这项研究是由一个赠款MHS和AG（SNF IZLIZ3_157230）巴塞尔科学基金会的大学和瑞士国家科学基金会的支持。我们感谢:塔尼亚里纳尔迪博士Burkat慷慨地提供基础设施;该Bettler小组讨论和评论的所有成员。我们感谢多恩格哈德（徕卡公司，瑞士）的专业和称职的安装全高清MC170摄像机（徕卡公司，瑞士）。

所有动物的程序遵循指南"实验动物的护理和使用"（NIH出版， 第 8版，2011）和巴塞尔的动物福利委员会（用于动物的护理和使用瑞士准则）获得批准。通过这些准则的动物协议被认为是"最严重的动物品位"的。 1.膨胀培养基的制备注:在无菌条件下的标准组织培养工作。 <ol><li>取两个15毫升管，加入5 ml L-谷氨酰胺的DMEM / F12（1:1）从500毫升培养基瓶到每个两个15毫升管中。到的前15毫升管中，添加50毫克人脱辅基转铁蛋白，腐胺1M的股票（0.1毫终浓度）的50微升和硒酸钠500μM的股票（最终浓度为30nM）的30微升。 <ol><li>通过0.2μm注射器过滤成原来的DMEM / F12瓶过滤。 </li></ol></li><li>到第二15ml试管中，添加12.5毫克胰岛素。加6 - 9滴1M的氢氧化钠。涡旋以完全溶解的胰岛素。通过0.2μm注射器过滤成原来的DMEM / F12瓶过滤。 注意:氢氧化钠是有毒和腐蚀性。使用防护手套，外套，和护目镜。 </li><li> 5毫升青霉素（10,000单位/ ml）/链霉素（10,000微克/毫升）/两性霉素B（25微克/毫升）添加到DMEM / F12培养基瓶。 </li><li>加入5毫升的200mM L-谷氨酰胺股票刚解冻或寡肽含有谷氨酰胺（200mM的L-丙氨酰-L-谷氨酰胺二肽）的DMEM / F12培养基瓶。 </li><li>摇匀。准备50毫升等分。 注意:膨胀介质可以在4℃贮存至少2周。相比保持在500毫升的瓶子分装中显示管少pH值的变化。 </li></ol> 2.传代培养基的制备<ol><li>以1升的Ca 2+ -和Mg 2 2+ HBSS媒体，添加990.85毫克葡萄糖（5毫摩终浓度）和840.10毫克碳酸氢钠 </sUB&gt;（10毫米终浓度）。调节pH至7.3，用HCl（1M的股票）。过滤消毒用0.2微米的过滤器和赢利50毫升分装。 注意:传代培养基可在4℃贮存至少4周。 </li></ol> 3.生长因子的制备<ol><li>制备无菌1×PBS中的1％BSA（PBS-BSA）的单独或与在4mM的PBS（PBS-BSA-HCl）的盐酸（HCl）。 注意:盐酸是腐蚀性和毒性，需要特殊的安全措施（大衣，护目镜，手套，头罩）。 </li><li>解散10微克/毫升重组人成纤维细胞生长因子2（rhFGF2）在PBS-BSA（10毫微克/毫升终浓度），10微克/毫升的重组人骨形态发生蛋白4（rhBMP4）在PBS-BSA（10毫微克/毫升的最终浓度），和20μg/ ml重组人转化生长因子β1（rhTGFβ1）在PBS-BSA的盐酸（40纳克/毫升最终浓度）。 <ol><li>不要过滤消毒。准备等分。 注:生长因子等份可以储存在-20℃，至少2年。 </li></ol></li></ol> 4.涂料细胞培养皿中<ol><li>溶解1,875毫克的聚L-鸟氨酸在500毫升（巴解组织）蒸馏水2 O准备了250X巴解组织的股票。过滤消毒用0.2μm注射器过滤器和将2毫升分装。 注意:这些等分试样可被储存在-20℃下至少2年。 </li><li>溶解1毫克牛纤维粘连蛋白在1毫升无菌蒸馏水2 O准备纤维连接蛋白1000倍的股票。不要涡旋因为这可能会凝结成块的黏蛋白。轻轻摇动用手10分钟。 <ol><li>孵育在室温下偶尔摇动1小时。检查完全溶解。温热该溶液至低于37℃，纤连蛋白的完全溶解。不要过滤，消毒。 注意:溶液可以被存储在4℃下长达6个月。 </li></ol></li><li>如需要使用的等离子预处理塑料细胞培养皿（100毫米或其它尺寸本实验）。为了评估迁移，使用35毫米菜肴500微米的网格。过夜用2ml 1X PLO在37℃下在5％CO 2的组织培养箱中孵育菜肴12小时。用2ml 1×PBS中洗两次。 </li><li>在37℃下加入2毫升1×纤连蛋白（1微克/毫升终浓度）孵育菜肴最少12小时的，在5％CO 2的培养箱中。刚电镀细胞之前删除纤维连接蛋白。 注意:纤连蛋白包被的培养皿可以在37℃下贮存至少2周。 </li></ol> 5.标准化的解剖和皮质下区的准备（SVZ） <ol><li>获得大鼠胚胎14.5天（E14.5，只SD）和小鼠E13.5（C57BL / 6）通过胚胎定时交配。交配18:00动物，直到第二天早上08:00。交配后的一天中午被认为是E0.5。 </li><li>麻醉孕动物，用5％异氟醚和0.8升/分钟的氧气流。通过检查用锋利的爪子迪斯响应压缩挠度镊子。斩首动物。避免二氧化碳窒息的CO 2会影响干细胞的恢复。 </li><li>检索剖腹产胚胎。 <ol><li>将动物仰卧位和消毒用70％乙醇的皮毛。使用外科镊子和剪刀使约8cm V形皮肤切口在子宫上方的下腹部。切开只有皮毛，同时保持肌肉的墙壁完好无损。 </li><li>使用新鲜镊子和剪刀切开肌肉和腹部肌肉壁进入腹膜。 </li><li>识别在下部后腹膜子宫。从周围组织被删除锐性分离子宫。 </li><li>洗用无菌1×PBS中的胚胎并置于冰冷的1×PBS中的子宫。 </li><li>用细尖的剪刀剪开子宫壁由胚胎解剖显微镜下释放胚胎（放大3倍， 图1B）。