我们感谢丹尼斯FLIPO用于图像处理和分析意见，戴维W在银鱼的实验室生成的Gata4p [5KB]-GFP的小鼠品系。研究在实验室皮隆是由CIHR，NSERC，FRQS和FRQNT资助。

伦理声明在老鼠的实验进行了以下的在医学研究中使用动物的照顾和处理动物护理指引加拿大议会。涉及动物的操纵协议已获魁北克蒙特利尔大学的伦理委员会（科米特Institutionnel去保护DES ANIMAUX;编号0512-R3-650-0513）。 1。胶原蛋白凝胶的制备在无菌的方式工作，在组织培养罩。 <ol><li>准备完整的5倍DMEM培养基（Eagle的修改必需培养基），包括标准的抗生素。溶解3.37克DMEM粉末和0.925克的NaHCO 3在20毫升水中。穿过一个0.22微米的过滤器进行消毒。添加2.5无菌100X青霉素/链霉素和25毫升无菌热灭活胎牛血清的溶液中。贮存于4℃。 </li><li>冰，混合800微升的I型胶原溶液（3.77 mg / ml的0.02N的乙酸，过滤灭菌的），600微升的完全DMEM培养液5倍和17微升1N的NaOH。用无菌水稀释至3毫升的最终体积。包括在组合有关的生长因子。我们通常使用的GDNF在10毫微克/毫升，以刺激肠道NCC迁移。 </li><li>存款约480μl，以单行的在24孔板中每个孔中。避免气泡。其余行可用于测试的其他生长因子对细胞迁移的影响。 </li><li>让胶原蛋白在37℃下聚合至少1小时，在无菌恒温箱</li></ol> 2。 16动物的解剖<ol><li>成立交配和检查阴道塞第二天早晨。一天E0.5是中午阴道塞被发现的当天，隔离女性和等待12天，直到E12.5。 </li><li>麻醉孕鼠与异氟醚和二氧化碳吸入安乐死。 </li><li>喷鼠标，用70％的乙醇。提起腹部s亲属，并与解剖剪刀打开腹部腔。 </li><li>取出子宫到玻璃培养皿中充满了冰冷的PBS（磷酸盐缓冲盐水）。切子宫横向个人蜕膜肿胀之间的隔离每个胚胎着床部位。 </li><li>每个植入位点在另一个玻璃培养皿充满冰冷的PBS分别工作。在解剖显微镜下，用细镊子取出子宫肌肉层。 </li><li>打开卵黄囊和羊膜揭示胚胎。切断血管加盟胚胎的胎盘/卵黄囊的时候，因为他们是交织在一起的发展肠子照顾。 </li><li>断绝胚胎的头颈部。 </li><li>插入一个封闭镊子在腹腔胚胎的空腔，就在暗红色色肝上，并让钳子打开的本身（停止施加压力），以使一个横向开口在腹部空腔中。抠开镊子下来，对胚胎完全打开腹部的后端。 </li><li>抢肝背后的结缔组织和拉的胆量了腹部，注意不要打破肠（大肠连接到肛门）。 </li><li>切断结肠，从胚胎的其余部分释放的胆量。切割可以在沿着结肠的任何位置进行。保留尾部后。 </li><li>梳理出从盲肠结缔组织，肠子然后休息。要小心，不要伤了肠子，而这样做。 </li><li>切掉肝和胃（在小肠的喙末端），以及中肾和生殖嵴，如果一些都存在。 </li><li>隔离小肠。再次，要小心不要夹到肠。从现在起，肠道组织的rosrrocaudal方向可以使用锋利的曲率目前在喙末端进行跟踪。留在小肠的PBS中于室温下尽可能短的时间尽可能烯前床上用品（见步骤3.3）。 </li><li>最后，记录尾部体节的每个胚胎的数目准确地确定胚胎发育的阶段。 </li></ol> 3。胚胎肠切片<ol><li>在继续进行胚胎解剖，熔体1.5克琼脂糖于100ml PBS（磷酸盐缓冲盐水），并保持在50℃。 </li><li>倒入琼脂糖融化成一个嵌入模具（ 例如闭2 ml离心管，已被切割纵向切除约1/4的管壁）。让琼脂糖冷却至约42-45℃，它应该是只稍微温暖的触感。 </li><li>就在琼脂糖固化之前嵌入胚胎肠（这可以用钳子提示进行评估，并会发生大约36-38℃）。保持由所述折叠喙端与钳肠道，通过沿着模具的长度琼脂糖拉得非常缓慢。这有助于保持肠道直而琼脂糖集。放开T只要它开始反抗移动的问题。跟踪肠道的喙，尾方向。 </li><li>放于冰箱中2-3分钟的模具，以确保琼脂糖完全凝固。 </li><li>取出琼脂糖从模具中（通过向外滑动打开的Eppendorf管中）。用刀片，取出多余的琼脂糖两端，使切口垂直于肠。 </li><li>胶水肠/延髓结束琼脂糖挡了下来与振动叶片切片机的金属舞台。修剪对肠/琼脂糖块两侧的过量的琼脂糖。 </li><li>安装在上述振动切片机室中的金属的阶段。如果有必要，调整阶段的角度，使肠道是尽可能垂直（因而垂直于叶片）。盖在冰冷的PBS试样。使切片机刀片向下到几毫米下的缓冲表面。 </li><li>使200微米vibratome横向切口尾最小肠，确保该电子ACH琼脂糖片包含一个完整的肠道部分。 </li></ol> 4。肠道外植体的培养<ol><li>轻轻的存放新鲜切肠/琼脂糖片平放在胶原凝胶用钳子，把一片鸡犬中间好。 </li><li>孵育3天37℃，在潮湿的5％CO 2的气氛中，以允许NCC的迁移出来的外植体。 </li><li>取小肠/琼脂糖片落胶原凝胶轻轻地用钳子。小心不要损坏凝胶下方。 </li><li>避免在为了随后的温育和洗涤步骤直接触摸胶原凝胶，以确保细胞迁移模式不受影响。修正了每孔1小时，在室温下500微升4％PFA（多聚甲醛）（在PBS）中。 </li><li>更换固定液，用500μL的DAPI（4&#39;，6 - 二脒基-2 - 苯基吲哚）溶液（5μg/ ml的PBS中），每孔，孵育10分钟，在室温下进行。 </li><li>每次洗好3倍用500μlPBS中5分钟。 </li><li>拍摄嵌入在胶原凝胶内各孔的荧光细胞（GFP和DAPI通道）。 </li></ol> 5。图像分析我们广泛利用ImageJ的17来处理和量化外植体培养后生成的图像。 <ol><li>首先，成像微米幻灯片在相同的放大倍数为荧光细胞的照片。测量以像素为单位一微米（用直线工具）的长度。 </li><li>设置比例（分析/设置刻度;像素/微米输入号码）。 </li><li>对于每个GFP荧光照片，图像格式改变为8位灰度（Image/Type/8-bit）。 </li><li>调整信号（图像/调整/亮度/对比度）的强度。 </li><li>减去背景噪音，如果有必要（工艺/减去背景;调整滚球半径）。 </li><li>设定阈值以突出细胞和细胞团块（图像/调整/阈值），则Ñ​​应用分水岭分割团块（工艺/二进制/流域）。 </li><li>分析粒子指定感兴趣区域（ROI）的区域，并生成细胞数量的统计数据（分析/分析颗粒;集大小最小排除剩余的像素）。 </li><li>设置测量选项包括费雷特直径（分析/设置测量）。 </li><li>集团的投资回报，并测量作为一个整体来确定的Feret直径，指示细胞扩散（分析/工具/投资回报率经理/更多/ OR，则投资回报率经理/添加和ROI经理/测量）。 </li></ol>

