Name: functional evaluation of biological neurotoxins in networked cultures of stem cell-derived central nervous system neurons
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这个工作是由过敏的国立卫生与传染病研究所（IAA数量AOD12058-0001-0000）和国防威胁降低局的国家机构 - 联合科技厅，医疗科技事业部（金人数CBM.THRTOX.01.10 .RC.023和CBM.THRTOX.01.RC.014）。这项研究进行，而PB举行的国防威胁降低局，美国国家研究委员会的研究会士奖和KH召开全国研究委员会研究会士奖。我们感谢安吉拉·阿德金斯和空间Kaylie Tuznik（USAMRICD）的技术援助;和Cindy Kronman（USAMRICD）的编辑协助。本文所表达的意见是作者的，并不反映陆军部，国防部，或美国政府的官方政策。

1.胚胎干细胞适应饲养细胞悬浮培养<ol><li>解冻的胚胎干细胞在37℃，直到冰的棉条仍然存在。 </li><li>使用P1000吸管，轻轻转1 - 2.5×10 6分离胚胎干细胞，以含10预热ESC培养基中的非组织培养处理的菜。孵育在湿润的组织培养箱20小时，在37℃和5％的CO 2。 </li><li> 20小时后，通过用10ml的血清吸管悬浮培养轻轻转移到15ml锥形管中洗涤细胞。颗粒胚胎干细胞3分钟，在200×g的。在旋转，背面加入10毫升新鲜ESC中低附着菜。 </li><li>吸上清，注意避免撞出细胞沉淀，并加入1 ml新鲜的ESC培养基细胞沉淀。使用P1000吸管转移​​细胞对细菌培养皿，并返回到培养箱中。 </li><li>每日观察细胞直至聚集体变得可见，以肉眼（通常4 - 8的天数（d）;聚集体将是0.2- 0.5mm）的。如果骨料是看不出来的后4天，重复步骤1.3。一旦聚集是可见的，离解，并在第2节所述维持胚胎干细胞。 </li></ol> 2. ESC传代和维护<ol><li>传送ESC聚集到15ml锥形管中，使用10ml无菌吸管，并允许聚集体沉降到一个紧凑的粒料（通常为3 - 5分钟）。吸出上清液，小心避免破坏细胞沉淀，并用5ml PBS洗涤聚集。虽然重力沉降建议，或者沉淀细胞，在100×g离心2.5分钟，而不是以节省时间。 </li><li>吸PBS，加入500微升胰蛋白酶。孵育聚集在胰蛋白酶3分钟的水浴中在37℃。加入500μlESC中稀释胰蛋白酶，轻轻地磨碎10次与P1000吸管分手聚集。计数使用血球细胞。 </li><li>粒料分离细胞3分钟，在200×g离心和重悬细胞，以1.0的最终浓度×10 7细胞/ ml的ESC培养基中。转移150微升（1.5×10 6个细胞）以10cm的细菌培养皿10ml新鲜ESC培养基并返回到组织培养箱48小时。过量的胚胎干细胞可以分化，冻存于1 - 5×10 6个细胞/ 90％ESC培养基毫升补充有10％的DMSO，或丢弃。 </li><li>通过汇总每48小时。聚集准备通过将清晰可见，直径超过0.5毫米。 注: - 1.4解冻冻存，悬浮适应细胞和文化每步1.2来完成。聚集将准备在传代48 - 72小时。 </li></ol> 3.神经元分化注意:进行步骤3.1 - 3.5前的中午和步骤3.7中午之后。的分化过程的概述示于图1A。 <ol><li>分离胚胎干细胞在步骤2.1 - 2.5。游离转移350微升（3.5×10 6）ð胚胎干细胞含有25毫升分化培养基10cm的低附着菜。放置在轨道摇床设定为30 - 45 rpm的组织培养箱内，在37℃，5％的CO 2。这是分化的第一天， 在体外 （DIV），称为日-8。 注:使用超低附着菜增加了方法的成本，而且产生比细菌培养皿稍大的产量，因为聚集偶尔可以坚持培养皿。如果不同盘尺寸是优选的，培养基体积和细胞数量可以相应地缩放。 </li><li> 48小时后（DIV -6），用25毫升移液管向分化细胞聚集体转移到50ml锥形管中。立即添加新鲜25毫升分化培养基的培养皿中。 </li><li>允许聚集解决超过2 - 5分钟，产生一个可见的颗粒是1 - 2毫米深。忽略单个细胞或小聚集体残留在悬浮液中。小心吸介质和传输CELL沉淀回使用P1000的培养皿。