用一对镊子除去胚胎船体（ <stroNG&gt;图1B）。保持冰在扩张中的胚胎。 </li><li>由显影大鼠神经系统5的图谱验证正确的发育阶段。正确的胚胎尺寸示于图1B。 </li><li>通过如在图1A中所示的鼠前肢（佛罗里达州）的开头位形成的存在下进一步检查正确的年龄。 注意:后肢（HL）尚未显示数字的分离（ 图1B）。 </li></ol></li><li>对于剥离，转移胚胎充满了冰冷的1X PBS无涂层的培养皿。使用高压细镊子立体解剖显微镜（3X到20X倍）下进行清扫。 注意:培养皿中防止组织与餐具表面的附着，与等离子涂层细胞培养皿可能发生。削尖的钨丝，针或类似也是夹层的某些步骤有帮助。 </li><li>取出头部皮肤及颅骨开始在INTE通过以获得显影神经管（ 图1C）前方和后方的两个镊子同时拉动显影端脑（TEL）和间脑（DI）（ 图1B）的rsection。 </li><li>确定中脑-后脑边界（MHB，黑色箭头图1B-D），分离从菱脑的中脑（MES）。切菱脑只是在或低于MHB（ 图1D，三角形箭头）。 注:在MHB额外的皮下空间有利于去除皮肤，而不伤害神经管。 </li><li>在断绝颅底与面部骨骼（ 图1E）端脑/间脑之间的连接。这导致含端脑，间脑和中脑（TEL-DI-MES）（ 图1F-H）的块。 </li><li>传送TEL-DI-MES块进入膨胀介质在冰上。保持完满成功伊利浑身膨胀介质在未涂布培养皿。握住块在一双镊子的脑，以免触及包含与神经干细胞皮质SVZ端脑。 </li><li>重复步骤5.5至5.8的所有的胚胎（ 图1F-H）。 </li><li>转移一个TEL-DI-MES块到新鲜的冰冷的1X PBS。沿于前脑（ 图1F-H）中的虚线切割，从间脑分离脑， 如图1I。 </li><li>通过沿图1F中的虚线切割从TEL分开的DI。参见图1J隔离端脑。 </li><li>拆分两个端脑半球，沿着图1J的虚线切割。这导致两个分离的半球， 如图1K所示。 注:左半球如图1L放大。 </li><li>确定内侧团伙lionic隆起（MGE， 图2A; *）和横向神经节隆起（LGE， 图2A +）通过椎间孔未来可见interventriculare。 <ol><li>使用镊子或针，沿着在MGE和LGE和前皮层（蚂蚁-CTX， 图2B）之间的交叉点的直线解剖。通过皮质，海马（臀部）和脉络丛（CP）的切割直线。这个分离前皮层包括来自神经节隆起嗅球（OB）（GE， 图2B，C）。 </li></ol></li><li>在尾神经节隆起（CGE， 图2C，D）和后皮层（ 图2C）的交叉点切的第二直线，通过皮层，海马和脉络丛再次切割。 </li><li>扁平化端脑。完全除去后极和嗅球（ 图2D）。鉴定含有海马和脉络丛（ 图2C）皮质下摆，如先前6,7限定。 注意:海马具有比皮质较薄神经上皮。该CP含红船。 <ol><li>从皮层分离皮质下摆，而使皮质相同于神经节隆起（ 图2D）的大小的大小。这导致皮质（靶组织）和神经节隆起（GE， 图2D）的块。 </li></ol></li><li>翻转与心室（内）侧与餐具表面皮层-GE块。 注意:半球的外表面将面对实验者（ 图2E）。上的外表面是血液的容器中，脑膜（ 图2E）。 <ol><li>针脚的GE用左手盘表面，并与在右侧（显性）手用镊子剥离脑膜关闭（ 图2F </str翁&gt;）。 注:这是技术上最苛刻的步骤，因为脑膜强烈坚持皮质。从皮层脑膜的分离是通过切割在步骤5.13.1和5.14完全直线（未锯齿状）促进。 </li></ol></li><li>重复步骤5.12到5.16的另一半球，所有的胚胎。切割沿LGE皮层在LGE（ 图2G，2H）一半的主直径的距离。解剖皮质跨越1.2毫米x 2.4英寸毫米（ 图2H）的面积，包括与神经干细胞在SVZ /生发层。 </li></ol> 6.准备外植体培养的<ol><li>切皮质成直径小于400微米（ 图2I）的外植体。使用定位在解剖培养皿下列参考（ 图2H和2I）400μm的格子（ 补充图1）。全部转让植到一个新的培养皿冰协LD膨胀介质。 </li><li>从组织培养皿（步骤4.4）拆下纤维连接蛋白。允许它干。 1毫升冷膨胀介质的添加到35mm培养皿以10毫米×10毫米（ 图3）的网格尺寸的中心。允许介质中以形成一个球形下降（ 图3）。 </li><li>插入多达8个外植体以液滴的中心。外植体应坐落于电网与迁移分析相互之间至少有3毫米的距离（见第8节）。 </li><li>孵化菜用于在细胞培养孵化器外植体的附着约1小时（37℃，21％O 2，5％的CO 2）。允许外植体沉降并附着在纤连蛋白涂覆的表面。不要摇晃的菜，因为植体分离和移动优化电网中心的。 </li><li>孵育1小时后（37℃，21％O 2，5％的CO 2），该盘填满至2ml膨胀介质的总体积加生长˚F行动者或感兴趣的物质的测试和孵化。 注意:无论是酶消化，也不是血清或离心所必需的外植体的准备。这是最佳的细胞完整性。 </li></ol> 7.准备NSC单细胞培养<ol><li>预热在37℃扩张和传代网上平台。 </li><li>传送解剖皮质片（从步骤5.17）放入15毫升管与冷膨胀介质（管A）。离心机皮质片5分钟，在1200×g的。吸出上清液。加入200μl预热的膨胀介质悬浮皮质件。 </li><li>加入700μl预热的传代中至15毫升管与P1,000头（管A）。轻轻重悬沉淀。将吸头在15ml试管的底部。通过与P1,000尖端抽吸三次轻缓地解离组织片。 注意:传代培养基促进细胞分离，并且需要避免使用酶。 </li><li>允许管坐30到60秒，大（重）未解离片底部定居。单离解的细胞将保留在上清液中。转约700微升的上清液至新鲜15ml试管（试管B）。 </li><li>重复步骤7.3和7.4以解离大块。 </li><li>重复步骤7.3和7.4的第二时间以解离较大的碎片。全部转移到上清新鲜15ml试管（管B）。 </li><li>膨胀介质添加到5毫升的总体积。算使用血球细胞。通过台盼蓝染色评估死细胞。 注:小神经干细胞容忍的步骤7.2至7.6比大细胞更好。从10胚胎期望约4×10 6个细胞，用10％的死细胞。 </li><li>为神经干细胞的扩张，板在8ml膨胀介质的体积每10cm纤连蛋白包被的组织培养皿1.5×10 6个细胞。对于标准的扩展，加入8微升的1000倍rhFGF2股票。添加8微升rhFGF2每天改变我直径隔日1次。 注:在这个发展阶段的皮层中含有大多数的神经干细胞，只有分化细胞​​的少数。分化的少数细胞不给rhFGF2处理响应，并扩大培养过程中死亡。 </li><li>通路扩大的神经干细胞在60％汇合。 <ol><li>要通过神经干细胞，取出扩张中。洗涤细胞迅速三次用5毫升传代网上平台。等待2 - 4分钟。不含Ca 2+和Mg 2+的细胞从表面慢慢脱落，向上舍入。验证阶段显微镜下的细胞脱离。再等几分钟，如果需要的话，直到表面视觉支队观察。 注意:单细胞就会完全分离，但多数仍细胞将坚持与餐具表面。需要分离的持续时间依赖于纤连蛋白涂层的持续时间。短纤维连接蛋白涂层（12小时或更少）导致更快的细胞脱落。对于较长的实验（&gt;; 1周）超过24小时的纤连蛋白涂层建议。 </li></ol></li><li>用10毫升吸管轻轻从表面分离细胞。收集与细胞中的5毫升传代培养基，并将其转移到新鲜的15ml试管。有一个额外的5毫升传代中的重复。 </li><li>离解在15ml试管中的细胞通过上下吹打三次，将10毫升吸管在管的底部。旋管15分钟，在室温下1200 xg离心。除去上清，悬浮于2ml膨胀介质中的颗粒。算使用血球细胞。使用台盼蓝评估死细胞。 注意:扩张至60％汇合后，期望4 - 5×10 6个细胞与死细胞计数在10％左右。 </li><li>板每10cm培养皿进一步通路8×10 5个细胞。 </li></ol> 8.迁移评估<ol><li>对于迁移评估，根据第5种子植于植隔离35毫米电网菜肴。电镀后一小时，添加FGF2（rhFGF2）。将在37℃培养箱菜2天。 注:为了获得最佳的外植体附着，避免移动的菜肴。 </li><li>由1000微升提示删除网上平台。由1000微升尖端起始于内圆（ 图3），加入2mL扩张介质。这降低了外植体的表面张力。 </li><li>添加生长因子单独或根据需要组合进行实验。使用FGF2 / BMP4 /TGFβ1作为阳性对照。注意:在这个实验中，每35mm培养皿2微升FGF2，2微升BMP4和4微升TGFβ1分别单独和组合（ 图5）中加入。 <ol><li>添加额外的因子和/或测试的物质。保留菜在37°C 2天后再次触及。 </li></ol></li><li>采取上倒置显微镜外植体的照片。 注:生活外植体产生更好的相衬图像比固定细胞。两者都可以使用。 </li><li>对于迁移评估，使用图形软件，使像素测量（ 如 ，斐济8）。 </li><li>测量为500μm距离像素的菜格，并用它作为内部参考。 </li><li>定义为外植体迁移起点的中心。测量外植体的中心和10个细胞的最外组之间的距离。 注:所有细胞迁移从植离心之遥。它们自发形成于下测试的情况下（ 图5）的周围的片。 </li></ol> 9.评估入侵<ol><li>根据协议第4制备纤连蛋白包被的组织培养的24孔板中。 </li><li>将300微升温暖扩展介质进入细胞侵袭室顶好。孵育室在37℃，30分钟和5％ 的 CO 2。 </li><li>除去从顶部以及膨胀介质和Boyden小室放置到涂覆24孔板。 </li><li>添加500微升与rhFGF2 0.5和0.5μlrhBMP4至底部以及温暖膨胀介质的。 </li><li>神经干细胞传代和第7节经文说明1至4计数都适合。 </li><li>板5×10 5细胞以300μl暖膨胀介质与rhFGF2 0.3微升并在boyden小室的顶部以及0.3微升rhBMP4的体积。 </li><li>培养24小时的神经干细胞种子。 </li><li>添加额外的因子和/或检验的物质的腔室和培养的细胞另外48小时。 注意:如果细胞是侵入性的，它们将通过从顶部以及通过基底膜层的底部很好。侵入细胞会附着到Boyden小室的底部。 </li><li>遵循由生产商提供的方案。用棉签（包含在侵袭测定试剂盒）通过清洗两次以除去在顶部以及非侵入性的细胞。 </li><li>从孔中除去培养基并加入500μl膨胀介质与质膜染色活细胞，用核染料DAPI到孔中。 </li><li>孵育在37℃下10分钟和5％ 的 CO 2。 注意:染料将分别纳入膜和侵入细胞的细胞核，以获得最佳的可视化8。选项​​:省略质膜染料如果多色免疫计划。 </li><li>与染料取出培养基并用扩张中洗两次澡。可视化和先前8演示了一个倒置荧光显微镜计数侵袭细胞。 注意:某些侵袭性细胞可能已经从腔室的底部分离的，并已下降到低于它们粘到涂布表面，其中所述孔的表面。有关完整的分析，包括孔表面的细胞。 </li><li>除去培养基并固定在冰 - 冷的新鲜的4％PFA的细胞10分钟。洗3次用1×PBS。上侵袭细胞进行免疫细胞化学，如前所述8-10。 </li></ol&gt;

