这项工作是有计划资助从威康信托AMA，一个EPSRC助学金，以PB-J和伊拉斯谟，STICHTING博士贡献资助由ERC高级研究员奖AMA（DAT，TB）。亨德里克·穆勒的Vaderlandsch全宗和Fundatie范·德·Vrijvrouwe面包车Renswoude TE的-Gravenhage到SCvdB。我们要感谢J.布里斯科，S.穆尼奥斯 - Descalzo，C施罗特和T·罗德里格斯，讨论和建设性的批评，华日Navascués的安装介质协议，双方AK Hadjantonakis和C Budjan分享他们的实验室的协议有关整个安装胚胎的染色。以前的bioRxiv预印本服务器29上提供的这篇文章的早期版本。

1.培养条件之前，聚合<ol><li>保持在ESLIF介质mESCs（见表具体材料和设备的配制）在加湿培养箱明胶涂层为 25cm 2的组织培养处理的烧瓶在37℃和5％CO 2 21-25共 。 </li><li>生长的细胞至少有两个通道，在这个协议中，使用前解冻后;细胞在较低的通道是一般更成功在整个实验重复产生可再现的特性（即，不超过15个传代培养），不同的ES细胞系之间然而细微变化是可以预期的（见表2所测试的细胞系）。 </li><li>培养细胞40-60％汇合。不要使用过度融合细胞的聚集。 </li></ol> 2.生成集料<ol><li>预温PBS（+ 钙 ，镁+ 2 +），ESLIF，N2B27 26,27 </sup&gt;和胰蛋白酶-EDTA在37℃水浴中。 </li><li>从组织培养瓶中吸出培养基（步骤1.3）。用5毫升的PBS轻轻漂洗烧瓶，两次。吸出PBS并加入预热的胰蛋白酶-EDTA（0.25％）的1〜2毫升以解离细胞。 <ol><li>放置在孵化器的烧瓶&lt;5分钟或直到细胞已经从烧瓶的表面完全脱离。移液器上下用1毫升吸管，以产生单细胞悬浮液计数准确和均匀的聚集体尺寸的形成。 </li></ol></li><li>胰蛋白酶中和5-10毫升ESLIF;洗下生长表面最大化细胞回收并转移到50毫升离心管中。除去1毫升等分试样从离心管中并计数细胞用血球计。 </li><li>确定悬浮液，得到10个细胞/微升所需的体积（整体的96孔板需要5×10 4个细胞在5ml的悬浮液）。细胞计数准确，大频偏从规定的细胞数目离子可以聚集体对刺激的反应产生不利影响。 </li><li>添加5×10 4个细胞到5ml预热的PBS在新鲜的50ml离心管中，并在旋转〜170×g离心5分钟。 </li><li>小心吸PBS的加入5 ml预温PBS轻轻的;不干扰沉淀在试管的底部。离心在〜170×g离心5分钟。 </li><li>小心吸PBS第二次。除去尽可能多PBS作为可能的，而不干扰颗粒为PBS携带可聚合产生不利影响。用P1000吸管，以产生均匀的细胞悬浮液，接着再加入N2B27至所需体积首先重悬沉淀在1ml温暖N2B27（为一个5×10 例如添加 4 ml的4个细胞/ 5ml混悬液）。 </li><li>细胞悬液转移到无菌的贮存器和吸管40微升液滴进入非组织培养处理过的各孔的底部，&#39;U&#39;底96瓦特使用多道移液器ELL板。覆盖96孔板，其相应的盖和确认细胞与一个倒置的台式显微镜（图1B）的存在。 注意:重​​要的是，这些板用于限制细胞粘附的可能性。别涂布96孔板用明胶，纤连蛋白或促进细胞粘着的任何其他涂层的底部。 </li><li>孵育细胞48小时，在湿润的培养箱中在37℃和5％CO 2的聚合。 </li></ol> 3.应用刺激与改变中<ol><li>以下的48小时温育期，观察mESCs的外观的96孔平板的每个孔中，以确认成功地聚集（图1E）。注意:聚集会出现球形性质，在直径约为150-200微米。请参阅故障排除部分，如果出现问题（ 表1）。 </li><li>0;添加150微升新鲜的辅助介质（3μMChi99021（中）在N2B27 19;股票在DMSO准备在10毫米）使用多道移液器每孔。吸移管以足够的力量，以清除可能已经开始粘附于孔的底部的任何聚集体。在37℃和5％的CO 2（图1A），在湿润的培养箱中孵育聚集在次级介质24小时。 注意:使用这种辅助介质中生成可重复的拉长和偏振聚集。其他次级介质组合物也可以根据需要和实施例显示在图3中的实验条件下使用。 </li><li>对于随后的介质的变化，使用以一定的角度（大约30°）保持多道移液器从每个侧面轻轻取出150微升次级介质的井。然后，吸管150微升新鲜N2B27到每口井有足够的力量，以清除可能有任何开始聚集版附着在孔的底部。 </li><li>重复点3 3，每24小时，直到时间过程是完整的（聚合培养实验的典型长度为120小时）。 