放置在旋转摇床上于组织培养箱。 </li><li>在DIV -4重复步骤3.2。小球会2 - 第4毫米深。更换补充有6微米的全反式视黄酸（RA）的陪替氏培养皿30毫升分化培养基。返回到旋转振荡器中的组织培养箱进行另外48小时。 </li><li>在DIV -2重复步骤3.3。沉淀将4 - 8的毫米深在此点。 </li><li>在DIV -1，准备电镀表面，在第4节。 </li><li>在DIV 0，解冻5毫升预等分并冷冻鼻咽癌胰蛋白酶消化培养基，在37℃下进行5 - 10分钟，发生在组织培养罩。用25毫升移液管，移分化聚集到50ml锥形管中。允许聚集定居，仔细吸媒体。洗涤用10毫升PBS中沉淀两次，使聚集到洗涤之间沉降。 </li><li>第二PBS洗涤后，加入5 ml NPC胰蛋白酶介质的颗粒，376; C为5分钟。轻轻拂去2.5分钟后的管中。 </li><li>加入5毫升0.1％的大豆胰蛋白酶抑制剂（STI）以灭活胰蛋白酶和通过颠倒混合。轻轻磨碎10 - 15倍，用10毫升血清移液管，直到一个相对均匀的细胞悬浮液制备。 </li><li>将细胞悬浮液慢慢转移到40微米或70微米的细胞滤网置于50ml锥形管的顶部。一旦所有的悬浮液被过滤，加入1 ml N2培养基通过过滤器以洗涤剩余的细胞与沉淀分离的细胞悬浮于6分钟，在200×g下。 </li><li>吸出介质，而不会干扰沉淀。用10毫升N2培养基洗涤细胞两次，制粒细胞5分钟，在200×g离心并用P1000洗涤之间捣碎。在此之前的第二洗涤，计数使用血球细胞。 </li><li>重悬的细胞在N2培养基以每毫升和板ESN信息1×10 7细胞以150,000细胞密度- 200000细胞/ cm 2。 </li><li>转让新开发平台编ESN信息向加湿组织培养培养箱中在37℃和5％的CO 2和维持，如第5。 </li></ol> 4.准备培养表面镀神经前体在DIV 0 <ol><li>准备组织培养处理过的菜至少1天前电镀。加入足够的聚乙烯亚胺（PEI;在无菌H 2 O 25微克/毫升）或聚维赖氨酸（PDL; 100微克/毫升无菌H 2 O），以支付组织培养处理的塑料菜和孵化O / N在37℃。 </li><li>电镀上午，两次洗碗用双蒸H 2 O和一次PBS。最后一次洗涤后，加入足够的N2培养基以覆盖培养皿（ 例如，每12孔培养皿的孔的1 ml或每6cm皿4ml）中。 </li><li>准备至少一天前神经元电镀18毫米盖玻片。清洁盖玻片通过等离子体清洁4分钟。 </li><li>紧接在一个底清洗盖玻片转移到乙醇洗涤封口膜大型无菌培养皿，并添加400微升的PEI或PDL的解决方案，准备在步骤4.1。在组织培养孵化器孵化O / N在37℃。 </li><li>在早晨，洗涤盖玻片三次水，并添加5微克/ ml的层粘连蛋白在PBS中1 - 3小时，在37℃。之前解离的神经元，吸层粘连蛋白，并立即盖玻片转移至12孔培养皿含有1ml鼻咽癌介质的，一定要保持经处理的一面朝上的孔中。 </li><li>商店菜肴和盖玻片，在37°C，直到NPC都准备好板。 </li></ol>神经元5.保养<ol><li>在DIV 1，抽吸培养基并用N 2培养基代替。 </li><li>在DIV 2和4所示，抽吸培养基并有B27培养基更换。 </li><li>在DIV 8，吸出培养基并替换为含有B27的培养基有丝分裂抑制剂，以消除污染的非神经元细胞。 </li><li>在DIV 12，更换B27培养基。 </li><li>请不要删除DIV从5％CO 2的12 + ESN信息，直到准备使用。 </li></ol>突触传递（MIST）分析的量化微型兴奋性突触后电流测量6.抑制 注意 :梭菌神经毒素是已知的最毒的物质，估计人类LD 50值低0.1 - 1纳克/千克。事先获得使用这些毒素和使用适当的预防措施，必要的批准。 <ol><li>仔细稀释肉毒毒素/ A到100倍的ESN培养基中所需的最终浓度和温暖至37ºC。加入适当量的毒素，以DIV 21 + ESN文化，摇动培养皿，并返回到孵化器。 </li><li>如果细胞将被分析中毒后超过4小时，加入毒素的菜和涡流而不偏离5％CO 2的去除，如直接在培养箱或在一个恒定的CO 2室中。 </li><li>在适当的时间点，aspiraTE ESN培养基和细胞外记录缓冲（ERB）洗两次。