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作者什么都没有透露。

我们展示了如何我们的体外外植体培养技术可以用来精确地量化肠溶NCC迁移潜力的GDNF的存在。这种精确的定量是通过使用200微米厚的vibratome肠组织切片而不是大块的近似大小，如先前所描述11,12,15大大便利。的确，这使得我们能与细胞中一种高度可重现设定合理数量的工作。值得注意的是，荧光肠溶NCC的小肠从该外植体条带被切断的尾最区域内的均匀分布还允许从一个单一的肠道?...

神经嵴细胞（NCC）是胚胎发育过程中形成了许多衍生工具的瞬态细胞类型特有的脊椎动物。该细胞群出现在神经板，相邻的非神经外胚层1的边界。在神经胚形成，为神经板的地方NCC沿形成神经管背侧边缘弯曲。 NCC再经过上皮 - 间质转化，分离和迁移远离神经管。 NCC殖各个胚胎结构，包括消化道那里形成整个肠神经系统（ENS），神经节的互连网络中嵌入肠壁。由于最近检讨2,3，许多基因都参与了这个复杂结构的发展。 大多数ENS的是从迷走神经管（ 即约准后脑/脊髓边界）一个小水池NCC原始的派生4。这些神经祖细胞到达前肠围绕胚胎一天（五）9.0小鼠，然后尾端迁移肠道间质内，直到约e15.0拓殖整个胚胎肠。结肠的神经祖细胞的未成年子集也由骶NCC，其侵入后肠道的相反方向到盲肠4提供。既迷走神经和骶NCC需要多次迁移，增殖，存活和分化，促进线索，以确保完全形成ENS的。在这方面，动物模型 - 尤其是转基因小鼠 - 是有助的在几个必要的细胞外配体的鉴定:GDNF（神经胶质细胞源性神经营养因子），内皮素-3，神经营养素-3，骨形态发生蛋白（骨形态发生蛋白），netrin诱导，以及声波和印度刺猬（Shh和Ihh信号）5-10。其中，GDNF通过酪氨酸激酶跨膜受体RET（转染过程中整理的）信号被确认为日Ë最关键的途径的吸引力和NCC的指导，并在胚胎肠道。 GDNF是由肠道间质细胞分泌，并形成了一个时空方式控制rosrrocaudal梯度是直接chemoattractive到肠道NCC，其表达RET 11,12。 除其他功能外，ENS通过与肠壁平滑肌相互作用调节消化道内运动。在肠道导致先天性巨结肠的末端区域神经节的缺席:受影响区段的强直性收缩导致堵塞，消化材料的上游积累和肠道和腹部的大规模扩张。先天性巨结肠症发生大约在5,000一只活产婴儿。肠道NCC的rostro-尾迁移模式被认为是主要的促进因素先天性巨结肠症的病因。从迁移NCC和b的最后部分的源结肠，最远owel被殖民统治，是最容易受到ENS中形成的缺陷。按照肠道NCC迁移的关键作用，GDNF / Ret信号的干扰是先天性巨结肠13的主要已知的遗传原因。 为了更好地研究华北克拉通和ENS的发展，我们产生了转基因小鼠线-命名Gata4p [5KB]-GFP 14 -在这种迁徙NCC都贴有绿色荧光蛋白（GFP）。我们接下来完善的体外细胞迁移测定中，由其它组11,12,15适于从发表的作品，即现在允许在各种生长因子，如GDNF的存在肠溶NCC迁移潜在精确定量。

<table><tbody><tr><td>DMEM powder</td><td>Wisent</td><td>219-010-XK</td><td>&amp;nbsp;</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Bioshop</td><td>SOB999</td><td>Biotechnology grade</td></tr><tr><td>Steriflip vacuum filtration system (0.22 micron)</td><td>EMD Millipore</td><td>SCGP00525</td><td>&amp;nbsp;</td></tr><tr><td>Penicilin-Streptomycin solution, 100x</td><td>Wisent</td><td>450-201-EL</td><td>&amp;nbsp;</td></tr><tr><td>Fetal bovine serum</td><td>Wisent</td><td>095-150</td><td>High quality grade</td></tr><tr><td>Collagen I</td><td>BD biosciences</td><td>354236</td><td>&amp;nbsp;</td></tr><tr><td>NaOH</td><td>Bioshop</td><td>SHY700</td><td>Diluted from 10 N stock then sterile-filtered</td></tr><tr><td>GDNF</td><td>Cedarlane</td><td>CLCYT305</td><td>&amp;nbsp;</td></tr><tr><td>Falcon 24-well Plate</td><td>BD biosciences</td><td>353047</td><td>&amp;nbsp;</td></tr><tr><td>Dissecting scissors</td><td>Fisher Scientific</td><td>089515</td><td>&amp;nbsp;</td></tr><tr><td>Glass Petri dish</td><td>VWR</td><td>89000-306</td><td>&amp;nbsp;</td></tr><tr><td>PBS</td><td>Sigma</td><td>P5493</td><td>Cell culture grade</td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>M125</td><td>&amp;nbsp;</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-20</td><td>&amp;nbsp;</td></tr><tr><td>Agarose</td><td>Bioshop</td><td>AGA001</td><td>Biotechnology grade</td></tr><tr><td>Surgical blade</td><td>Feather</td><td>21</td><td>&amp;nbsp;</td></tr><tr><td>All Purpose Instant Krazy Glue Pen</td><td>Krazy Glue</td><td>KG824</td><td>&amp;nbsp;</td></tr><tr><td>HM 650V Vibrating-Blade Microtome</td><td>Thermo Scientific</td><td>920110</td><td>&amp;nbsp;</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>&amp;nbsp;</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9564</td><td>&amp;nbsp;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Table of specific reagents and equipment.&lt;/p&gt;</td></tr></tbody></table>