注意:确保聚集体自由移动以下介质的变化，以确保内，每个96孔板之间的再现性和一致性。平台必须每天改变以下聚合期间。 </li></ol> 4.准备骨料的免疫染色分析激光共聚焦显微镜<ol><li>固定和初级抗体孵育 注意:由AK Hadjantonakis 28所描述的协议已被修改，以适应卓制粒料免疫染色。在我们的研究和它们的稀释液所用的典型的抗体，参见先前发表的作品19,21,24,25,29和表3中。 <ol><li>使用以一定的角度举办了多道移液器（约30℃），轻轻从侧面Ø去除150微升媒体f各自孔的96孔板的。吹打150微升PBS到每口井洗两​​次。留下几个每次洗涤之间的分钟，以使聚集体沉降。 </li><li>设置P1000的吸移管以分配200微升，附上对应的枪头和切断约3毫米，从与一对无菌剪刀的尖端的末端。在96孔板的每个孔中绘制的PBS的一部分的短的方式进入尖端排出它以搅拌聚合。 </li><li>使用相同的尖端井内包括骨料和吸管放入小（直径30毫米）玻璃果蝇夹层以及绘制出整个卷。传送从96孔板，将经历相同的免疫染色体制成相同的玻璃夹层以及所有的聚集体。 注:从不同的实验条件转移聚集的时候，以防止聚集结转使用新大切的枪头。 </li><li>放置包含骨料玻璃井ES到解剖镜下。摇动玻璃以及迫使聚集体的中心，并吸出PBS从井一面用玻璃巴斯德吸管。使用显微镜，以确保聚集体不吸出。留出一个小体积的PBS中的玻璃以及以覆盖聚集体，以防止它们干燥。 </li><li>加入1毫升新鲜制备的甲醛（4％）溶解在PBS中孵育2小时，在4℃在定轨摇床设定为低速。注意:多聚甲醛是已知的过敏，致癌和毒性。穿戴合适的保护，同时处理。 </li><li>下列固定，吸中相同的方式的甲醛溶液如第4 1 4中所述，用1毫升的PBS三次，每次洗涤洗聚集体，用10分钟，在定轨摇床设定为低速。 </li><li>吸出PBS作为第4. 1. 4描述的和用含10％胎牛血清和0.2％三执行另外三个，10分钟洗涤吨X-100（PBSFT）。上执行轨道摇床设定为低速的10分钟洗涤。 注意:在一个明确的洗涤缓冲液使用胎牛血清（FBS）的结果，减少了协议期间如果使用牛奶，否则将失去的聚集体的数目。要注意的是以下固定术，免疫组织化学程序，可以在除下面详述的其他各种方式执行，并且应当确定和由研究者优化是重要的。 </li><li>方框在轨道摇杆设定为低速，在4℃在PBSFT聚集体1小时。该协议可以在此时被暂停，并聚集阻止O / N，并持续搅拌在水平回转摇杆在4℃。 </li><li>吸出PBSFT如先前所述（见节4. 1. 4）和孵化用500μl稀释在PBSFT O / N所需初级抗体的聚集体在4℃下于轨道摇床设定为低速。用石蜡膜覆盖，以防止蒸发。 </li></ol></li><li>二级抗体孵育<ol><li>吸出第一抗体溶液并用1ml PBSFT以下述方式清洗（4℃）:5分钟两次;三次15分钟;和四到七倍1小时。执行在4℃和在定轨摇设置为低速每个洗涤步骤。注:冰寒洗使用中之前。不要在冰上进行清洗，因为这可能会导致聚集到片段。 </li><li>吸出最后一次洗涤介质和孵育在500μlPBSFT O / N所需的二级抗体的聚集体，在4℃在黑暗中在水平回转摇杆。加入核染色，如赫斯特（如果需要）。 </li></ol></li><li>共聚焦显微术安装和成像<ol><li>如第4节1.4描述了4℃PBSFT洗涤聚集体。最后一次洗涤后，冲洗骨料与含有1ml的PBS 0.2％FBS和Triton X-100（PBT）以下列方式:5分钟两次;三次15分钟。执行洗涤在RT在定轨摇避光。 </li><li>吸出介质如先前所述（部分4 1 4）和温育30分钟，在黑暗中以1:1的溶液甘油:PBT（1毫升），接着由第二孵育30分钟以7:3的溶液甘油:PBT（1毫升）中。 注意:在使用旋转器混匀甘油和PBT溶液在4℃下。 </li><li>吸出最后的甘油:PBT解决方案，并添加1 ml封固剂24。该协议可以在此时安装前被暂停（如果暂停，密封该孔用石蜡膜并存储染色孔，在4℃）。 </li><li>吹打它们在17微升液滴的切口P20前端（图2）安装在显微镜载片上的聚集体。折叠双面胶带回到它自身上四次，以产生隔板和附加到幻灯片的各端。放置顶部盖玻片（22毫米×60毫米）在这些隔离物（ 图2）。 注:这是必不可少的切尖在此阶段使用，以便不损坏样品。隔板防止盖玻片压碎骨料。 </li><li>一旦聚集安装，使用图像使用协议共聚焦显微镜的样品前文所述19,21,24,25。一旦放置在显微镜下，离开不受干扰舞台上的滑动几分钟以使聚集到安装平台内沉降。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

