）。 </li><li>运行在步骤4.2中制备的单色彩控制和调节的光电倍增管（PMT）电压在必要时（即负人口过高和/或阳性细胞关闭刻度）。在软件的补偿窗口进行颜色补偿。 </li><li>运行FMO控制准备在步骤4.2（LEX-FITC FMO控制，CD24-PC7 FMO控制和Ax647结合的EGF配体FMO控制），并绘制分配门（ 图1）。排序将细胞直接入100微升培养基在1.5ml微型管。 </li></ol> 6.准备细胞显微镜<ol><li>板新鲜排序细胞以1:1的密度秒- 3×10 3个细胞/孔于聚-D-赖氨酸包被的96孔μ-板用300μl培养基。 </li><li>之前的视频显微镜，孵育培养板在37℃和5％的CO 2的至少1小时，以允许细胞粘附。 </li></ol> 7.显微镜设置和图像采集<ol><li>在37℃下的CO 2的气氛中的5％上附连到一个倒置的恒温室中的共聚焦激光扫描显微镜系统:执行使用计划的Apo虚电路20×DIC物镜（0.75 NA）的实时成像。 </li><li>定位96孔μ-板的预热和平衡恒温室中，并用一个恒温盖更换盖子。 </li><li>打开NIS-Elements软件，然后单击菜单栏上的"收购/并购控制/ ND收购"来选择延时选项（长，舞台的位置，共聚焦Z-路段，...），"收购/并购控制/钛垫221;选择目标和"收购/并购控制/ A1plus设置"，选择菜单栏中的每个荧光的PMT水平。选择一个文件夹来保存数据文件。 </li><li>使用ND采集窗口，设置中心每个以及舞台的位置，并选择大图片选择7×7平方毫米。这将创建一个围绕每个中心的马赛克图像良好。对于大马赛克图像的重叠设置为5％。拍照每20分钟24小时。选择计划阿婆VC 20X DIC物镜（NA:0.75）中的钛板窗口。 </li><li>在A1plus设置窗口，获取使用高速共振扫描仪图像在512×512像素格式的1.26微米/像素的分辨率。明使用可视化的细胞形状。在FUCCI红小鼠的情况下，激发红色荧光在561 nm和收集用五十零分之五百九十五纳米过滤器。在FUCCI绿小鼠的情况下，激发绿色荧光在488nm和收集用四十分之五百三纳米过滤器。确定最佳的PMT水平，偏移和激光功率对于每个波长。 注意:我们建议使用自动对焦功能的明路，让软件自动对焦在每次收购之前，每个阶段的位置。提示:甲计划的Apo虚电路20×DIC物镜（NA:0.75）中用于其无需油优异的分辨率。其他目标可根据所需的光学分辨率被使用。 </li><li>选择ND采集窗口上的"立即运行"按钮，开始采集。 提示:按照电脑工作1循环，以确保一切都在正常工作。 </li></ol> 8.图像处理和分析<ol><li>数据直接在NIS-Elements软件通过单独跟踪的细胞分析。提示:为了争取时间，使用NIS-Elements软件保存为.AVI格式的每个位置，并分析了电影ImageJ的。 </li><li>在ImageJ的软件，追踪经历至少2个部门（ 即一个单元单个单元格四细胞集落）。裁剪一个小范围内的小区周围，并选择"图像/复制"。 </li><li>在菜单栏中选择"图像/栈/制作蒙太奇"，使一个蒙太奇。指定要被包括在帧图像的大小和蒙太奇保存为.tif文件为最佳分辨率。 </li><li>要计算的第一个SG / M期长（ 图2C，D），选择一个红色的荧光细胞（在G中1），然后设置T = 0（S期将开始，一旦红色荧光关闭）。计数帧的数目，直到细胞分裂来估计对SG 2 / M的长度。 注:计算的时间取决于每个帧之间的时间间隔。 </li><li>计算以下G1期（图2C，D）中，继续跟踪，只是除以细胞。如果分割单元进入G1期，将有CDT1红色荧光蛋白的积累。计算的帧的数量，直至红色荧光关闭阿加在对每个小区。 提示:为g 1日开始的红色荧光，可能是弱所以选择G 1期的发病帧中小区划分了避免近似的。 </li></ol>