<ol>
	<li>Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-Mesenchymal+Transitions+in+development+and+disease:+old+views+and+new+perspectives.">Epithelial-Mesenchymal Transitions in development and disease: old views and new perspectives.</a> Int J Dev Biol. 53, 1541-1547 (2009).</li><li>Sauka-Spengler, T., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+regulatory+network+orchestrates+neural+crest+formation.">A gene regulatory network orchestrates neural crest formation.</a> Nat Rev Mol Cell Biol. 9, 557-568 (2008).</li><li>Barriere, G., Fici, P., Gallerani, G., Rigaud, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Transition:+a+double-edged+sword.">Mesenchymal Transition: a double-edged sword.</a> Clin Transl Med. 4, 14 (2015).</li><li>Zawadzka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS-resident+glial+progenitor/stem+cells+produce+Schwann+cells+as+well+as+oligodendrocytes+during+repair+of+CNS+demyelination.">CNS-resident glial progenitor/stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination.</a> Cell Stem Cell. 6, 578-590 (2010).</li><li>Paxinos, G., Ashwell, K. W. S., To&#776;rk, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of the developing rat nervous system. , (1994).</li><li>O'Leary, D. D., Chou, S. J., Sahara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Area+patterning+of+the+mammalian+cortex.">Area patterning of the mammalian cortex.</a> Neuron. 56, 252-269 (2007).</li><li>O'Leary, D. D., Sahara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+arealization+of+the+neocortex.">Genetic regulation of arealization of the neocortex.</a> Curr Opin Neurobiol. 18, 90-100 (2008).</li><li>Sailer, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+neural+stem+cells+become+invasive+in+vitro+by+combined+FGF2+and+BMP4+signaling.">Non-invasive neural stem cells become invasive in vitro by combined FGF2 and BMP4 signaling.</a> J Cell Sci. 126, 3533-3540 (2013).</li><li>Sailer, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP2+and+FGF2+cooperate+to+induce+neural-crest-like+fates+from+fetal+and+adult+CNS+stem+cells.">BMP2 and FGF2 cooperate to induce neural-crest-like fates from fetal and adult CNS stem cells.</a> J Cell Sci. 118, 5849-5860 (2005).</li><li>Sailer, M., Oppitz, M., Drews, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cellular+contractions+in+the+human+melanoma+cell+line+SK-mel+28+after+muscarinic+cholinergic+stimulation.">Induction of cellular contractions in the human melanoma cell line SK-mel 28 after muscarinic cholinergic stimulation.</a> Anat Embryol (Berl). 201, 27-37 (2000).</li><li>Wicki, A., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+role+of+podoplanin+in+tumour+invasion.">The potential role of podoplanin in tumour invasion.</a> Br J Cancer. 96, 1-5 (2007).</li><li>Wicki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+invasion+in+the+absence+of+epithelial-mesenchymal+transition:+podoplanin-mediated+remodeling+of+the+actin+cytoskeleton.">Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton.</a> Cancer Cell. 9, 261-272 (2006).</li><li>Mishima, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+podoplanin+in+malignant+astrocytic+tumors+as+a+novel+molecular+marker+of+malignant+progression.">Increased expression of podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression.</a> Acta Neuropathol. 111, 483-488 (2006).</li><li>Motomura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+analysis-based+proteomic+subclassification+of+newly+diagnosed+glioblastomas.">Immunohistochemical analysis-based proteomic subclassification of newly diagnosed glioblastomas.</a> Cancer Science. 103, 1871-1879 (2012).</li><li>Kono, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+detection+of+the+lymphatic+marker+podoplanin+in+diverse+types+of+human+cancer+cells+using+a+novel+antibody.">Immunohistochemical detection of the lymphatic marker podoplanin in diverse types of human cancer cells using a novel antibody.</a> Int J Oncol. 31, 501-508 (2007).</li><li>Raica, M., Cimpean, A. M., Ribatti, D. <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+podoplanin+in+tumor+progression+and+metastasis.">The role of podoplanin in tumor progression and metastasis.</a> Anticancer Res. 28, 2997-3006 (2008).</li><li>Panchision, D. M., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+neural+stem+cells+by+morphogenic+signals.">The control of neural stem cells by morphogenic signals.</a> Curr Opin Genet Dev. 12, 478-487 (2002).</li><li>Lamouille, S., Xu, J., Derynck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+epithelial-mesenchymal+transition.">Molecular mechanisms of epithelial-mesenchymal transition.</a> Nat Rev Mol Cell Biol. 15, 178-196 (2014).</li><li>Gonzalez-Perez, O., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendrogenesis+in+the+subventricular+zone+and+the+role+of+epidermal+growth+factor.">Oligodendrogenesis in the subventricular zone and the role of epidermal growth factor.</a> Brain Res Rev. 67, 147-156 (2011).</li><li>Hu, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDGF-AA+mediates+B104CM-induced+oligodendrocyte+precursor+cell+differentiation+of+embryonic+neural+stem+cells+through+Erk,+PI3K,+and+p38+signaling.">PDGF-AA mediates B104CM-induced oligodendrocyte precursor cell differentiation of embryonic neural stem cells through Erk, PI3K, and p38 signaling.</a> J Mol Neurosci. 46, 644-653 (2012).</li><li>Galvao, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+quiescent+adult+oligodendrocyte+precursor+cells+into+malignant+glioma+through+a+multistep+reactivation+process.">Transformation of quiescent adult oligodendrocyte precursor cells into malignant glioma through a multistep reactivation process.</a> Proc Natl Acad Sci U S A. 111, E4214-E4223 (2014).</li><li>Liu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+double+markers+reveals+tumor+cell+of+origin+in+glioma.">Mosaic analysis with double markers reveals tumor cell of origin in glioma.</a> Cell. 146, 209-221 (2011).</li><li>Das, S., Srikanth, M., Kessler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+and+glioma.">Cancer stem cells and glioma.</a> Nat Clin Pract Neurol. 4, 427-435 (2008).</li><li>Modrek, A. S., Bayin, N. S., Placantonakis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+stem+cells+as+the+cell+of+origin+in+glioma.">Brain stem cells as the cell of origin in glioma.</a> World J Stem Cells. 6, 43-52 (2014).</li><li>Sampetrean, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+precedes+tumor+mass+formation+in+a+malignant+brain+tumor+model+of+genetically+modified+neural+stem+cells.">Invasion precedes tumor mass formation in a malignant brain tumor model of genetically modified neural stem cells.</a> Neoplasia. 13, 784-791 (2011).</li><li>McNeill, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+astrocytoma+pathogenesis+in+vitro+and+in+vivo+using+cortical+astrocytes+or+neural+stem+cells+from+conditional,+genetically+engineered+mice.">Modeling astrocytoma pathogenesis in vitro and in vivo using cortical astrocytes or neural stem cells from conditional, genetically engineered mice.</a> JoVE. , e51763 (2014).</li><li>Lara-Padilla, E., Caceres-Cortes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+nature+of+the+tumor-initiating+cell.">On the nature of the tumor-initiating cell.</a> Curr Stem Cell Res Ther. 7, 26-35 (2012).</li><li>Busch, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP-2-dependent+integration+of+adult+mouse+subventricular+stem+cells+into+the+neural+crest+of+chick+and+quail+embryos.">BMP-2-dependent integration of adult mouse subventricular stem cells into the neural crest of chick and quail embryos.</a> J Cell Sci. 119, 4467-4474 (2006).</li><li>Thiery, J. P., Acloque, H., Huang, R. Y., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions+in+development+and+disease.">Epithelial-mesenchymal transitions in development and disease.</a> Cell. 139, 871-890 (2009).</li><li>McDonald, O. G., Wu, H., Timp, W., Doi, A., Feinberg, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+epigenetic+reprogramming+during+epithelial-to-mesenchymal+transition.">Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition.</a> Nat Struct Mol Biol. 18, 867-874 (2011).</li><li>Rahimi, R. A., Leof, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+signaling:+a+tale+of+two+responses.">TGF-beta signaling: a tale of two responses.</a> J Cell Biochem. 102, 593-608 (2007).</li><li>Xu, J., Lamouille, S., Derynck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta-induced+epithelial+to+mesenchymal+transition.">TGF-beta-induced epithelial to mesenchymal transition.</a> Cell Res. 19, 156-172 (2009).</li><li>Suva, M. L., Riggi, N., Bernstein, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+reprogramming+in+cancer.">Epigenetic reprogramming in cancer.</a> Science. 339, 1567-1570 (2013).</li><li>Sullivan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+possible+new+focus+for+stroke+treatment+-+migrating+stem+cells.">A possible new focus for stroke treatment - migrating stem cells.</a> Expert Opin Biol Ther. , 1-10 (2015).</li></ol>

