作者感谢批判手稿Lwigale实验室的成员。 SLG是支持国家眼科研究所（F32 EY02167301）由露丝属Kirschstein NRSA奖学金。爱国青年同盟的支持，国家眼科研究所（EY018050）。

1。孵化小鸡和鹌鹑蛋所需的阶段 HH9胚胎，典型的孵化时间范围从29-33小时，在38°C的63 <ol><li>鸡蛋用温水洗净任何碎片。 </li><li>横向排列托盘上的鸡蛋。用铅笔标记面朝;这将对应的胚胎将局部区域。孵化鹌鹑蛋的钝端。 </li><li>在38个地点°C间湿润的孵化器。打开摇摆功能。 </li></ol> 2。准备鸡蛋的窗口和解剖<ol><li>从孵化器中删除的鸡蛋，并用70％乙醇消毒的上衣。这是最好的喷乙醇和用纸巾或Kimwipe的避免任何乙醇的吸收，通过蛋壳迅速擦掉。 </li><li>放置到一个单独的鸡蛋持有人（我们使用培养皿内衬折叠纸巾，例如，“Kimwipes”的）鸡蛋。使用AA钳，挖掘小HOLE蛋壳的蛋尖底上表面。 </li><li>删除1.5 3ML 18½Ğ皮下注射针和5毫升注射器从鸡蛋白蛋白的光。这是最好的，面临的锥尖底的蛋（图2A），成孔垂直插入针。通过这种方式，被撤回白蛋白时，是通过吸蛋黄意外损坏的风险不大。丢弃的白蛋白。这一步将降低内蛋黄和胚胎，以便在蛋壳打开一个窗口。 </li><li>用70％乙醇擦拭孔如上所述，并用透明胶带密封。重要的是，孔周围的蛋壳是完全缺乏白蛋白和干或不密封胶粘剂。 </li><li>与机管局钳，点选另一个洞，不是很显着的水平鸡蛋上表面到顶点。小心，不要让镊子去太远通过蛋壳，以避免损坏蛋黄或胚胎。 </li><li>在沙地成孔平行鸡蛋壳的弧形光圈镊子的一侧。捏壳上，工作中的圆周运动，打破1〜2厘米直径窗口，在鸡蛋壳。丢弃删除蛋壳。另外，包括用包装带鸡蛋上表面，并用一把剪刀剪出窗口。这可以最大限度地减少落入鸡蛋的蛋壳碎片的机会。胚胎应该会出现蛋黄上的不透明光盘。丢弃任何未受精的蛋（蛋黄表面上的白色小点，或胚盘的情况下确定）。 </li><li>注入少量墨汁（稀释在无菌林格氏液包含“钢笔/链球菌”的解决方案1:10抗生素：终浓度为100微克/ mL青霉素和100μg/ mL链霉素）协助举办胚胎的胚下方。用1 mL注射器和26½Ğ皮下注射针，锥朝上弯曲成45°角进针的基地，另外，马y使用一把拉住玻璃巴斯德吸管口移液设备（注射器针头弯曲或打破玻璃针时戴上保护眼睛的）。穿刺外胚外围的卵黄膜和滑动针尖下的胚胎，蛋黄表面接近，但不是在胚层（图2B）。注入足够的墨水勾勒出胚胎，然后小心地撤出针（图2C）。注入墨水过多，可导致胚胎死亡。重要的是，不注入任何气泡下方的胚胎，因为这可以是一个污染源。注：在此过程中使用的所有林格氏液消毒，并包含上述定义的画笔/链球菌。无菌磷酸盐缓冲液（PBS）也是一个可接受的替代林格在解决本协议的所有步骤。 </li><li>根据汉堡包和汉密尔顿63阶段的胚胎和记录的永久性墨水或铅笔在蛋壳上的阶段。下立方米，是最好的评估阶段omicroscopy，与光纤的“鹅颈”光源，其中有一个有限的热负荷。 </li><li>适用于2-3滴温暖无菌林格氏液胚胎的表面，以防止脱水和污染。暂时封住封口膜在蛋的表面伸出的窗口。 </li><li>重复步骤开始之前移植实验与所有鸡蛋2.2-2.9。 </li><li>由于鹌鹑胚胎只用于捐助者组织， 在OVO步骤2.2-2.9可以省略。为前囊胚OVO准备，鹌鹑蛋的孵化钝边，弯钳在这一地区开揭示了胚胎。用解剖剪刀，削减通过卵黄膜和胚胎外胚盘（确保所有削减满足）在一个正方形的形状。使用弯曲的虹膜钳，轻轻抓住一切缘和电梯从鸡蛋胚胎，并将其转移到林格在培养皿中的解决方案。另外，可以使用曲线ð虹膜镊子解除下，或胚胎勺或塑料移液管，通过广泛的气缸部分削减，可用于切除胚胎转移的胚胎蛋。 ＃5镊子轻轻取出卵黄膜。如上收集剩下的鹌鹑胚胎，在解剖显微镜下举行。 </li></ol> 3。