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R., 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=Continuous+live+imaging+of+adult+neural+stem+cell+division+and+lineage+progression+in+vitro.">Continuous live imaging of adult neural stem cell division and lineage progression in vitro.</a> Development. 138, 1057-1068 (2011).</li><li>Ponti, 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=Cell+cycle+and+lineage+progression+of+neural+progenitors+in+the+ventricular-subventricular+zones+of+adult+mice.">Cell cycle and lineage progression of neural progenitors in the ventricular-subventricular zones of adult mice.</a> Proc Natl Acad Sci U S A. 110, 1045-1054 (2013).</li><li>Sakaue Sawano, A., 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=Visualizing+spatiotemporal+dynamics+of+multicellular+cell+cycle+progression.">Visualizing spatiotemporal dynamics of multicellular cell cycle progression.</a> Cell. 132, 487-498 (2008).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay.">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> Journal of visualized experiments JoVE. , (2010).</li><li>Daynac, M., 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=TGFbeta+lengthens+the+G1+phase+of+stem+cells+in+aged+mouse+brain.">TGFbeta lengthens the G1 phase of stem cells in aged mouse brain.</a> Stem cells. 32, 3257-3265 (2014).</li><li>Roccio, M., 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=Predicting+stem+cell+fate+changes+by+differential+cell+cycle+progression+patterns.">Predicting stem cell fate changes by differential cell cycle progression patterns.</a> Development. 140, 459-470 (2013).</li><li>Walker, T. 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The authors declare that they have no conflict of interest.