在这个手稿中描述的技术有效地和可重复性地产生小鼠胚胎干细胞（mESCs），该显示组织对称破和伸长19的聚集体，结合两种由Marikawa 等人 14和EB形成所述悬滴培养。这些聚集体继续发展的轴向伸长，补充从ES细胞前器官的产生如光学杯11和大脑皮层13现有的方法，和含有细胞与三种胚层，其显示类似于原肠胚形成过程中处理的标识如快速的细胞运动19,21。 有趣的是，推测在对称破事件的性质，因为它发生在没有外部图案形成组织和合子不对称用的杆19。一个基本轴的自发形成可能来自预先存在的heterogeneities在细胞中，其形成的基础不对称的发展的起始培养。事实上，细胞ESLIF条件下培养的种群显示自我更新和分化细胞的混合物，其中的相对比例可从一个集合到另一个会有所不同。此外，在我们的实验室初步研究表明，从细胞的多能全人口聚集显示图案发育迟缓和缺陷，暗示异质性组织模式形成一定的作用（DA特纳A.马丁内斯阿里亚斯，准备中）。这也是值得考虑的一个反应扩散机制可能产生的模式，与抑制剂的扩散远离骨料的表面上。 Warmflash 等 41认为，这种机制可能是负责开发的径向不对称贴壁培养，使得它在三个维度一个可能的候选人图案。 在初始L 48小时的聚集期，细胞达到类似于植入后外胚层，中，他们有能力从辅助介质19,21,24响应信号中的状态。典型地，这里描述的方案为细胞提供次级介质的脉冲，其中包含的因素（如Wnt基因/β连接素激动剂），提供足够的伸长的信号。由兰开斯特等人所述先前培养的方法。（使用人ESC 13）和 Eiraku 等（与mESCs 11）用于聚合的低粘附力的96孔板之前凝集细胞转移到基质胶液滴为导通短时间去文化。虽然聚集和基质胶插入之间的时间是研​​究间不同，在这两种情况下，被用于形成聚集体（约3,000-4,500个细胞）大量的细胞。相反，这里描述的协议19使用少得多的细胞（约400细胞每个聚集体）已被确定为是绝对必要的伸长，对称破和被观察到19的偏振的过程。此外，这些细长结构能够在更短的时间周期中产生，而不在人工基质包埋:由兰开斯特等人比较限定的大脑区域的产生（&gt; 20-30天）从Eiraku 13和光学杯 。 等（〜11天）11与所需的偏振细长结构的产生的5天。 一与然而这里描述的培养方法的局限性在于，它们只能被培养约5-6天柱镀层直到它们的大小使得它们主管在重力作用下沉入到孔的底部，并且甚至在非强制的粘合包被的塑料制品。使用人工基质如前面提到的那些进一步的实验中，可以允许我们增加的周期观察和实验，并工作仍在进行中，以确定限制聚集体以这样的方式的效果。这种技术的另一个限制是，在为了改变介质不去除聚集体，小体积的培养基中必​​须留在井的底部。化学遗传学方法的后果是需要考虑这种稀释，并从此前媒体的任何残余的影响:在3微米志脉冲的一天，2.37微米的解决方案是实际交付，以及随后的媒体变化导致的0.499结转μM（72-96小时）和0.105的时间点之间微米（96-120小时）。目前的工作并没有稀释校正并假定每日介质变化将最大限度地减少残留的影响。 有许多在协议关键步骤，以确保这两个区域内和跨实验的再现性。首先，mESCs的起始文化必须保持良好，并没有结束合作nfluent，和介质应该总是良好的质量即 ，新鲜和充分混合（图5A，B）。差的培养条件的聚集（图5Bi）产生不利影响。细胞的精确数量，以获得一个〜400个细胞/ 40微升细胞悬浮液是必需的，因为较大的聚集体无法自我组织，而且更容易形成各孔（图5Bii）内双聚集而较小的聚集体是不能达到正确的密度在那里他们能够对刺激（图5Biii）作出响应。一键优化步骤是列入第二PBS洗涤，去除污染物血清细胞悬浮液（步骤2.6）的;这提高了聚集次数加速聚集体的发展由约24小时。接着，确保介质改变施加足够的力来移去具有粘附到井的表面上的电势的任何聚集体;如果不这样做地区环境部门一道LTS的集合体"崩溃"（ 图5Biv）。此外，和如前面提到的（及以下），它是必不可少的，以确保聚集体保持在悬浮培养并附着于任何表面被阻止。一旦聚集一直坚持，基因表达和其潜在的延伸的格局被打乱。 由于该技术是相对简单的，并且在具有良好的组织培养技术的个人的职权范围很好，大多数的与聚合有关的问题，可以相对迅速地，如果遵循这些关键步骤解决;的故障排除全表可用（ 表1）。一旦掌握，该技术可以被用来研究轴向展开，对称破和细胞决定事件。更具体地，这些聚集体具有以产生细胞的池迄今一直无法在培养，如脊髓和摩托的电位- [R神经元。 道德注:应注意适当谨慎，这一协议对人类ES或iPS细胞培养的转换可能带来的实验者接近十四天限制对人类胚胎研究，原条即产生的法律依据，详见2008年人类受精与胚胎学法案（英国）42。由于该法案适用于所有活的人类胚胎，无论创作自己的方式，超越了原始的，条纹状阶段人类gastruloids文化将超出许可的研究范围。