准备主机接受移植的胚胎<ol><li>从主机（鸡）胚胎取出封口膜，并用削尖的钨针或＃5镊子，撕裂卵黄膜的小孔，在神经管（图3A）的理想地区。 </li><li>加入林格氏液对胚胎的下降。注意保持完全淹没在林格在整个过程的解决方案，以防止脱水的组织的胚胎。保持胚胎的表面如果有必要通过转移移液器加入几滴林格氏液。 </li><li>使用拉玻璃针，仔细延髓和CAudal相应的兴趣背神经管的长度和地区的横向切口。 （拉玻璃针从硅玻璃毛细管针拉仪器已在热拉。）切口单侧移植只应延伸到背神经管的管腔，并为双边移植，使切口跨越整个背神经管。然后削减双边之间的背神经管和近轴中胚层。 </li><li>仔细分离切除外植体，然后从神经管从胚胎中取出吸成玻璃微（图3B）。 </li></ol> 4。准备捐赠移植组织<ol><li>选择一个阶段匹配的鹌鹑胚胎。同机管局钳下来的胚胎，去除卵黄膜，消费的神经管类似大小的区域，如上所述（3.3-3.4）（图3A&#39;-B“）。根据实验的需要，这个地区可能是一个主机（鸡）神经管或从不同地区沿rostro尾轴互补的区域。 （注：对于较大区域的移植，包括完整的神经管移植，研究人员可能希望在这一步中添加蛋白酶消化，以消除任何附着捐助组织移植前骨髓间充质组织，但对小背神经管移植，在早期阶段。蛋白酶消化，这里详述，没有必要为神经管很容易被从邻近的间质分离，严格的手术技巧。） </li></ol> 5。移植的组织<ol><li>含有林格氏液的玻璃微吸管吸成捐助外植体。要小心移液器不引入任何气泡。 </li><li>转让的外植体切除鸡主机地区毗邻。使用拉玻璃针，外植体的方向，轻轻地引导到消融区（图3C）。 </li><li>仔细林格氏液添加几滴胚胎，以防止脱水。照顾，以避免下降液体直接移植网站上，因为这可能撞出移植。 </li><li>包装胶带封住窗口。确保胶带密封的整个地区的窗口蛋。这可以防止脱水和污染的胚胎，在随后的培养。 </li><li>返回蛋的孵化器，直到所需的阶段。确保“摇摆”功能被关闭，同时孵化的嵌合体。鸡蛋可以轻轻打开手一天两次，因为这可能会增加他们的生存能力，如果以后的发展阶段有针对性。 </li></ol> 6。准备嵌合胚胎切片<ol><li>在实验终点，磁带覆盖窗口使用细剪刀剪去。 </li><li>把握胚盘边缘的弧形光圈钳（最容易做的锯齿提示），然后切从蛋黄中的胚胎离4大幅削减外胚边界。 </l我&gt; <li>胚胎转移到培养皿中含有林格氏液。照顾，以尽量减少暴露在空气胚胎为了避免脱水的组织。 </li><li>在立体显微镜下，轻轻分开使用＃5镊子胚卵黄膜。 </li><li>精心安排在菜的胚胎，使胚胎在同一方向，因为它是在鸡蛋周围奠定了平膜。 </li><li>用解剖针从钨丝64干净地从胚胎中取出安排在胚胎和胚外组织的边界针，钨丝平行盘底部的长度，周围的胚。使用温和的锯议案通过削减组织。 </li><li> ＃5镊子小心取出羊膜。在这一点上，你可能会想无论是进一步剖析你感兴趣的地区，或留下的胚胎整体。 </li><li>胚胎转移到冰冷的4％paraformaldehyde和岩石在4°C过夜</li><li>准备样品和嵌入冷冻或石蜡切片。 </li></ol> 跟踪主机内的胚胎移植的组织 。鹌鹑组织内的鸡胚，包括苏木素染色检测鹌鹑核仁（鹌鹑核有非常大的，颜色较深的比小鸡核染色包裹），Feulgen反应Rossenbeck反应的，吖啶橙或商务苯甲酰染色确定有几个技巧结合电子显微镜，或鹌鹑特定抗原的免疫标记3,47,65,66。这里我们使用QCPN的抗原和免疫技术标准的整体安装或部分，以确定鹌鹑鹌鹑鸡嵌合体（图4）神经嵴细胞。这项技术在实验设计提供了最大的灵活性，作为QCPN免疫可能也可以与其他抗体相结合，以确定来自捐助（鹌鹑）组织的分化细胞。使用standa路全安装或部分免疫荧光技术标记鹌鹑源性干细胞在嵌合体。 7。代表结果嫁接的神经管区域的重新孵化后的6H代表形象（HH11）预计嫁接捐助（鹌鹑）到主机（鸡）神经管（图4A）组织成立。