The authors declare that they have no competing financial interests.

在这项研究中描述了EMT的分析利用神经干细胞的标准化系统（总结于补充图3）。标准化保证再现性（ 表1和2）。的神经干细胞被从显影皮质衍生的，通常不经历EMT的组织。这是有利的为在EMT早期步骤的分析。在EMT初始步骤不能充分研究中已积累的遗传改变，并且可以已经通过EMT的特征的肿瘤细胞。此外，原发肿瘤的样品是不理想的理解EMT因为大多数恶性肿瘤是异质含有侵入性和非侵入性的细胞。所提出的协议提供EMT的研究人员用一种新的模型来研究EMT诱导的早期步骤。这里，我们表明，瑞伯和扭家庭的关键EMT诱导剂被依次激活:在第一阶段ZEB1和ZEB2被上调，在第二阶段期间TWIST2（FIGURË6）。此前，我们已经表明，SNAIL1是BMP4曝光后8上调已经好几个小时。我们还显示，在BMP4曝光从中央皮层神经上皮干细胞分化成间充质细胞，积极为平滑肌肌动蛋白（SMA）和调宁9。上述结果完成先前的研究中表明，所有的三个已知的键EMT调节中涉及的系统。此外，我们观察到TGF-β1可以单独显著增强FGF2 / BMP4的迁移效果。这些结果指出EMT诱导过程中FGF-，BMP-和TGFβ-信号之间的网络。 对神经干细胞的EMT和迁移分析成功地分离出最关键的步骤是:正确识别胚龄，确定胚胎发育的解剖标志，准备新鲜的培养基和（至少过夜）涂层板，用细尖的仪器和亲每显影皮质的中央部的清扫建立皮层外植体。 所呈现的技术可通过一些修改来增强。如上面所建议的，多个外植体铺板于单个菜的网格。对于多种化合物的筛选，但是，它可能会更方便放置单个植于96孔平板的每个孔。此外，该模型系统可以细化用于大规模药物发现。如上所述，PDPN是因为PDPN在侵入性的神经干细胞8找到的新获得的迁徙功能的有价值的标记。一个PDPN记者在EMT诱导前的正常神经干细胞转染。作为结果PDPN表达细胞可以作为内部控制细胞转化，因为PDPN中不正常的神经干细胞（ 图4和表1）表示。另外还有几个NSC标记可用，如巢，被诱导EMT后8丢失。作为结果，两个初始状态下，"非迁移/非侵入性/巢蛋白+"表示，最后的状态，"迁徙/侵袭性/ PDPN +"，可以被监测，从而使自动药物发现。在具有多个孔（ 例如 ，96孔板，以及较大的）初始细胞大细胞培养板可被接种和既定的药物或没有已知功能的化合物进行处理。因此，多孔板可以自动扫描PDPN或其他迁移/侵袭标志物的表达。如果抑制剂是屏幕的主要对象，PDPN表达的消失可以帮助确定推定有趣的小说抑制化合物。 期间的新物质的试验外植体可能无法显示的迁移和/或外植体可向上舍和从盘分离。在这种情况下，最好是每天（代替隔日完全介质变化）仅改变媒体的一半。这种表面张力与新鲜培养基更换时能够减轻压力。该植可能不是最初电镀后附加。在这种情况下，纤连蛋白必须在与餐具表面接触至少36小时。用于涂布纤连应该不暴露新鲜配制到塑料管中超过10分钟，因为纤连蛋白可能粘到塑料。此外，将外植可能定居在网格之外。在这种情况下，最好是漩涡以缓慢圆周运动的菜。这允许外植体，以重新定位到由向心力（参见图3）的中心。 有强有力的证据表明胶质瘤来自神经干细胞衍生23,24神经干细胞和/或衍生的OPC。从INK4 / ARF缺陷小鼠Sampetrean 等人分离的神经干细胞和强制的H-的Ras 25的过表达:作为结果其他团体已NSCs的基础上建立胶质瘤生长模型。高档的恶性肿瘤导致的结果显示增殖和侵袭移植后25。 MCN奥尼尔等人用转基因小鼠也孤立的神经干细胞（两侧装接loxP皂苷Rb1，NF1，KRAS，PTEN的单独和组合）26。所述基因通过Cre的病毒感染灭活和神经干细胞移植26。这两个模型系统具有侵入可在体内和潜在的新的抗侵袭药物被监控的优点是可以进行测试。他们的主要缺点是，入侵的发病没有明确定义，目前尚不清楚该细胞是否显示胶质瘤典型EMT入侵（EMT基因）或仅局部迁移。进一步只有一种药物可以每只动物进行测试和分析需要几个星期。以确定一个新的抗侵袭药，医药公司需要（a）至筛选几千化合物或多个一次，（b）向复制试验和（c）尝试不同的药物浓度。要准备几千移植的动物，用不同的药物对待他们，最后测试组织化学或生物发光效果分析是非常消耗资源的。此处被描述为EMT相关的入侵的新型基于NSC-模型系统，使数以千计的化合物的成本有效筛选。 虽然此模型使化合物的高通量筛选，它仅仅是一个初级筛选平台。一旦感兴趣的化合物在该模型中被确定，他们将需要额外的验证。 体内测试是必要的用于识别的任何化合物的安全性和功效参数和移植模型按上述方法变得重要25,26。该模型也被事实，即它是基于啮齿动物细胞的限制。虽然该模型可用于研究大鼠或小鼠EMT，如果相同的机制是在人细胞或人患者的地方，目前尚不清楚。还需要进一步的实验来调查，如果神经干细胞不仅在体外侵袭，而且移植后。若干条证据支持神经干细胞的作用在神经胶质瘤的形成。胶质瘤进展已链接到下列基因:韧粘素C，Hey1，SPARC SNAIL1和Snail2，FGFR + BMPR1A，EGFR，PDGFRβ，SOX2，Podoplanin，的Gli3和P75NGFR 8。所有这些基因的正常的非侵入性的神经干细胞的侵入细胞间质8的转型过程中也表达。当时之中，目前还不清楚是否神经干细胞可以转化成由唯一的外部生长因子的肿瘤。 从不同的肿瘤肿瘤起始干细胞（国际信托）已被分离并用作模型来理解肿瘤进展27。国际信托，然而，不适合于EMT分析，因为EMT并已经发生在国际信托27。理解的EMT诱导早期和晚期也步骤，就需要一个初级非肿瘤细胞群。理想的情况是这个人口并不意味着首先要经历EMT。它先前已表明，胚胎神经干细胞是合适的人选为此9,28。也就是说，迁移和侵袭可能由FGF2和BMP4 9,28神经干细胞被诱导。 FGF2 / BMP4诱导的迁移是有关单EMT相关基因，然而，缺乏完整的证据，因为关键EMT家庭瑞伯和扭尚未研究8。上述结果表明，四种因子的特定组合，FGF2，BMP4，TGFβ1与胰岛素导致非常强的和完整的EMT诱导，而不是之前观察到。目前的研究表明，Zeb-和扭家庭的关键EMT基因也上调。因此，本研究首次提供了证据表明，没有单个基因的选择性上调，但结果表明，所有关键EMT家庭都在建议的细胞培养系统中有效。 EMT也已在脑的外上皮癌，如肺癌，乳腺癌，结肠癌和胃癌29观察到。一些模型来研究EMT在使用这些肿瘤30-32，这来，但是，与显著局限性:（1）携带多个遗传和表观遗传改变33转化的肿瘤细胞系的使用;识别EMT的抑制剂早期癌症的阶段，晚期癌症细胞是不够的; （b）在含有各种不确定生长因子的血清依赖性培养;这使它的关键因素识别非常困难。此外，实验重复性受损，因为血清质量一批不同于一批; （三）的血清含有抑制剂和酶的活性可能失活潜在的有用的外源性化合物; （四）需要降解细胞表面分子酶细胞传代的; （五）以识别EMT激动剂促进生理EMT，需要正常细胞，以测试诱导。肿瘤细胞模型不足以发现，在正常干细胞促进肝细胞再生的物质。 还需要迁移正常过程，如伤口​​愈合和REGENE受伤的大脑18的口粮。创伤性脑损伤和中风后，神经干细胞是必要的，迁移到病灶部位参与再生4,34。当前系统也可用于鉴定促进迁移和侵袭的物质。如上图所示，迁移启动时用BMP4治疗引起的。如果细胞不暴露于骨形成蛋白，而是新的化合物，这些可以取代的BMP动作，这将有助于确定新的BMP激动剂。与报告系统，表明PDPN表达，对于新物质大规模测试变得可行;如果一种新的化合物可以替代的BMPs，PDPN表达细胞可被自动识别。 所提出的系统也进行调查细胞信号的相互作用是有用的。我们的研究结果表明，对迁移的BMP作用可被激活TGFβ1增强。结果发现BMP-和TGFβ-之间的相互作用添加剂信号。然而，更多的信号传导途径，如Notch的，Wnt-或EGFR-/ MAPK-和其它信号级联可能参与。因此，需要对BMP /TGFβ-相互作用和串扰其他途径进一步调查。此外，响应中央皮质可以从转基因小鼠中，其可以有助于阐明驱动EMT基本机制中分离出来。总之，这里所描述的EMT模型系统可以是在癌症研究中的干细胞生物学和再生的领域有帮助的，以及。它可以用于药物文库筛选抑制或增强迁移和侵袭的物质。

