这项研究导致这些结果根据出让协议已收到的资金来自欧盟第七框架计划（FP7/2007-2013）N°323267，来自法国电力公司（EDF）和L&#39;法新社国立德拉RECHERCHE - 桑特 - 环境在等桑特-劬劳（ANR-SEST，Neurorad）。

1，动物规程该协议已经被设计符合欧共体理事会指令11月24日，1986（86/609/EEC），并已通过了动物福利（CETEA-CEA DSV IDF）机构委员会。 <ol><li>注射用EdU /的BrdU和E14.5孕鼠照射（ 图2A）。 <ol><li>制备的EdU的溶液在1毫克/毫升和BrdU在5 mg / ml的PBS中。执行腹腔注射100微升的溶液的EdU使用25号针头成孕鼠（IP）。 1.5小时后，照射小鼠（全身照射），在0戈瑞或2戈瑞（0.6格雷/分钟）。紧接在后，用25号针头进入孕鼠注射（IP）的200微升的BrdU溶液。 </li></ol></li><li>制备胚胎的大脑冠状切片。 <ol><li>在1小时或4小时照射后，通过二氧化碳安乐死的孕鼠。 </li><li>打开腹腔CAVITY超过用剪刀3厘米。通过切片角的两个末端从子宫的其余部分分开的两个子宫角。转移子宫角到含有PBS 0.6％葡萄糖的培养皿中。为了分离胚胎打开子宫角用钳子。删除使用镊子每个胚胎的羊膜囊。 </li><li>解剖胚胎头与钳和4％多聚甲醛（PFA，pH值= 7.4）O / N修复它们浸泡在4°C。 </li><li>在4℃下冲洗胚胎头在PBS中的至少一个夜头可以保持在PBS中数天。 </li><li>使用真空浸润处理器 ，如表1所示在石蜡中嵌入的头。头嵌入也可以根据化学罩在一个65℃的烘箱中的石蜡浴进行乙醇和xylen浴，然后。 </li><li>准备5微米厚的冠状切片，用切片机。 </li><li>安装到多聚赖氨酸包被的载玻片。&lt;/ LI&gt; <li>幻灯片可以保持在室温（RT）下持续数月。 </li></ol></li></ol> 2，教育/的BrdU染色<ol><li>脱蜡和抗原修复<ol><li>通过幻灯片浸泡在甲苯5分钟的3浴Deparaffinize石蜡包埋脑切片。 </li><li>补充水分在2浴室降低乙醇浓度的各溶液中3分钟，如以下：乙醇100％，95％乙醇，70％乙醇和5分钟的H 2 O。幻灯片可以保持在去离子水中几个小时。 </li><li>煮沸的切片中的柠檬酸溶液（10mM，pH为6）10分钟，然后孵育在去离子水中5分钟。 </li></ol></li><li>染色的EdU <ol><li>根据制造商的方案进行的EdU检测。透化的细胞用0.5％的Triton X-100的PBS中为10分钟。 </li><li>通过稀释于去离子水中的10倍的溶液制备1X的EdU缓冲添加剂。此溶液应新鲜制作，并于同达使用年。 </li><li>准备的EdU反应的鸡尾酒，包括的EdU的Alexa Fluor 488叠氮化物。添加的成分如下表2中所列的顺序，否则该反应将不会进行优化是非常重要的。内准备15分钟用埃杜反应鸡尾酒。 </li><li>除去透化缓冲液（步骤2.1.1），然后用PBS洗两次。 </li><li>添加150微升的EdU反应混合物，把盖玻片，并温育在黑暗中在室温下30分钟。 </li><li>删除埃杜反应鸡尾酒，然后用PBS洗一次。 </li></ol></li><li>的BrdU染色<ol><li>制备饱和缓冲液含7.5％山羊血清和PBS中的7.5％的胎牛血清。加入150μl的饱和缓冲液对脑切片，放盖玻片，孵育1小时在室温阻断非特异性抗体结合。 </li><li>取出盖玻片。加入150μl稀释在饱和缓冲液1/300鼠抗BrdU一抗，把盖玻片和孵化的O /n在4°C。 </li><li>取出盖玻片，然后在PBS中洗涤3次。 </li><li>加入150μl在饱和缓冲液中稀释为1/400的山羊抗小鼠Alexa594第二抗体，将一个盖玻片，并孵育1小时，在室温下。 </li><li>取出盖玻片，然后洗一次在PBS。 </li><li>核染色：加150微升4&#39;，6 - 二脒基-2 - 苯基吲哚（DAPI）在1微克/毫升的PBS中，把盖玻片并孵育在室温下2分钟。 </li><li>在安装介质安装滑轨。 </li></ol></li></ol> 3。分析<ol><li>检查脑切片用20X物镜或激光共聚焦显微镜，荧光显微镜下，在3个通道（488纳米，594纳米和UV）获得的图像作为分开的文件。 </li><li>堆栈用Photoshop软件映像。 </li><li>在背内侧脑壁的标准部门分析脑切片。这个扇区是100微米的在其内侧 - 外侧尺寸和被分成10μ18箱（或更多）使用叠加在图像的网格（ 图1），在其径向尺寸米高度先前描述31。 <ol><li>网格对齐诸如第一bin是否在心室表面，其平行于心室边界长轴。然后按数字标记EDU，的BrdU和/或细胞核固缩（对应于凋亡细胞）在每个箱。号位于在当中载有其较大的部分，或者在较低的垃圾桶，当原子核占据了两个垃圾桶内相等面积的bin两个垃圾桶的边界上的核心。 </li></ol></li><li>统计分析。 </li></ol>分析每个胚胎的至少两个皮质切片。对来自不同窝的至少3个胚胎重复实验。结果应以平均值（SEM）的平均值±标准误差。统计分析与GRAPHPAD棱镜采用双因素方差分析和Bonferroni多重比较进行事后检验进行。

