我们要感谢马克·沃兹尼亚克视频援助。我们也想承认在得克萨斯大学西南肖恩·莫里森的培养大鼠数控细胞的原始协议。这项工作是支持范德比尔特大学医学中心的学术计划支持，由国立卫生研究院（HD36720，HD036720-11S109）和的果酸11GRNT7690040的为PAL，从AHA（0615209B）和国立卫生研究院（NS065604）博士前奖学金不结盟运动，和ERP赠款由美国国立卫生研究院培训资助T32HD007502支持。

1。准备板<ol><li>在任何时候使用无菌技术。 </li><li>纤维连接蛋白（FN）Dulbecco的PBS（DPBS）在3.3毫升的稀释成终体积100μL人体血浆FN的股票准备。终浓度为30微克/毫升，这可以在4°C保存1周。 </li><li>覆盖的底部，并让每个无菌组织培养与FN的解决方案4孔板以及坐了15分钟。确保覆盖整个表面。在这段时间内（步骤2和3）媒体。 </li><li>删除FN的解决方案，并让板干燥。轻轻冲洗井与500μL的DMEM培养液，去除培养液，添加500μL的自我更新（SR）中（见下文）。在37°C的潮湿含5％ 二氧化碳培养箱中孵育。完成这个约一小时前清扫，使板块将使用前干燥。 </li></ol> 2。准备简中等<ol><li>为迷走神经和主干数控文化从10个克隆胚胎（约一乱抛垃圾，取决于遗传背景的鼠标线），准备25毫升的SR培养基。结合低血糖12.5毫升培养液，7.5毫升的Neurobasal中等，25μL维甲酸（117微米的最终浓度），25μL2 - 巯基乙醇（50毫米终浓度）。拌匀。 </li><li>加入3.75毫升鸡胚提取物，250μL的氮盐的补充，500μL，B27的补充和250μL的青霉素，链霉素（终浓度1％）。通过0.22微米的过滤器的过滤介质。 </li><li>加入10微升无菌IGF1的（20μg/ mL的终浓度）和20μL无菌碱性成纤维细胞生长因子（20μg/ mL的终浓度）。混合相。保存于4°C。 </li></ol> 3。准备洗净中等<ol><li>约10胚胎准备清洗液50毫升。结合35毫升低糖DMEM培养液，15毫升的Neurobasal中等，和500μL的青霉素，链霉素（终浓度1％）50毫克，牛血清白蛋白。 0.22无菌过滤微米过滤器。 </li></ol> 4。准备胶原酶/ Dispase酶<ol><li>加入50μL100 mg / mL胶原酶/ Dispase酶5毫升DPBS。拌匀。 </li><li>注射器用0.2微米的过滤器过滤和添加到三个井的12孔板每1.5毫升。吸取约1毫升，其余井洗介质。存储整个冰板，直到准备好消化解剖组织。 </li><li>削减1 P20和P1000移液器过滤嘴用无菌刀片的技巧。削减略低于针尖的斜面边缘。削减了P1000将用于转移整个胚胎，而P20将用于传输孤立的组织块。 </li></ol> 5。从9.5 DPC胚胎分离迷走神经干的神经管<ol><li>与对于超时怀孕，阴道塞坝被认为是在中午0.5 DPC上午插头观察。牺牲和删除9.5 DPC的子宫​​。 </li><li>删除从子宫蜕膜和轻轻ŕemove的胚胎从蜕膜。一旦该协议经历，隔离在无菌DPBS 3-4 9.5 DPC胚胎。使用无菌技术和消毒清扫工具。仪器可以灭菌消毒，热杀菌或乙醇的培养。 </li><li>对于前迷走神经数控:使用胰岛素针，切中期耳基板的神经管。再次削减在第 4 届体节的后边缘。修剪消除咽弓和心脏神经管腹侧组织。 </li><li>为主干数控:使用胰岛素针，取出部分之间的神经管体节16-22（或如果胚胎发育早于22体节期的最后体节）。 </li><li>保持卵黄囊和任何剩余胚胎的基因分型组织。 </li></ol> 6。拆除非神经外胚层和中胚层<ol><li>将神经管胶原酶/​​ Dispase酶分割成包含在室温下10分钟第立即洗涤液洗。 </li><li>返回组织无菌DPBS。使用无菌胰岛素针，轻轻地从组织中删除非神经外胚层远离神经管分离体节。最后剩下的部分中胚层体节组织可以去掉研制过程中轻轻用截止P20尖。