胚胎出现后reinc​​ubation的完整集成的移植，或颅地区或体节的不对称发展，应该被丢弃。 通过嫁接地区跨越标示HNK-1迁移横向距离的神经管在HH11秀NCC的部分。鹌鹑细胞，促进NCC的迁徙流，神经管，都清楚地标示与QCPN（图4B）。 在稍后阶段，鹌鹑华北源性干细胞可以追溯到他们的最终目标的组织。 QCPN穿插标记细胞内的鸡胚间质E5公司（图4C）的上颌过程。 QCPN抗体可以很容易地与其他抗体结合检查鹌鹑源性NCC的主机环境中的分化。鹌鹑华北派生三叉感觉神经元由QCPN和Tuj1抗体（图4D）的标记。 <img alt="图1" src="/files/ftp_upload/3622/3622fig1.jpg" /> 图1。概述实验程序和必要的手段。）主机（鸡）和捐助国（鹌鹑）胚胎阶段匹配。标注的NCC流，有助于三叉神经节和颌面下颌地区，HH9是理想的。在HH9，神经管开始关闭在延髓区域，但尚未完全密封。在图中的灰色线代表中线神经管闭幕。点缀的白线，表明切除脑区的神经管的右侧，从主机和捐助的切割线，EMbryos。主机胚胎切除的地区将被丢弃，产生嵌合胚胎移植的供体组织。 b）必要的手段包括：18）5毫升注射器半Ğ皮下注射针，b）1 26毫升注射器半Ğ皮下注射45°角，Ç）印度墨针弯曲，ð）透明胶带，é）玻璃口移液吸管机管局钳，仪器，F）60毫米培养皿，G）H）带锯齿的提示，我的弧形光圈钳）＃5镊子，j）的罚款剪刀，K）削尖的钨丝，L）拉玻璃针封口膜，M）正方形。 <img alt="图2" src="/files/ftp_upload/3622/3622fig2.jpg" /> 图2。鸡蛋卵子和胚胎的制备A）横断面图，其中包括理想的插入点18½G为白蛋白的光撤出注射器针头。 b）在退出〜3毫升光白蛋白，蛋黄和胚胎降低蛋内，使研究人员能够减少在T的一个“窗口”（箭头）他蛋壳访问胚胎。墨汁，在无菌林格氏液1:10稀释，然后注入胚下方提供的胚胎容易分期的对比。 c）在墨鸡胚。 <img alt="图3" src="/files/ftp_upload/3622/3622fig3.jpg" /> 图3。原理和实例农户阶段9捐助和主机胚胎移植手术的每一步。）小鸡主机胚胎。图中虚线表明卵黄膜应撕下来访问颅地区进行嫁接。一旦撕裂卵黄膜三角瓣应尾端去皮。 （BC）的使用，在图像框表示插图。 A&#39;）的鹌鹑捐赠胚胎。图片是从卵切除移植组织解剖，并放置到培养皿的捐赠胚胎。 （B&#39;），用于在图像框表示插图。虚线示意图（A&#39;），表明卵黄和卵黄膜应削减，以消除ţ他从鸡蛋胚胎。 B）与主机单方面脑区神经管切除（白色箭头），等待移植的胚胎。在原理图中的虚线表示，削减应在中脑区域神经管切除。 B“），供体胚胎神经管背侧移植组织切除（移植组织是由黑色箭头表示）。图中的虚线表示削减应在鹌鹑胚胎消费的捐助组织作出单方面的脑区的神经管嫁接。 c）嵌合胚胎后鹌鹑背神经管植嫁接到鸡脑区域。 <img alt="图4" src="/files/ftp_upload/3622/3622fig4.jpg" /> 图4。鹌鹑具体捐助者源性细胞在嵌合胚胎核抗原QCPN的免疫检测 ）全安装HH11（，HH9嫁接）嵌合体显示红色QCPN的染色，染色HNK-1（迁移NCC的标志形象）在绿色。 10X的放大倍率，背视图。二）横截面在（A），通过胚胎神经管和洄游华北HNK-1（绿色）联合染色QCPN积极鹌鹑细胞。 40X放大。 c）跨节通过上颌E5公司嵌合体的过程（培养后移植3天），显示QCPN阳性华北源性干细胞（红色），嫁接地区，他们的最终位置在胚胎神经管迁移。 10X的放大倍率。 d）第通过E5公司鹌鹑嵌合体显示派生华北三叉神经节（红色）和分化成TuJ1阳性神经元（绿色）的贡献。 40X放大。 *，NCCS，上颌过程MP;新台币，神经管;（TG），三叉神经节。

