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;

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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?...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BMP4, rhBMP4</td>
			<td>RnD Systems&#160;</td>
			<td>314-BP-01M</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine pancreas insulin</td>
			<td>Sigma&#160;</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 1:1000 .</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>LifeTechnologies</td>
			<td>D1306</td>
			<td>Stock at 5mg/ml. Use at 1:10000. 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&#160;</td>
			<td>F4759</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax supplement&#160;</td>
			<td>Gibco Invitrogen&#160;</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&#160;</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&#160;</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 1:2000, 4% PFA fixation, Triton X100 at 0.1%</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Fungizone</td>
			<td>Gibco Invitrogen&#160;</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&#160;</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma&#160;</td>
			<td>P5780</td>
			<td>Skin and eye irritant. Appropriate protection required.</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma&#160;</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。

此EMT模型系统是基于对NSCs都为单细胞或作为从显影神经管的特定区域的外植体的标准化的隔离，中央皮质（ 图1和2）。对于量化考核，在植500微米格培养皿中（ 图3）的中心接种的权利。从中央皮质植体首先暴露于FGF2两天，随后在生长因子（ 表1）的不同组合的其他两天。我们开始与它比显影神经管的其它区域更大的皮?...

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

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

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 ...

科研

发育生物学

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

预测与基因调控元件的验证激活过程中维甲酸诱导胚胎干细胞分化

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

人诱导多潜能干细胞衍生肝细胞的药物发现方法

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

P2X7 受体实时活细胞流式细胞仪检测成人神经祖细胞中的钙影响、毛孔形成和吞噬作用

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

人类诱导多能干细胞衍生神经细胞治疗移植小鼠脑损伤控制皮质影响模型

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

从多能干细胞中无血清自由生产三维人类肝球

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...