本文所述的细胞分拣技术允许静止的NSCs，激活神经干细胞和它们的性质的其后代能够研究和动力学在成人脑9之间可靠歧视。再加上FUCCI技术，其允许在活细胞23的细胞周期进展的可视化，我们开发了一种快速和有效的技术，以跟随对G 1和SG 2 / M期的细胞周期从年轻成年和老年鼠脑细胞。 在这个协议中使用的细胞分拣技术是标记允许五个主要神经源性群体的纯化从SVZ 9的第一验证组合。这也是第一个验证的技术使静态和激活神经干细胞的区别。值得注意的是，该技术不要求使用转基因小鼠本身 ，当适于转基因小鼠模型ス这是必要的CH为FUCCI。从那时起，Codega的等人 10已经使用了GFAP-GFP / CD133 / EGFR三重标记组合来从他们的活化的对应物区分开的静态的神经干细胞，但它不能被适于FUCCI技术，因为它要求使用GFAP-GFP转基因小鼠。密歇根州等 11个国家制定了GLAST / EGFR / CD24三联标记策略共享，在这项研究中9所使用的技术共同作用的结果。事实上，在Daynac 等人已经发现lex和GLAST神经干细胞标志物之 ​​间的高相关性，研究9。然而，在美国密歇根州等人的技术中使用的GLAST抗体偶联藻红蛋白，因此不能适用于与使用FUCCI红小鼠。 还有需要注意排序前SVZ细胞几个重要的技术点。首先，将解离的步骤是非常重要，因为脑组织必须被解离成单细胞，同时保留结构在完整性用于抗体的标记策略的蛋白质。 0.05％胰蛋白酶-EDTA已被证明是非常有效的解离SVZ组织27，但LEX抗原被认为是高度敏感的，因为几乎所有的LEX-FITC免疫荧光丢失了（数据未显示）。因此，木瓜蛋白酶，使用，因为它是更有效的和比对脑组织的其他蛋白酶破坏性较小。此外，无论是LEX也不表皮生长因子受体CD24也受到影响，木瓜蛋白酶处理。应当指出的是，抗体标记被更改，如果木瓜蛋白酶治疗超过15分钟。我们得到的最佳结果与机械分离（吸管上下20倍）相关的10分钟木瓜蛋白酶处理。 聚-D-赖氨酸涂层允许贴壁细胞的培养，并在培养数天形成菌落。现在人们普遍认为 ，在体外实验带有其局限性28,29。我们建议确定只有第一个细胞周期经历至少一个后续分裂细胞停留尽可能接近到体内细胞表型。 值得注意的是，提及的是，Geminin（绿色荧光）和CDT1（红色荧光）的蛋白质用于设计FUCCI 系统 23以前证明是大量由神经祖细胞表达在早期神经发生在小鼠中30和在成人脑组织9,25 。虽然mKO2-hCdt1（一百二十〇分之三十）构建体主要用在本研究遵循与红色荧光G1期，同时使用构建体[m​​KO2-hCdt1（一百二十零分之三十零）和MAG-hGem（110分之1） ]可以设想，以允许细胞周期的主要阶段的可视化（G 1和SG 2 / M）以及对G 1 / S转换23。双彩色成像的主要缺点是荧光的有限相容集，仍然可以用于细胞标记。一个解决方案是使用separati在列。例如，我们已经成功地从贫使用分离柱细胞CD24阳性细胞级分之前分类和活成像，因为我们使用的CD24-PE抗体共享比FUCCI -红色荧光25相同的发射波长。 很少有研究调查成年小鼠SVZ人口的细胞周期的长度。与我们的技术获得的总细胞周期长度接近一个通过Costa等人的 21估计在体外 ，但我们能够首次来区分不同的细胞周期阶段。 在体内研究中，蓬蒂等人 22使用胸苷类似物的掺入，以确定不同SVZ细胞群的增殖动力学。然而，它们既不能进行连续监测的细胞周期也不跟踪细胞在单细胞水平。这可能是因为它表明一个非均质性强存在机智的问题欣给定的SVZ细胞群体22,25。我们在这里描述的体外技术的替代，容易设置，它允许不同的成人神经源性群体的细胞周期阶段的实时成像在单个细胞水平。 了解神经干细胞和祖细胞的细胞周期的调节仍然是新的治疗方法中的老化或脑病症的上下文中的发展是一个挑战。因此我们的协议可具有广泛的应用。在老龄化的背景下，这种技术也被证明有助于了解对神经干细胞分化衰老的作用。事实上，这是可能的，以确定进入分化的神经干/祖细胞作为它们红色荧光强度是鲜明高出 26。最后，它也可能是感兴趣的利用连续实时成像在一个单细胞水平来研究负责谱系亲细胞内在的和外在的过程回归成人神经干细胞。