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该作者有没有利益冲突。

这里所描述的基础上的EdU 1.5小时掺入实验设计照射前和BrdU的掺入允许照射后立即NSPCs能够激活S期和G2 / M期检查点，但第1小时过程中没有在G1 / S检查点之后的一个示范基因毒性应激在胎鼠大脑。我们进行了在其中的EdU已经注入在不同时间照射和BrdU后，才刚刚1小时小鼠牺牲其他的实验中，让我们特别欣赏进入S期的速度。这些实验表明，NSPC不激活G1 / S期阻滞在体内 22,23基因?...

在胚胎脑发育，在脑室区产生的大脑皮层的投射神经元中，假复层上皮的不同类型的神经干细胞和祖细胞（NS​​PC）所组成的行侧脑室。其中NSPC，径向胶质细胞（RGC），其作为神经干细胞，经过interkinetic核迁移（INM）：它们在心室和心室区的基极限的S-相的表面进行有丝分裂（VZ） 1，2,3。它们可分为两种对称地产生两个RGC或不对称，以生成一个RGC和一个神经元或中间祖细胞（IPC）的4，5。工控机迁移到上覆的增殖层称为脑室下区（SVZ），其中最后一个对称分裂后，便产生两个不成熟的投射神经元6-8。相反，研资局，IPC不进行INM（9审查）。新生成的神经元MIGR吃了径向沿径向纤维通过中间区（IZ）到达其 ​​最终目的地，皮质板（CP）10，8。所有这些事件的最佳时机是正确的皮质发育至关重要。例如从扩散切换到神经祖细胞群分化是由G1期持续时间11，12控制。 G1期的延长，细胞分化从而关联。 基因毒性应激，如电离辐射，严重危害大脑的发育（13审阅）。我们和其他人已经表明，NSPC是极易受辐射诱导的细胞凋亡14，15,16。电离辐射诱导DNA双链断裂是对增殖细胞的最严重的损害。 DNA损伤反应（DDR）的循环细胞的一个重要组成部分是细胞周期检查点在G1 / S期或G2 / M期的转换或S在激活相（内S检测点）17-21。他们阻断细胞周期进程，为DNA损伤修复或消除过于受损细胞的提供时间。因此，细胞的死亡，以及延迟细胞周期进程可能改变大脑发育响应于电离辐射曝光22-24。因此，这是有趣的，开发一种方法，以评估在NSPC细胞周期检查点在被照射的小鼠胚胎脑的活化。 在细胞周期的进展是经常随后使用的胸苷类似物，5 - 溴-2&#39;-脱氧尿苷（BrdU）标记的掺入。在细胞周期中，当DNA被复制的S期的BrdU掺入。使用针对的BrdU抗体，该抗体的允许细胞，是在S期的BrdU的脉冲期间的随后的检测。 一种新颖的胸苷类似物，5 - 乙炔基-2&#39;-脱氧尿苷（EDU）由叠氮化物的荧光检测。不同的方法来检测的EdU和乙<html> RDU不交叉反应的25，使同时检测两个胸腺嘧啶的类似物，它是细胞周期进程的研究是有用的。通常细胞却先脉冲用EdU，然后用脉冲的BrdU，其中两个法团之间的时间持续两小时25，26。在含有的EdU培养基另外的BrdU导致优先掺入的BrdU到DNA用EdU的排除，而同时加入的EdU 25与等摩尔或等摩尔的一半的BrdU向媒体结果中只BrdU掺入27。这简化了通过消除通常须从培养基中取出的第一个标签之前加入第二标记物的洗涤步骤的双标记方法。这也就是， 在体内研究中，在洗涤步骤是不可能的，以确定进入S期或细胞群体的出口处的准确定时的特别的兴趣。 吨“&gt;最近，已开发了在使用的EdU和BrdU 23，24,22胚胎小鼠大脑的双脉冲标记的方法来分析细胞周期进程和NSPC的INM后在子宫内的辐射，而且它已被证明的是，在S期，小鼠细胞首先复制常染色质区域，然后在臂间异染色质28,29,30。有趣的是，聚集在interphasic核不同染色体臂间异染色质形成异染色质灶也被称为chromocenters和DAPI染色不易察觉的亮灶。因此，常染色质和chromocenters的差的EdU和BrdU染色有助于我们分析更精确地NSPC S期的进展。 这种方法允许的明显缺乏G1 / S期检查点在NSCP 22-24，这是相当令人惊讶，因为这个检查站被认为是对基因组稳定性的关键示范。几个EXP基于对的EdU和BrdU脉冲的各种组合erimental设计已经用于分析细胞周期进程中的腹侧和背侧端脑。这里，我们给协议允许NSPC 在 E14.5小鼠胚胎的子宫内的辐照后的急性DNA损伤的前4个小时内的学习的例子。