作者没有透露。

鹌鹑神经管移植到主机鸡胚这里描述的是一个简单和廉价的技术跟踪特定亚群的迁移沿rostro尾轴21,67-69来自不同地区产生的净捐助国。这种技术易于访问禽流胚胎（如哺乳动物胚胎相比）的优势，可与其他技术相结合，如组织消融，注射抑制分子，或通过电表达质粒的基因操纵，实验研究具体华北迁徙种群的反应不同的发展线索，在胚胎47,69。 鹌鹑，鸡嵌合体...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>鸡蛋</td><td>不同的 - 我们使用得克萨斯州A＆M大学家禽科学系，德克萨斯州。 </td><td></td></tr><tr><td>鹌鹑蛋</td><td>不同的 - 我们使用奥沙克蛋公司，MO。 </td><td></td></tr><tr><td>蛋孵化器（数字读数运动员孵化器1502 W /湿度110-120伏交流） </td><td> <a href="http://www.poultrysupply.com" target="_blank">www.poultrysupply.com</a> </td><td> 1502 </td></tr><tr><td>杜蒙AA钳，Inox公司环氧涂层</td><td>罚款科学工具</td><td> 11210-10 </td></tr><tr><td>透明胶带</td><td>任何办公用品商店</td><td></td></tr><tr><td>弧形光圈钳</td><td>罚款科学工具</td><td> 11065-07 </td></tr><tr><td>我NDIA油墨</td><td>任何艺术供应商店</td><td></td></tr><tr><td> PEN /链球菌（青霉素，链霉素）解决方案</td><td> VWR国际</td><td> 101447-068 </td></tr><tr><td>透明胶带</td><td>任何办公用品商店</td><td></td></tr><tr><td>针拉仪器</td><td> narashige，日本</td><td> PE-21 </td></tr><tr><td>拉玻璃针，从1.5-1.8×100mm的硼硅玻璃毛细管</td><td>金布尔大通</td><td> 34500 99 </td></tr><tr><td>拉玻璃吸管，从5¾“巴斯德吸管</td><td> Fisher Scientific则</td><td> 13-678-6A </td></tr><tr><td>口吸管装置（用于校准的微毛细管吸管吸气管总成） </td><td> Sigma-Aldrich公司</td><td> A5177-52A </td></tr><tr><td>杜蒙＃5镊子</td><td>罚款科学工具</td><td> 11251-30 </td></tr><tr><td>墩gsten线，0.1毫米直径</td><td> VWR国际</td><td> AA10404 - H2 </td></tr><tr><td>针持有人（镀镍针座） </td><td>罚款科学工具</td><td> 26018-17 </td></tr><tr><td> QCPN血清</td><td>发展研究杂交瘤细胞银行，美国爱荷华大学</td><td> QCPN </td></tr><tr><td>的Alexa Fluor二次抗体（如的Alexa Fluor 594山羊抗鼠IgG1） </td><td> Invitrogen公司</td><td> A21125 </td></tr><tr><td>林格氏液（2L）： <ul><li> 14.4克氯化钠</li><li> 0.34克氯化钙2 </li><li> 0.74克氯化钾</li><li> 0.230克的Na 2 HPO 4 </li><li> 0.04克KH 2 PO 4 </li><li> DDH 2 O至2L </li><li>过滤器和高压灭菌器</li></ul></td><td> Fisher Scientific则所有试剂</td><td><ul><li> 7647-14-5 </li><li> 10043-52-4 </li><li> 7447-40-7 </li><li> 7558-79-4 </li><li> 7778-77-0 </li></ul></td></tr></tbody></table>

analysis of neural crest migration and differentiation by cross-species transplantation