静态的神经干细胞（NSCs）是源成年神经1和可以转化成它们的增殖"激活"的形式表达EGFR（aNSCs）2。一旦被激活，它们产生短暂扩充细胞（的TAC）3，然后成神经细胞，为了嗅球（OB）通过星形胶质细胞的管迁移，最终分化成神经元4,5。神经干细胞和它们的后代被组织在专门的"利基市场"架构沿侧脑室，牵连因素控制其扩散6万千。 SVZ神经原细胞的分离和纯化是必要的，照亮其扩散的复杂分子的规定，但仍然很长一段时间了挑战，由于缺乏特异性标记物，并适于技术。 使用流量的新方法术已使可能的神经干细胞和兵卫的隔离- [R后代从成人的SVZ 2,7-11。利用干细胞标记法12以及成神经细胞标志物CD24 13和荧光EGF对增殖细胞2标签 EGFR，我们最近开发出一种FACS策略允许五个主要SVZ神经源性人口的净化:静态和激活的神经干细胞，总可捕量，不成熟以及迁移成神经细胞9。在这里，我们详细描述这种细胞分选技术，以及如何一个LEX / EGFR / CD24三重允许首次染色两者的静态和激活的神经干细胞的分离。 虽然神经发生成年时期仍然存在，生产新的神经元在大脑老化14急剧下降。大多数研究同意逐步减少在SGZ和SVZ 15-20增殖祖细胞的数量。其后果是不是无害的年龄有关的下降，神经发生在SVZ引发n的缩减ewborn神经元在老年人的大脑的嗅球，最终导致在嗅觉辨别减值老年小鼠18。阐明神经祖细胞的细胞周期动力学是了解衰老过程中成年神经的演变的基本机制的关键步骤。最近的研究已经调查了细胞周期成人神经干细胞和谱系进展体外 21 和体内22但它们都没有利用了细胞分选技术和基因编码的荧光细胞周期的探针可视化分离的细胞的细胞周期阶段在单细胞水平。 这里，我们描述一个协议，取，其中将细胞在其细胞周期荧光转基因FUCCI小鼠的优势，使1克和SG 2 / M期23之间的区别。这个协议显示神经干细胞和它们的后代的自成人FUCCI英里如何准隔离策结合的时间推移的视频显微镜允许细胞周期动力学的在单细胞水平的研究。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>96-well uncoated glass bottom culture plates&#160;</td>
			<td>MatTek Corp.</td>
			<td>P96G-1.5-5-F</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Plates 96-well</td>
			<td>Ibidi</td>
			<td>89626</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Millipore</td>
			<td>A003E</td>
			<td></td>
		</tr>
		<tr>
			<td>NeuroCult NSC Basal Medium</td>
			<td>STEMCELL Technologies</td>
			<td>5700</td>
			<td></td>
		</tr>
		<tr>
			<td>NeuroCult NSC Proliferation Supplement&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>5701</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>STEMCELL Technologies</td>
			<td>7980</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Millipore</td>
			<td>GF144</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-2</td>
			<td>Millipore</td>
			<td>GF003</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003119</td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>Invitrogen</td>
			<td>24010-043</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>C-7352</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5M EDTA, pH 8.0</td>
			<td>Promega</td>
			<td>V4231</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>D5025-15KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor type II</td>
			<td>Sigma</td>
			<td>T9128</td>
			<td>Ovomucoid</td>
		</tr>
		<tr>
			<td>DMEM:F12 medium</td>
			<td>Life Technologies</td>
			<td>31330-038</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;m filter&#160;</td>
			<td>BD Medimachine Filcon</td>
			<td>340622</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microtubes 3810X</td>
			<td>Sigma</td>
			<td>Z606340-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A1595</td>
			<td>Bovine serum albumin solution</td>
		</tr>
		<tr>
			<td>CD24 phycoerythrin-cyanine7 conjugate [PC7]</td>
			<td>Life Technologies</td>
			<td>A14776</td>
			<td>Mouse IgM; clone MMA. 1:50</td>
		</tr>
		<tr>
			<td>CD15/LeX fluorescein isothiocyanate [FITC] - conjugated</td>
			<td>BD Biosciences</td>
			<td>332778</td>
			<td>Rat IgG2b; clone M1/69.&#160; 1:50</td>
		</tr>
		<tr>
			<td>Mouse anti-human LeX antibody</td>
			<td>BD Pharmingen</td>
			<td>559045</td>
			<td>Mouse IgM; clone MAM.&#160; 1:50</td>
		</tr>
		<tr>
			<td>Alexa647 - conjugated EGF ligand</td>
			<td>Life Technologies</td>
			<td>E35351</td>
			<td>1:200; Ax647-conjugated EGF ligand in the text</td>
		</tr>
		<tr>
			<td>CompBeads</td>
			<td>BD Biosciences</td>
			<td>644204</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS LS separation columns&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>separation columns (in the manuscript)</td>
		</tr>
		<tr>
			<td>Anti-mouse IgM microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-047-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33258</td>
			<td>Sigma</td>
			<td>861405</td>
			<td>HO (in the manuscript)</td>
		</tr>
		<tr>
			<td>INFLUX cell sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>FACS sorter (in the text)</td>
		</tr>
		<tr>
			<td>Nikon A1R confocal laser scanning microscope system attached to an inverted ECLIPSE Ti</td>
			<td>Nikon Corp.</td>
			<td></td>
			<td>confocal laser scanning microscope (in the manuscript)</td>
		</tr>
		<tr>
			<td>NIS-Elements AR.4.13.01 64-bit software</td>
			<td>Nikon Corp.</td>
			<td></td>
			<td>NIS-Elements software (in the manuscript)</td>
		</tr>
		<tr>
			<td>Plan Apo VC 20x DIC objective (NA: 0.75)</td>
			<td>Nikon Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ECLIPSE Ti thermostated chamber</td>
			<td>Nikon Corp.</td>
			<td></td>
			<td>thermostated chamber (in the manuscript)</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>RBS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>Tree Star, Ashland, OR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FUCCI mice</td>
			<td>RIKEN BioResource Center, JAPAN</td>
			<td></td>
			<td>Sakaue-Sawano et al. 2008</td>
		</tr>
	</tbody>
</table>