研究和细胞命运决定的理解早期哺乳动物的发展可以利用胚胎干细胞培养（胚胎干细胞），从有能力自我更新和分化成生物体的所有类型的细胞囊胚派生无性系种群的IE浏览器。，他们是多能1,2。虽然这些培养物已经并继续对细胞命运决定的分子基础的理解是有用的，它们不能重现一些胚胎原肠胚形成过程中所产生的空间配置与全局行为。在胚胎，原肠胚形成的过程中可以将一个单一的上皮细胞层到三个不同的胚层，并赋予具有明显的前后组织3-5个胚胎。企图概括这些事件体外已经基于对胚胎干细胞的三维聚集体的产生，被称为胚体（EB），并且对它们吨Ø分化条 ​​件6,7。这些聚集体可以被诱导以分化成许多不同的细胞类型，其中一些是要么不能获得或诱导低效率在贴壁培养或不能在所有产生; 例如 ，血液8和原始生殖细胞9。在使用胚的局限性然而，就是他们无法显示的形态发生的行为，胚层的分布和轴向组织，是胚胎发育的主要特征，导致空间的解体6,10。在一个报告，处理的EB与Wnt信号导致基因表达弱极化在一些聚集，但没有明确的形态观察7。在最近的报告中，已经培养了延长的时间段的EB培养前的结构，如视网膜，皮层和内耳感觉细胞，其模拟它们的胚胎的对应而开发没有轴向organizat的上下文离子11-13。 通过报告Marikawa 等 14，同时用鼠标P19胚胎癌（EC）的悬滴法形成细胞的聚集工作，报告中胚层起源让人想起了与exogastrulae在两栖类动物中观察到的伸长的细长结构的出现和海胆胚胎和凯勒外植体15-18。因为这并没有被观察到小鼠胚胎干细胞（mESCs），我们试图再现与使用mESCs 19的聚集体P19胚胎细胞中观察到的行为和我们报告的培养条件，导致它们的对称破和轴向伸长。从与P19细胞工作的一个重要的区别是，这里描述的聚合协议在96孔板中，类似于由Eiraku 等人 20所述进行的，而不是悬滴。这种变化导致了增加的效率在骨料的回收和在这两个方面的区域内和跨实验的重复性。重要的是，保持聚集在单个孔确保聚集体之间的融合（共同汇集从悬滴聚集体时）不会发生。此外，该协议的主要特点是24小时暴露在GSK3抑制剂CHI99021（池），Wnt基因/β-catenin信号通路的有效激活，下面聚集。 这里所描述的方法提供了理解自我组织，轴向组织和胚层规范的在培养的方法中，允许在体内 19,21关于轴向展开进行推断的基础。由于这些原因，该方法具有潜在的，以允许可能难以在胚胎学过程的详细机理分析。此外，一个潜在的应用是在组织和器官中没有的贴壁培养很容易获得的产生是由于缺乏一个结构蜂窝利基如脊髓前体21和运动神经元。三维聚集体培养提供了一个物理结构和信令环境，可能无法实现通过常规方法，导致对胚细胞系的以空间有组织的方式推导的新方法。