在胚胎发育的几个阶段，上皮细胞失去强粘附到彼此（ 例如，紧密连接），并获得在这个过程被称为上皮至间质转变（EMT）1迁徙表型。 EMT需要形成额外的细胞类型，如间充质神经嵴细胞，一个人口从神经上皮2偏析。 EMT是不在胚胎阶段只有必要的，但还需要在成年生命的后期阶段保持在成年生物体的生理过程，如伤口愈合3和中枢神经系统（CNS）的再生在脱髓鞘病灶4。 上皮性肿瘤是众所周知的激活EMT作为迁移，侵袭和转移引发步骤，最终导致癌症的发展1,3。 EMT确实集中连接到强大的迁移1,3。 C的细胞的步骤onditioning，发起，接受和维持EMT尚不完全清楚，需要进一步调查。 在此，提出了一种基于神经干细胞的标准化体外 EMT模型系统，以确定生长因子和媒体（无血清和无酶的使用）。这个模型系统是在EMT工作的科学家的相关性。蜗牛，瑞伯和Twist蛋白家族已被证明是既在发育和疾病1为EMT的关键。蜗牛，瑞伯和扭曲的家庭也参与所提出的系统。该系统是基于通常不经历EMT提供一个特别的优点为初始事件的EMT诱导时研究的前脑的特定区域。 该模型系统可能被用于研究在CNS外上皮EMT，由于关键的EMT诱导剂，如蜗牛，瑞伯和扭曲的蛋白质，也都EMT期间在CNS外组织系统中发现的。该型号System使神经干细胞的分离标准化来自发展中国家皮质学习一般干细胞的功能和EMT尤其如此。使用该系统，我们分离的神经干细胞，诱导的EMT和研究FGF2和BMP4的作用下随后的迁移。我们观察到，FGF-和BMP-信令相互作用与TGFβ-信令以促进细胞迁移，从而验证该模型的系统。