<table border="0" cellpadding="0" cellspacing="0" width="727">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:228px;">EdU</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">A10044</td>
			<td style="width:163px;">1 mg/ml</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">BrdU</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">B5002</td>
			<td style="width:163px;">5 mg/ml</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:228px;">IBL 637</td>
			<td style="width:212px;">CIS BIO International</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Tissu Tek VIP</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Microtome</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;">RM 2125 RT</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Triton X-100</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td align="right" style="width:125px;">93443</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:228px;">Click-iT EdU Alexa 488 imaging kit</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">C10083</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:228px;">Anti-BrdU</td>
			<td style="width:212px;">GE Healthcare</td>
			<td style="width:125px;">RPN202</td>
			<td style="width:163px;">1/300</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:228px;">Goat anti mouse-Alex594</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">A11001</td>
			<td style="width:163px;">1/400</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Fluoromount</td>
			<td style="width:212px;">SouthernBiotech</td>
			<td style="width:125px;">0100-01</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Polysine slide</td>
			<td style="width:212px;">Thermo scientific</td>
			<td style="width:125px;">J2800AMNZ</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Paraformaldehyde</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">P6148</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:228px;">PBS</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">20012-068</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:228px;">DAPI</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">D9542</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Microscope BX51</td>
			<td style="width:212px;">Olympus</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Confocal microscope SPE</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Prism software</td>
			<td style="width:212px;">Graphpad</td>
			<td style="width:125px;"></td>
			<td style="width:163px;">Version 5.0c</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Photoshop software</td>
			<td style="width:212px;">Adobe</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
	</tbody>
</table>

assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress

从不同类型的神经干细胞和祖细胞（NS​​PC），它形成一个假复层上皮衬里胚胎脑的侧脑室脑发育过程中产生的大脑皮层神经元。遗传毒性压力，如电离辐射，对发育中的大脑与NSPC的高灵敏度非常有害的影响。所涉及的细胞和分子机制的阐明依赖于这些特定类型的细胞中，DNA损伤反应，需要一个准确的方法通过细胞周期来确定受损组织NSPC进展的表征。这里显示的基础上的EdU和BrdU对孕鼠和进一步检测在胚胎大脑冠状切片这两个胸腺嘧啶核苷类似物的连续腹腔注射的方法。的EdU和BrdU都在复制细胞的DNA在S期注册成立，并通过两种不同的方法（叠氮化物或检测的特异性抗体，分别），这有利于它们的同时检测。的EdU和BrdU染色，然后在其从心室率在背端脑的标准区域距离的函数确定每个NSPC核。因此，这种双标记技术允许区分细胞，通过细胞周期进展从那些在DNA损伤反应已经激活细胞周期检查点，导致细胞周期阻滞。 实验的一个例子给出，其中的EdU被照射和BrdU前后立即注入和分析的4小时内下照射进行。这个协议提供了NSPC在细胞周期在它们已经被照射的相位的函数的急性DNA损伤反应的精确分析。这种方法是很容易的转座到许多其他系统，以确定在生物体组织上的细胞周期进展的特定治疗的影响。

在图2中描述的实验中，埃杜给药1.5小时照射和BrdU刚照射后前。四种类型的细胞，然后在1或4小时后照射制备的皮质切片区别，根据任一的EdU或的BrdU，双方或无的掺入（ 图2A和2B）。重要的是，既没有的EdU也不BrdU掺入改变辐射诱导的细胞凋亡（数据未示出）的电平。此外，该染色方法允许在S-期DNA复制过程中EDU和BrdU的唯一的检测中，而没有相应的灵敏度来检?...

Watch this Scientific Journal Video about Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress at JoVE.com

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

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

胸苷两个类似物管理，埃杜和BrdU，在孕鼠允许在神经细胞和祖细胞在胚胎小鼠大脑细胞周期进程的分析。这个方法是有用的，以确定基因毒性应激，包括电离辐射，脑发育过程中的作用。

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 ...

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15.2K 次观看.  CEA DSV iRCM SCSR. 胸苷两个类似物管理，埃杜和BrdU，在孕鼠允许在神经细胞和祖细胞在胚胎小鼠大脑细胞周期进程的分析。这个方法是有用的，以确定基因毒性应激，包括电离辐射，脑发育过程中的作用。 

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

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

科研

神经科学

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

神经细胞集落形成细胞分析检测，以区分善意的神经祖细胞的神经干细胞

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

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 ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

神经母细胞含量：从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

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 ...

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

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

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. ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.