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Gibco/Invitrogen</td><td>31350-010</td><td></td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; Cell Culture Flask</td><td>Grenier Bio-one</td><td>690 175</td><td></td></tr><tr><td>Benchtop Centrifuge</td><td>MSE</td><td>MSB020.CX1.5</td><td></td></tr><tr><td>BES Buffered Saline (BBS)</td><td>Sigma-Aldrich</td><td>B2891</td><td>BBS+CaCl&lt;sub&gt;2&lt;/sub&gt; made by disolving 50 mM BES Sodium Salt, 280 mM NaCl, 1.5 mM Na2HPO4 in distilled water with 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt; adjusted to pH 6.96 with 1 M HCl&amp;nbsp;</td></tr><tr><td>Centrifuge tubes (15 or 50 ml)</td><td>Greiner Bio-One&amp;nbsp;</td><td>118271 or 227261</td><td></td></tr><tr><td>CHIR 99021 (Chi)</td><td>CSCR</td><td></td><td>Bought through the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute. Stock concentration: 10 mM in DMSO</td></tr><tr><td>Dissection Well (30 mm Internal Diameter)</td><td>Fisher Scientific</td><td>12678586</td><td></td></tr><tr><td>ESLIF</td><td></td><td></td><td>500 ml GMEM, 5 ml Sodium Pyruvate, 5 ml Non-Essential Amino Acids, 5 ml Glutamax, 1 ml beta-mercaptoethanol, 50 ml FBS and 550 &amp;micro;l LIF (1,000 units).</td></tr><tr><td>Foetal Bovine Serum (FBS)</td><td>Biosera</td><td>FB-1090/500</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G1890-100G</td><td>Dissolved in distilled water to a 1 % (w/v) solution, autoclaved and diluted to 0.1% in PBS (see below) for individual use.</td></tr><tr><td>Glasgow&#039;s Minimal Essential Medium (GMEM)</td><td>Gibco</td><td>11710-035</td><td></td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Improved Neubauer Haemocytometer</td><td>Hawksley</td><td>AS1000</td><td></td></tr><tr><td>Leukaemia Inhibitory Factor (LIF), Recombinant</td><td>CSCR</td><td></td><td>Produced in house by the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute.</td></tr><tr><td>n-Propyl-gallate</td><td>Sigma-Aldrich</td><td>02370</td><td></td></tr><tr><td>N2B27/NDiff 227</td><td>Stemcells, Inc.</td><td>SCS-SF-NB-02</td><td>&lt;a href=&quot;http://www.stemcellsinc.com/_literature_40366/Technical_Specifications_Ndiff&quot;&gt;http://www.stemcellsinc.com/_literature_40366/Technical_Specifications_Ndiff&lt;/a&gt;</td></tr><tr><td>Non-Essential Amino Acids</td><td>Gibco/Invitrogen</td><td>11140-035</td><td></td></tr><tr><td>Paraformaldehyde powder</td><td>Sigma-Aldrich</td><td>P6148</td><td>Dissolved in water to form a 8% Formaldehyde stock solution. Diluted with BBS&lt;sup&gt;+&lt;/sup&gt;CaCl&lt;sub&gt;2&lt;/sub&gt; to give a 4% working solution</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>D8662</td><td>With Calcium and Magnesium</td></tr><tr><td>Sodium Pyruvate</td><td>Invitrogen</td><td>11360-039</td><td></td></tr><tr><td>Sterile Reservoir</td><td>STARLAB</td><td>E2310-1010</td><td></td></tr><tr><td>Sterile, Non-Tissue Culture Treated, U-Bottomed 96-well Plate</td><td>Grenier Bio-one</td><td>650185</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td></td></tr></tbody></table>