a quantitative cell migration assay for murine enteric neural progenitors

神经嵴细胞（NCC）是一个短暂的和多能细胞群源于背神经管和广泛地在整个发展脊椎动物胚胎迁移。除了提供周边神经胶质细胞和神经元，NCC生成的黑素细胞，以及大部分的颅面骨骼。 NCC迁移和分化是由它们的轴向原点沿神经管和他们接触到不同的区域外线索的组合控制。细胞外配体的这种贡献是特别明显的肠神经系统（ENS），神经节的一个复杂的相互连接的网络，本地控制（其中包括）肠道肌肉运动和肠蠕动的形成过程中。在吻端到尾鳍时尚 - - 准肠道的整个长度大多数ENS是从华北克拉通，为了殖民承接了漫长的旅程一个小的初始池派生。其中已知的多条信号通路影响肠道定植NCC，GDNF / Ret信号被认为是最重要的。事实上，RET配体GDNF的时空方式控制分泌肠道间质，主要是负责吸引和RET表达肠NCC指导，并在胚胎肠道。在这里，我们描述的体外细胞迁移测定，利用转基因小鼠系具有荧光标记的NCC，这使得在各种生长因子，包括GDNF的存在肠溶NCC迁移潜在精确定量。

下面的结果是代表什么可以与这里描述的（ 图1）的技术来获得的。使用生长因子（ 即 GDNF）刺激的表达GFP的肠溶NCC出肠道外植体并进入胶原凝胶（ 图2）的迁移。虽然一些小区出来的外植体在无生长因子，这些大多不是GFP标记和表示被动条目。有必要从胶原凝胶取出肠切片，以便记录的结果，因为这个组织仍然是人口稠密通过荧光细胞和本来隐藏的细胞趴在胶原?...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

我们提出了一个先体外后体内的细胞迁移测定法，允许在各种生长因子的存在下肠道神经嵴细胞迁移的潜在精确定量。

定量细胞迁移检测方法的鼠肠神经前体细胞

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

a-quantitative-cell-migration-assay-for-murine-enteric-neural

Research

JoVE Journal

Neuroscience

9.2K 次观看.  UQAM. 我们提出了一个先体外后体内的细胞迁移测定法，允许在各种生长因子的存在下肠道神经嵴细胞迁移的潜在精确定量。 

视频: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

科研

生物工程

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

一种酶和无血清神经干细胞培养模型EMT研究适合药物发现

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

发育生物学

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

使用琼脂糖凝胶为基础的微流体装置的评估神经干细胞的运动

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

生物学

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 ...

神经科学

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

一个的趋电含量为神经前体细胞迁移的动力学分析外部施加直流电场

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

人脑神经前体细胞神经发育表型的快速检测 (npc)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

神经干/祖细胞在小鼠EAE的慢性全身性注射

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

免疫与感染

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

鼠神经母细胞核转染到研究神经细胞迁移<em&gt;在体外</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

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 ...

可视化发育中的小鼠大脑中的神经元和神经胶质细胞动力学

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

作者聚焦：探索发育中的大脑中的细胞迁移和基因作用

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

胚胎小鼠脑切片中迁移神经元和神经胶质祖细胞的延时成像

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...