cell sorting of neural stem and progenitor cells from the adult mouse subventricular zone and live-imaging of their cell cycle dynamics

在侧脑室的脑室下区神经干细胞（NS​​Cs）（SVZ）维持整个生命的嗅觉神经发生在哺乳动物的大脑。他们先后产生短暂扩充细胞（总可捕量）和神经母细胞分化成神经细胞，一旦他们整合嗅球。新兴的荧光激活细胞分选（FACS）技术允许NSCs的隔离以及它们的子代，并开始阐明基因调控网络在成年神经源性龛的光。我们在这里报告一细胞分拣技术，它允许按照从成年SVZ用LEX / EGFR / CD24三重染色区分上述细胞群的细胞周期动力学。然后分离的细胞铺板以贴壁细胞，探讨在由时间推移的视频显微镜详细它们的细胞周期进程。为此，我们使用转基因荧光泛素细胞周期指示符（FUCCI）小鼠中，细胞是红色荧光durin的G 1阶段，由于A G 1特异性红CDT1记者。这种方法最近发现，神经干细胞增殖过程中的老化逐渐延长它的G 1期，从而导致神经损伤。这种方法是很容易转座到其他系统，可能是对脑细胞在脑病理学的上下文中，细胞周期动力学的研究极大的兴趣。