小心不损害神经管。密切观察这期间研磨。 </li><li>通过第二个和第三个30洗介质的第二洗，将孤立的神经管。 </li><li>在SR培养基洗一次，孤立的神经管放入一个FN涂层以及准备（步骤1.3）的中心。加湿缺氧室，无菌水菜。菜放入低氧室和冲洗室至3％的O 2（含1％O 2 6％CO 2，93％N2混合使用坦克）的混合气体。 </li><li>总是处理室非常仔细，以确保THAţ植原状，并留在井的中心。 </li><li>在37°C。步骤5-6摘要如图1所示。 </li></ol> 7。去除神经管<ol><li>培养24小时后，取出神经管神经管细胞迁移使用无菌胰岛素针距边缘轻轻逗​​弄。从中期的神经管在步骤6.2使用无菌P20停产删除和丢弃和更换介质，用新鲜的SR中（ 图2）。这是最容易使用倒置显微镜。 </li></ol> 8。代表结果经过24小时的潜伏期在37°C在缺氧条件下，数控细胞迁移的神经管在一个近乎纯的人口（ 图3a）。有时，小于理想的文化，不会产生强大的副产物。例如，它是可能的后24小时内ţ蜷缩着他的神经管会根据本身和数控不会迁移从神经管（ 图3b）。偶尔，神经管不会附加到纤维连接蛋白涂层板。 次优数控迁移或神经管附件的问题，在我们的经验，可以产生不利影响常氧条件或纤维连接蛋白的浓度，分别为。胶原酶/ Dispase酶的酶活性略有不同批次和消化时间必须适当调整，但不超过十五分钟消化组织不再。过量也会导致含有胶原酶/ Dispase酶在组织神经管缺陷的副产物。如果体节的组织是不会轻易从神经管胶原酶/​​ Dispase酶在孵化后取出，神经管可以培养超过10分钟。神经管有时会不重视基板。如果是这种情况，仔细检查fibronec锡浓度和缺氧的条件。 虽然常氧条件下可用于文化野生型数控，缺氧条件下，更加紧密地模仿体内环境29,30。根据我们的经验，缺氧条件下成为关键的时候培养突变数控。例如，当突变数控Foxd3在常氧条件下培养，主干数控大大降低细胞生长与对照组相比。这一产物大小悬殊被删除时，外植体在缺氧条件下培养（ 图4）。此外，野生型神经管外植体时，在常氧培养，凋亡阳性细胞数大于缺氧培养的类似植（​​数据未显示）。维持在缺氧的所有NC文化，比较可以更方便地进行动态控制和突变文化之间。 <img src="/files/ftp_upload/4134/4134fig1.jpg" alt="图1" /> <strong&gt;图1。数控隔离的整体示意图。甲）从胚胎解剖感兴趣的地区。乙）为10分钟（不超过十五分钟）胶原酶/ Dispase酶消化，神经管。 c）清洗在清洗介质。四）解剖了非神经外胚层和中胚层。英法）洗涤液洗两次。 G）的自我更新介质板。在37°C孵育在3％O 2和缺氧条件下。 <img src="/files/ftp_upload/4134/4134fig2.jpg" alt="图2" /> 图2。神经管植逐步去除。神经管的，必须拆除后24-48小时，以防止污染与非数控细胞。一）注意神经管和NC的产物（实线）之间的边界。 b）切割神经管的边缘，随着胰岛素的针。三）丢弃的神经管，并更换新鲜的自我更新介质的介质。虚线表示程度的产物。缩写:新台币，神经管。 图3。代表结果的例子。一）培养24小时后典型的外植体在缺氧条件下的自我更新介质的产物（虚线表示程度的产物）。 b）放大后，48小时在文化的数控产物。 c）不太理想的文化产物产量低。 A和C在相同条件下进行培养。图片展示文化鲁棒性的自然范围。这可能会影响神经管隔离，FN的浓度，缺氧条件下，胶原酶/ Dispase酶消化时间效率。 <img src="/files/ftp_upload/4134/4134fig4.jpg" alt="图4" /> 图4。 在体外常氧与缺氧数控植文化分析。控制（野生型）数控从神经管植在常氧培养条件下48小时后的细胞迁移。相比之下，Foxd3突变数控大大降低细胞在常氧副产物（红色标记边缘轮廓的数控副产物）。可比植体缺氧条件下生长时，Foxd3突变数控植可比增长控制，使后续分析。请注意，此行为与与Foxd3突变数控体内的行为。