generation of aggregates of mouse embryonic stem cells that show symmetry breaking, polarization and emergent collective behaviour in vitro

我们已经开发的协议提高电流胚体（EB）文化，让自我组织的研究中，对称性破缺，眼轴伸长及悬浮培养用小鼠胚胎干细胞（mESCs）的聚集体细胞命运规范。少量mESCs的在非组织培养处理，U型底96孔板，之后他们有能力为实验信号作出反应聚集在培养基48小时。处理后，这些聚集体开始显示偏振光基因表达的标志和逐渐改变来自一个球形团细胞的其形态向细长，良好的组织结构在没有外部不对称线索。这些结构不但能够显示三种胚层的标记物，但积极地显示原肠胚样动作，通过从聚合的，其中关键的是发生在长形结构的一个区域的单个细胞的定向脱出证实。这protoc醇提供用于再现地形成这些聚集体的，其刺激诸如的Wnt /β-Catenin的活化和BMP抑制和由单个时间点或时移荧光显微镜分析其信号的具体方法。此外，我们描述修改当前整体贴装小鼠胚胎染色程序的固定聚集体中的特定标志物免疫细胞化学分析。在形态，基因表达和聚集体长度的变化可以定量测定，提供了有关如何信号可以改变轴向命运信息。据设想，该系统可以既适用于早期发育事件的研究如轴向展开和组织，以及更广泛地，自组织和细胞的决策的过程。它也可能提供一个适当的利基存在于从常规贴壁培养不能取得，胚胎细胞类型的产生，如脊髓和运动神经元。