到成年小鼠SVZ干细胞谱系的不同细胞群之间可靠地鉴别的能力是原始研究其功能特性。为了这个目的，我们开发了一个LEX / EGFR / CD24三标记策略，允许从成年SVZ特定细胞群的净化:静态神经干细胞（LEX 亮），激活神经干细胞（LEX + EGFR +），总可捕量（EGFR +）的，不成熟的和迁移的成神经细胞（CD24 +的EGFR +和 CD24 +，分别）9（ 图1）。施加到FUCCI转基因小鼠，所述细胞分拣技术允许的不同神经源性群体在单细胞水平25的细胞周期阶段的实时成像。 FUCCI红小鼠用于按照G 1期与红色荧光使用时间推移的视频显微镜（ 图2A）。 FUCCI红阴性细胞代表该细胞在S-G2 / M期的细胞周期，FUCCI红POS细胞为G 1细胞和FUCCI红光亮细胞被细胞退出或离开细胞周期（图2B）-23,26-。 LEX + EGFR +和从年轻成年（图2C）和中年小鼠（ 图2D）的EGFR +细胞铺板作为聚D-赖氨酸包被的培养板中的贴壁细胞。第一SG / M期被 ​​确定在单细胞一旦红色荧光关闭，直到细胞分裂。如先前所观察到的SG 2 / M期长度表明，无论是在年轻或中年小鼠LEX + EGFR +和EGFR +细胞之间没有差异。然后，我们计算从第一部的下一个G 1期的长度，直到红色荧光关闭。有趣的是，G 1期延长的LEX + EGFR +细胞，但不老化过程中被发现在EGFR +细胞25（图2C，D）。结果，神经球电镀后第5天获得的是在从老年小鼠获得如图2E LEX + EGFR +细胞小。 FUCCI绿小鼠也可用于可视化对SG 2 / M期的绿色荧光（图3A，B），但细胞必须使用分离柱为LEX-FITC抗体预排序共享相同的发射波长比FUCCI-绿色荧光（图3C）。的FUCCI绿色荧光的第一SG 2 / M期的年轻成年LEX + EGFR +和EGFR +细胞的一个例子示于图3D。 <img alt="图1" src="/files/ftp_upload/53247/53247fig1.jpg" /> 图1:通过FACS小区选择的策略（A）的细胞首先根据吨选定采用侧向散射（SSC）与前向散射（FSC）参数继承人形态。有关形态门选择更多详细信息，请参阅Daynac 等 9（A&#39;）使用HO标记死细胞进行标记和排除的选择。（A&#39;&#39;）双峰是使用脉冲宽度参数排除。为了建立分配门，荧光减一（FMO）的CD24-PC7控制（LEX-FITC / FUCCI红/ EGF-Ax647标签; B）和 LEX-FITC（FUCCI红/ EGF-Ax647 / CD24 -PC7标签; B&#39;）中 ，使用（C，C&#39;）然后，分配门在LEX-FITC / FUCCI红连同FMO控制/ EGF-Ax647 / CD24-PC7标记的管进行了测定。总的细胞的平均百分比的栅极内表示。如果选择CD24 +细胞（神经母细胞），要注意排除CD24 亮表达细胞的大门。事实上，CD24 明亮相当于室管膜CELLS 2,9，C&#39;代表CD24阴性细胞（黑方块C语言）。仅LEX + EGFR +和EGFR +（C&#39;）分配门被用于本研究。 <img alt="图2" src="/files/ftp_upload/53247/53247fig2.jpg" /> 图2:细胞周期的使用FUCCI红小鼠实时分析（A）中 FUCCI红细胞周期的示意图，其中细胞SG 2 / M期23（B）的FACS的代表性过程期间G1期红色荧光和无色。 FUCCI-红色荧光的SVZ细胞（C，D）视频显微镜用LEX + EGFR +和EGFR +细胞分选的年轻（C）和中年（D）FUCCI红小鼠允许G 1期与追踪红色荧光，而SG 2 / M期可以推断磨片n个红色荧光关闭。连续图像示与 D中提出了一个时间刻度。菌落（E）的代表性图像（神经球）中得到铺板后5天。比例尺:10微米（C，D），30微米（E） <a href="https://www.jove.com/files/ftp_upload/53247/53247fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53247/53247fig3.jpg" /> 图3:策略细胞周期的使用FUCCI绿小鼠活分析（A）中 FUCCI绿细胞周期，其中细胞绿色SG期间中G 1期23 2 / M期和无色的示意图（B）的 FACS表示的FUCCI绿荧光:FUCCI绿阴性细胞代表G中的电池 1相的细胞周期而FUCCI格力ÑPOS细胞是在SG / M 23。（C）在为了遵循绿色荧光SG 2 / M期LEX + EGFR +及EGFR +细胞 ，SVZ细胞必须被预排序以分离柱。细胞用抗小鼠LEX抗体和Lex +馏分保留使用抗小鼠微珠的柱（参见协议4.）。然后，LEX +莱克斯-组分被洗脱，并标有EGF-Ax647和CD24-PC7抗体流式细胞分选（D）LEX + EGFR +的视频显微镜和EGFR +细胞分选的年轻FUCCI绿小鼠允许的跟踪。神光2 / M期绿色荧光。连续图像示与 D中提出了一个时间尺度。比例尺:10μm的（D）中 。

Watch this Scientific Journal Video about Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics at JoVE.com

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

cell-sorting-neural-stem-progenitor-cells-from-adult-mouse

Research

JoVE Journal

Neuroscience

14.1K 次观看.  CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967. We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

视频: Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

脑室下区恩的脸:Wholemount染色和室管膜流量

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

神经干细胞和祖细胞在小鼠脑开发基因毒性应激后的评估细胞周期进程

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

成人皮层的器官型培养作为人类干细胞移植和验证的 离体 模型

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

用于神经干细胞和少突胶质细胞祖细胞分离的大鼠"脑挤奶"

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

作者聚焦:使用新型脑挤奶方案从活体动物中收集神经干细胞和祖细胞 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

从活大鼠中分离神经干细胞和少突胶质细胞祖细胞的"脑挤奶"方法

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...