<ol>
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C., Keith, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+oxygen+availability+in+embryonic+development+and+stem+cell+function.">The role of oxygen availability in embryonic development and stem cell function.</a> Nat. Rev. Mol. Cell Biol. 9, 285-296 (2008).</li><li>Ivanovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+or+in+situ+normoxia:+The+stem+cell+paradigm.">Hypoxia or in situ normoxia: The stem cell paradigm.</a> J. Cell Physiol. 219, 271-275 (2009).</li></ol>

我们什么都没有透露。

小心应注意的胚胎发育阶段，以确保这种方法的成功。计数小鼠早期胚胎的体节是匹配的胚胎内的枯枝落叶，并确定正确地区神经管隔离阶段的关键。一个或两个胚胎体节之间的变化是在合理的范围内的发展时机，取决于进行试验的决议。 DPC 9和9.5之间的胚胎将有17和25体节之间。如果胚胎拥有超过25体节，是进一步推进数控发育时间，数控副产物，将在文化，那么强劲。分期根据胚胎体节数，便?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养基（低糖） </td><td> GIBCO / Invitrogen公司</td><td> 11885 </td><td></td></tr><tr><td>的Neurobasal中等</td><td> Gibco公司</td><td> 21103 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A3912-10G </td><td></td></tr><tr><td> DPBS </td><td> Gibco公司</td><td> 14190-144 </td><td></td></tr><tr><td> IGF1的</td><td> BD公司</td><td> 354037 </td><td>储存在50微克/毫升，分装在-20°C </td></tr><tr><td>碱性成纤维细胞生长因子</td><td> BD公司</td><td> 354060 </td><td>在25微克/毫升分装保存在-20°C </td></tr><tr><td>纤维连接蛋白</td><td> Gibco公司</td><td> 33016-015 </td><td>存储在为1mg/mL人iquots在-20°C。 </td></tr><tr><td>维甲酸</td><td>西格玛</td><td> R2625 </td><td>乙醇在-20°C重组后，在35微克/毫升分装店</td></tr><tr><td> 2 - 巯基乙醇</td><td>西格玛</td><td> D - 5637 </td><td></td></tr><tr><td> 氮的补充</td><td> Gibco公司</td><td> 17502-048 </td><td></td></tr><tr><td> B27的补充</td><td> Gibco公司</td><td> 17504-044 </td><td></td></tr><tr><td> steriflip 0.22微米的过滤器</td><td> Millipore公司</td><td> SCGP00525 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Invitrogen公司</td><td> 15140122 </td><td></td></tr><tr><td> 0.20微米的过滤器</td><td>康宁</td><td> 431219 </td><td></td></tr><tr><td>注射器（过滤） </td><td> BD公司</td><td> 301604 </td><td></td></TR&gt; <tr><td>四孔板</td><td>赛默飞世尔科技</td><td> 176740 </td><td></td></tr><tr><td>胶原酶/ Dispase酶</td><td>罗氏公司</td><td> 269​​ 638 </td><td>活动由一批不同。储存在-20°C 100毫克/毫升等分</td></tr><tr><td>胰岛素针 （29半规） </td><td>贝克顿·迪克森</td><td> 309306 </td><td></td></tr><tr><td>缺氧商会</td><td>比卢普斯-罗滕伯格</td><td></td><td></td></tr><tr><td>氧分析仪</td><td>比卢普斯-罗滕伯格</td><td></td><td></td></tr><tr><td>镊子＃5 </td><td>罚款科学工具</td><td></td><td>为消除子宫和蜕膜。 </td></tr><tr><td>胰蛋白酶-EDTA（0.25％） </td><td> Gibco公司</td><td> 25200 </td><td></td></tr></tbody></table>

isolation and culture of neural crest cells from embryonic murine neural tube

胚胎神经嵴（数控）是一个多能祖人口，背神经管方面的，经历了一个上皮间质转化（EMT）和迁移整个胚胎，从而引起不同的细胞类型1-3。数控也有独特的能力，影响靶器官4-6的分化和成熟。 在体外 explanted时，数控祖细胞进行自我更新，迁移和分化成各种组织类型，包括神经元，神经胶质细胞，平滑肌细胞，软骨和骨。 数控多能首次描述7-9禽流神经管的外植体。数控细胞体外分离，有利于数控加工动力学研究，包括细胞增殖，迁移和多能。在禽流感和大鼠系统的进一步工作表明，explanted数控细胞保留其数控潜力，移植胚胎10时-13。因为这些固有的保存explanted数控祖细胞特性，神经管外植体检测提供了一个有吸引力的选择研究在体外数控。 为了达到更好地了解了哺乳动物的数控，许多方法已受聘隔离数控人口。数控源性祖细胞培养胚胎和成年后洄游数控祖11,14-20研究动态，可以从迁移后的位置然而数控祖细胞的分离，因为他们从神经管移民提供最佳的保护， NC细胞的潜力和洄游特性13,21,22。有些协议采用荧光激活细胞分类（FACS）来隔离丰富的NC为11,13,14,17尤其祖人口。然而，与早期胚胎开始时，细胞数量足够用于分析是很难获得与流式细胞仪，复杂的隔离早期数控人群的从单个胚胎的NS。在这里，我们描述的方法，不依赖流式细胞仪和结果约96％的纯数控1 WNT1-Cre重组酶激活血统记者23人口。 这里介绍的方法是改编从大鼠数控11,13文化优化的协议。以前的方法相比，这种协议的优点是:1）细胞生长饲养层，2）没有需要获得一个相对纯净的的数控人口，3）premigratory数控细胞分离和4流式细胞仪）的结果很容易量化。此外，该协议可用于数控隔离任何突变小鼠模型，促进数控特点与不同的基因操作的研究。这种方法的局限性是数控的胚胎，这是众所周知的影响数控2,24-28的存活，迁移和分化的背景下删除。

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube at JoVE.com

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

胚胎神经嵴的神经管分离，便于使用<em&gt;在体外</em&gt;学习迁移，自我更新和多能神经嵴的方法。

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

17.1K 次观看.  Vanderbilt University Medical Center. 胚胎神经嵴的神经管分离，便于使用在体外学习迁移，自我更新和多能神经嵴的方法。

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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

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 ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

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 ...

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

在小鼠全胚胎培养的神经管封闭

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

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 ...

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 ...

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

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

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. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养:一个快速议定书"

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

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

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

从胚胎前脑内皮细胞的分离和培养

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