鸡胚胎提供一个独特的平台，为研究许多脊椎动物发育过程中，由于蛋内的胚胎方便。嵌合胚胎禽流感，鹌鹑捐助组织移植到鸡胚大毛 ，结合由禽流胚胎提出的操纵方便了不可磨灭的细胞群的遗传标记的权力。 鹌鹑鸡嵌合体是一个追踪迁徙的神经嵴细胞（NCC）的1-3古典的工具。 NCC的是胚胎细胞在一个短暂的迁徙人口，这源于在区域发展中的神经管4背。他们接受的上皮间质转化，并随后迁移到其他地区的胚胎，它们分化 ​​成各种细胞类型，包括软骨5-13，11,14-20黑色素细胞，神经细胞和神经胶质21-32。 NCC的是多向的，他们的最终命运是influenced由1）该地区的神经管中，他们沿着源自胚胎11,33-37，2 rostro尾轴）从邻近的细胞信号作为他们迁移38-44，和3），其最终的微胚胎内的目标45,46。追踪从他们的起源点在神经管细胞，其最终的胚胎内的地位和命运，图案和器官的发育过程的调节提供了重要的线索。 捐助者的神经管（伦嫁接）或不同地区的捐助者的神经管（异位嫁接）互补地区移植可以揭示沿rostro尾轴2,47 NCC的预规范的差异。这种技术可以进一步适应移植的神经管单方面车厢，一边是从供体组织取得，对侧遗体在宿主胚胎，易建联泰然自若elding 2,47在同一样品的内部控制。它也可以适应在以后的胚胎移植脑段，HH10后，当前神经管已经关闭了47。 在这里，我们报告通过神经管移植，允许从离散的神经管段的候鸟NCC的跟踪产生的鹌鹑鸡嵌合体的方法。具体物种的鹌鹑，的具体QCPN抗体48-56捐助源性干细胞的标记，允许研究者区分捐助国和宿主细胞在实验终点。这种技术很简单，价格低廉，并有许多应用，包括命运的映射，细胞谱系的追踪，并确定预图案，沿rostro尾轴45。由于易于访问禽流胚胎，鹌鹑鸡移植技术可以与其他操作相结合，包括但不限于镜头消融40，注射抑制分子57,58，或通过基因表达质粒59-61电操纵，特别是在胚胎的发育程序的扰动NCC的迁徙流识别的响应。此外，这种嫁接技术也可能被用来产生其他如鹌鹑，鸭嵌合体间的嵌合胚胎研究华北贡献颅面形态，或鼠标鸡嵌合体小鼠遗传学的力量结合禽流胚胎操纵方便62

Watch this Scientific Journal Video about Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation at JoVE.com

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

描述的分析禽流鹌鹑鸡嵌合胚胎的神经嵴细胞的迁移和最终命运的方法。此方法是一种简单明了的技术追踪神经嵴细胞迁移和分化的过程中，否则难以区分，在未添加鸡胚。

跨物种移植神经嵴迁移和分化的分析

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

analysis-neural-crest-migration-differentiation-cross-species

Research

JoVE Journal

Neuroscience

22.7K 次观看.  Rice University. 描述的分析禽流鹌鹑鸡嵌合胚胎的神经嵴细胞的迁移和最终命运的方法。此方法是一种简单明了的技术追踪神经嵴细胞迁移和分化的过程中，否则难以区分，在未添加鸡胚。

视频: Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

小鼠神经嵴干细胞的培养鸡胚提取物的生产

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

科研

神经科学

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

由运动神经元调制生理，形态学和神经化学的表征

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

从人类毛囊神经嵴干细胞的分离和培养的

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

T-迷宫强迫交替和评估工作，并参考在小鼠记忆的左，右的歧视任务

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

躯干神经嵴细胞迁移使用一个修改过的Zigmond室法分析

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

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 ...

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 ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

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 ...