细胞外观之后立即电镀和聚合后立即镀后，人们可以观察到在96孔板与标准倒组织培养显微镜（图1B）的各孔中的单个细胞的存在。内镀层的8小时，这些细胞将开始下降到井由于底部重力和将已开始聚结成离散簇（图1C）。 24小时后，将聚结的细胞将完成初级聚集，并且将已压缩成定义良好的，但粗糙的聚集体，其中个别的细胞是从彼此（图1D）模糊。由第二天（48小时），聚集体应该看起来"干净"，的端部具有吸收的所有细胞内的孔（图1E）;井的底部可以具有某些细胞已流下从聚合在这个阶段。提供该聚集体已被聚集在N2B27，在这个时间点上，它们应是球形的，大约150-200微米的直径和在井内移动自如（即，未附着到孔的底部）。聚集在其他媒体（即，ESLIF或N2B27与特定因子）具有改变了初始聚合形态和响应相继的刺激的电位（Turner等，准备中）。 代表性的形态变化加入含48至72小时（ 图1A，5A）驰辅助介质的24小时脉冲的产生明确的，显示偏光式的胚层19,21的特异性标志物细长的聚集。紧接蚩脉冲（72小时）后，聚集体将开始脱落细胞，并开始显示其响应于朝此天（图3A）的端这些信号。这些反应通过基因表达的变化和形态，这两者都是依赖于聚集体已收到以下在N2B27（图3B）的初始48小时聚合期间治疗者表现。如果只要求终点分析，在最佳时间图像聚集体是在约96-120小时。在这一点上的聚集体将发展明确形态和基因表达模式（图3A），以及它们的尺寸和质量是仍然足够低，以便从P200的吸管该介质的喷射是足以移除任何可能已附着。未能防止附着聚集到井将聚集体形成（图5Biv）产生不利影响。聚集体的形态和基因表达模式可以根据处理来改变。用于与任一单一治疗或因素的组合持续或脉冲制度代表性的结果示于BMP4，Dorsomorphin H1（乙MP受体抑制剂），ActivinA，SB43（激活素/交点抑制剂）和bFGF或PD03（MEK抑制剂）（图3B）。 聚合成像和定量图像分析聚集体是适合于成像由任一广角镜（主要为时间推移成像19），或固定和免疫染色共焦成像19,21（ 图2）。高于目前的协议和成像说明允许定量信息，从这些总量积累。以下共焦或宽视场图像捕获，长度和沿着所述聚集体的直径或脊柱（球形或拉长分别; 图4A，B）的相应的基因表达可以被测量。这些分析也直接适用于时间推移的图像，其中一个可以得到的不同条件下的聚集体的生长，大小和伸长率的信息（ 图4 </strong&gt;）。 <img alt="图1" src="/files/ftp_upload/53252/53252fig1.jpg" /> 图1.典型的时程和早期形态。（一）典型的时程进行聚合实验。细胞被聚合48小时在N2B27含有小鼠胚胎干细胞的悬浮液（10个细胞/微升）在一个96孔板（P1，P2）。（B）的立即镀（吨=〜5分钟）后，从个人细胞可以是看出，在悬浮液中，并已开始形成团块由8小时（C）（D）的 24小时后的细胞是否已形成在每个孔即大约100微米直径的单集合体。（E）的 48小时后聚合，聚合直径范围为150-200微米。在这点上，聚集介质（N2B27）是除去，被改变在b之前一个次级介质加入或者在实验（p1的部分A）中的其余部分，或24小时ACK给N2B27（P2在A部分）。比例尺为100μm。 <a href="https://www.jove.com/files/ftp_upload/53252/53252fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53252/53252fig2.jpg" /> 图2.安装聚集到显微镜载玻片上。一旦聚集体已被固定，免疫染色，并放置在安装平台，每个聚集体吸移到载玻片作为17微升的液滴（A，B，B&#39;）。足够的空间留给聚集水滴，防止它们之间的合并。间隔件均采用双面胶带，并放置在所述显微镜载片（A，A&#39;，B）的每一个角。正是基于这些，一个玻璃盖玻片放置。 <a href="https://www.jove.com/files/ftp_upload/53252/53252fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3.不同处理对聚集的ES细胞的形态的影响。继N2B27 2天，ES细胞已经形成聚集物。另外的具体因素既可以作为1天脉冲（A）或连续地（B）的可以改变聚集体的表型相对于基因表达，伸长潜力，或它们的整体形状的极性。在该图中的例子是120小时后成像和处理的指示从任文胸形成的聚集体:绿色荧光蛋白30（A）或 SOX1 :: GFP的27（B）的小鼠ES细胞。智:CHIR 99021 月31日; SB43:SB 431542 32; DM:DorsomorphinH1 33,34; PD03:PD0325901 <a href="https://www.jove.com/files/ftp_upload/53252/53252fig3large.jpg" target="_blank">请点击这里查看次的放大版本</a>是的身影。 <img alt="图4" src="/files/ftp_upload/53252/53252fig4.jpg" /> 集料图4.定量分析。从文胸形成的典 ​​型聚集::下面志的24小时脉冲成像在120小时GFP mESCs 25,30。亮场和GFP通道的合并图像被示出具有分段的线标记从点A的聚集体的"脊柱&#39;到B（A）中 ，沿其聚集体的荧光和长度可以测量（B）。的长度（C）和来自同一实验中24聚集体"圆度"（D）中被示出。形状描述符，如圆度（D）的 ，圆形度，周长和面积可以使用从图像分析软件斐济35的图像分析食谱插件来测量。意味着由水平线在每个时间点指示;误差棒表示标准偏差;在（A），比例尺为200微米。由于有报道细胞系之间的细微差别，因此预计蚩的脉冲后，平均最大长度的聚集体的平均和最小圆度会有所不同。不同的细胞系中，我们发现，平均最大长度可以是〜400〜800微米，0.4和0.6之间的平均最低圆度的范围内。 <a href="https://www.jove.com/files/ftp_upload/53252/53252fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53252/53252fig5.jpg" /> 图中的聚集体形成的失败5.实施例。以生成可再现的聚集体（A）中的能力取决于关键因素，例如（Bi）的新鲜和充分混合次级介质，（BII，BIII）的准确性计数初期n细胞和（起搏）确保聚集体棕土不形成粘附的菌落，即 ，"崩溃"进的井的表面。示出在聚集体形成的每个所提到的条件（B）中的误差的典型例子。缩放栏所示;见故障排除表的详细信息（表1）。 <a href="https://www.jove.com/files/ftp_upload/53252/53252fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/53252/53252table1.jpg" /> 表1.指南故障排除。与集合协议相关的典型错误给出建议，以它们的分辨率。 <img alt="表2" src="/files/ftp_upload/53252/53252table2.jpg" /> 表2表的细胞系测试了AGG形成regates。若干19,22,27,30,36-40已经表征为聚集体而形成的，虽然细胞系之间的细微差别，预计，并已观察到的细胞系，上述所有线显示在条款相似动力学伸长率和形态。在基因表达而言，表达模式是特定于基因表达，但每个细胞系中，该表达模式是基本一致的在若干通道。为了保持一致性，我们生成了没有文化超过15代的细胞聚集。 <img alt="表3" src="/files/ftp_upload/53252/53252table3.jpg" /> 表3列出在这些研究中使用的代表的抗体。一个选择的抗体和用于骨料免疫染色稀释因素。