<table><tbody><tr><td>BMP4, rhBMP4</td><td>RnD Systems&amp;nbsp;</td><td>314-BP-01M</td><td></td></tr><tr><td>Bovine pancreas insulin</td><td>Sigma&amp;nbsp;</td><td>I1882</td><td></td></tr><tr><td>Boyden chamber, CytoSelect cell invasion assay</td><td>Cell Biolabs</td><td>CBA-110</td><td>24 well plate system</td></tr><tr><td>Cell culture dish with grid</td><td>Ibidi 500 mm dish, 35 mm</td><td>80156</td><td></td></tr><tr><td>CellMask Orange</td><td>Life Technologies</td><td>C10045</td><td>Plasma membrane dye, use at 1:1,000.</td></tr><tr><td>DAPI</td><td>LifeTechnologies</td><td>D1306</td><td>Stock at 5 mg/ml. Use at 1:10,000. Cancerogenic. Appropriate protection (gloves, coat, goggles) required.</td></tr><tr><td>DMEM/F12 1:1 medium bottle</td><td>Gibco Invitrogen</td><td>21331-020</td><td></td></tr><tr><td>FGF2, rhFGF2</td><td>RnD Systems</td><td>233-FB-01M</td><td></td></tr><tr><td>Fibronectine, bovine</td><td>Sigma&amp;nbsp;</td><td>F4759</td><td></td></tr><tr><td>Glutamax supplement&amp;nbsp;</td><td>Gibco Invitrogen&amp;nbsp;</td><td>35050-061</td><td></td></tr><tr><td>Graphics software with pixel measurement feature</td><td>Fiji</td><td>fiji.sc/Fiji</td><td>version 2.0.0-rc-30/1.49s</td></tr><tr><td>HBSS media</td><td>Sigma&amp;nbsp;</td><td>H9394</td><td></td></tr><tr><td>Human apo-Transferrin</td><td>Sigma</td><td>T1147</td><td>Possible lung irritant. Avoid inhalation. Use appropriate protection.</td></tr><tr><td>L-glutamine</td><td>Gibco Invitrogen&amp;nbsp;</td><td>25030-024</td><td></td></tr><tr><td>Nestin, Mouse anti Nestin antibody</td><td>Genetex</td><td>GTX26142</td><td>Use at 1:100, 4% PFA fixation, Triton X100 at 0.1%</td></tr><tr><td>Olig2, Rabbit anti Olig2 antibody</td><td>Provided by Hirohide Takebayash</td><td>Personal stock</td><td>Use at 1:2,000, 4% PFA fixation, Triton X100 at 0.1%</td></tr><tr><td>Penicillin/Streptomycin/Fungizone</td><td>Gibco Invitrogen&amp;nbsp;</td><td>15240-062</td><td></td></tr><tr><td>Podoplanin, Mouse anti Podoplanin antibody</td><td>Acris</td><td>DM3614P</td><td>Use at 1:250, 4% PFA fixation, avoid Triton X100</td></tr><tr><td>Poly-L-ornithine</td><td>Sigma&amp;nbsp;</td><td>P3655</td><td></td></tr><tr><td>Putrescine</td><td>Sigma&amp;nbsp;</td><td>P5780</td><td>Skin and eye irritant. Appropriate protection required.</td></tr><tr><td>Sodium selenite</td><td>Sigma&amp;nbsp;</td><td>S5261</td><td></td></tr><tr><td>Sox10, Rabbit anti Sox10 antibody</td><td>Millipore Chemicon</td><td>AB5774</td><td>Use at 1:200, 4% PFA fixation, Triton X100 at 0.1%</td></tr><tr><td>TGFb1, rhTGFb1</td><td>RnD Systems</td><td>240-B-010</td><td></td></tr><tr><td>Uncoated Petri dishes</td><td>Falcon Corning</td><td>351029</td><td></td></tr></tbody></table>

an enzyme- and serum-free neural stem cell culture model for emt investigation suited for drug discovery

上皮至间质转变（EMT）描述的上皮转分化成间充质的过程。 EMT是也通常发生在胶质母细胞瘤，最常见的恶性脑肿瘤胚胎发育过程中的基本处理。 EMT也已在脑外多种癌包括乳腺癌，肺癌，结肠癌，胃癌观察。 EMT是通过促进迁移，侵袭和转移形成集中挂恶性肿瘤。 EMT诱导的机制尚未完全清楚。在这里，我们描述了皮层神经干细胞（NSCs）和随后的EMT诱导的标准化隔离的体外系统。这个系统提供了以使用单细胞或外植体培养的灵活性。在这个系统中，大鼠或小鼠胚胎前脑的神经干细胞是在限定的培养基中培养，不含血清和酶。的神经干细胞表达Olig2的和SOX10两个转录因子在oligodendroc观察YTE前体细胞（OPCs的）。使用这个系统，FGF-，BMP-和之间的相互作用的TGFβ信号传导涉及ZEB1，ZEB2，观察TWIST2哪里的TGFβ-活化显著增强细胞迁移，这表明有协同BMP-/TGFβ-相互作用。结果指向FGF-，BMP-和TGFβ-信令网络，以参与EMT诱导和维持。这个模型系统相关的体外研究EMT。它是具有成本效益的和显示出高再现性。它也允许对不同的化合物相对于它们的迁移响应（定量距离测量），和化合物抑制或增强EMT（定性测定）的高通量筛选的比较。因此，该模型是非常适合于测试药物库影响EMT物质。

Watch this Scientific Journal Video about An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery at JoVE.com

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

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 ...

an-enzyme-serum-free-neural-stem-cell-culture-model-for-emt

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.9K 次观看.  University of Basel. 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.

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

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES细胞源性神经上皮细胞培养

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

科研

生物学

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

基于发育毒性测定化学品安全筛选和系统生物学数据生成人类多能干细胞

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

发育生物学

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

培养小鼠神经干细胞的前体

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

一个人类神经干细胞系的三维文化，微小RNA的分析和推测的靶基因分化

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

神经科学

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

一个鼠标，两种文化:隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

生物神经毒素在茎的网络文化功能评估细胞衍生的中枢神经系统神经元

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

研究脑癌和神经退行性疾病的人类神经器官

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...