Watch this Scientific Journal Video about Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro at JoVE.com

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

小鼠胚胎干细胞的聚集体的产生，显示对称破缺，极化和突发集体行为<em&gt;体外</em

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

generation-aggregates-mouse-embryonic-stem-cells-that-show-symmetry

Research

JoVE Journal

Developmental Biology

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

17.6K 次观看.  University of Cambridge. We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

视频: Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

在微孔聚集尺寸优化人类多能干细胞的基于悬浮心脏分化

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

科研

发育生物学

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

在体外分化的小鼠胚胎干细胞（MES）使用悬滴法

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

生物学

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

使用微模式和定量成像绘制哺乳动物细胞的新兴空间组织图

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

基于细胞基和细胞外矩阵的Murine睾丸器官生成

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

o 型血管哺乳动物细胞产生均匀集料的轨道晃动培养

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

生物工程

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

小鼠胚胎干细胞向皮质 Interneuron 前体细胞的分化

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

神经科学

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Source: Mao, X., et al. <a href="https://app.jove.com/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent culture.

一种从小鼠胚胎干细胞生成神经祖细胞的技术

1. Mouse embryonic stem cell culture

	Prepare 0.1% gelatin coated cell culture dishes or plates.
	
		Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population

Source: Hubbard, K. et al., <a href="https://app.jove.com/v/52361">Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for embryonic stem cell differentiation into a networked population of neurons with functioning synapses. It outlines the steps involved in embryonic stem cell differentiation into neurons, induction of neuronal network formation, and enrichment of the culture with networked neurons.

将小鼠胚胎干细胞分化为联网神经元群

1. Neuronal Differentiation
NOTE: Conduct steps 1.1 - 1.5 before noon and step 1.7 after noon. An overview of the differentiation procedures is presented ...

小鼠胚胎干细胞的聚集体的产生，显示对称破缺，极化和突发集体行为

摘要

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