添加4毫升再培训局辅以5.0微米河豚毒素和10μM荷包牡丹，阻断动作电位和拮抗GABA 受体的活性，分别。 </li><li>转让菜电钻机。是必需的MIST测定既不灌注也不温度控制。 </li><li>使用微量拉马产生5-10毫欧的电阻的记录电极和填充细胞内记录的缓冲拉硼硅玻璃。轻轻沾在之前录制的硅化试剂填补记录电极。 </li><li>使用空气填充的注射器，提供正压力作为记录吸管下降到ERB。后轻轻地降落在记录吸管上要被记录的神经元的胞体，除去正压并形成千兆欧密封。降低保持电压至-70毫伏。小心施加负压打入全细胞构型。 </li><li>切换至电流钳来监控并记录静息膜电位。如果不调整液体接界电势，静息电位将介于-67到-82毫伏。 </li><li>执行连续-70 mV的电压钳记录为4 - 5分钟，检测微型兴奋性突触后电流（mEPSCs）。 </li><li>分析使用尖峰检测软件使用以下设置记录的数据为mEPSC检测为4分钟:阈值，5;期间搜索局部最大值，万微秒;基线高峰前的时间，5000微秒;期间搜索的衰减时间，20000微秒;峰分数找一个衰减时间，0.37;期间平均基线，1000微秒;面积阈值，20;点平均峰值，1号;峰方向，为负。 </li><li>收集并保存上检测到事件的信息。由240分在4分钟检测到的事件的数目，以确定mEPSC频率以Hz。 </li><li>收集mEPSC频率为8 - 12续ROLS和8 - 12肉毒毒素处理的样品每次曝光条件。分析对年龄和很多匹配的对照频率。确定抑制％使用单向ANOVA测试和Dunnett的事后检验突触活性的统计学显着性。 </li></ol>

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The U.S. Government has filed two international patent applications that incorporate methods described in this article: PCT/US2014/22042, titled &#8220;Methods of detecting neurotoxin using synaptic activity&#8221;; and PCT/US13/40641, titled &#8220;Toxin detection using stem cell-derived neurons.&#8221;

目前的黄金标准CNT检测，血清型的决心和定量的工作重点。所述MLA询问主机的整个范围:基板所需中毒发生在体内毒素相互作用（ 例如，毒素结合于细胞表面受体，该毒素-受体复合物，LC易位到细胞质的内化，液相色谱-介导的裂解和抑制突触神经传递）18。然而，虽然提供了MLA中毒的生理相关模型，它是资源密集型的，可以通过污染物被混淆，并涉及大量的小鼠的死亡作为终点。可替代地，基于细胞的测定法的BoNT检测和定量被限制在初级神经元培养物，这也需要使用动物，或神经母细胞瘤的细胞系，这不能形成突触和通常表现出敏感性差向神经毒素8。迄今为止，最次中毒测量ESE细胞为基础的平台上都依赖于检测SNAP-25，VAMP1 / 2或突触-1 11的蛋白裂解的。这是有问题的，因为SNARE蛋白切割位已被证明是非线性与突触抑制体内相关，因此可能无法准确地表示中毒或恢复从中毒19,20。 对于CNT检测一个理想的基于细胞的模型系统将（ⅰ）是神经系; （二）被突触加上neurotypic电反应; （三）对于所有CNT血清型或亚型高度敏感;及（iv）提供可扩展性，吞吐量和测定倍相当于或改良过的工作重点，以较低的成本，并且不需要使用动物。为了满足这些要求，我们开发了一种方法，从市售的鼠标ESC线产生大量高浓缩铀，网络谷氨酸的文化和GABA能神经元。然后，我们开发了MIST试验量化突触抑制响应中毒与帐篷，肉毒毒素/ A- / G。而MIST试验可以在任何突触神经元活动的人群（ 如初级神经元或脑切片）进行，目前ESN信息是唯一的体外派生神经元模型的重复性开发新兴的网络行为，因此适合于MIST检测。 生产足够量的定义沿袭CNT研究神经元需要一些创新的ESC文化和分化。首先，通过选择和培养的胚胎干细胞，可以存活悬​​浮适应，我们消除了由饲养细胞，以及需要从胚胎干细胞纯化饲养细胞在分化的起始污染的可能性。虽然底层悬浮适应的分子机制尚不清楚，悬浮适应的胚胎干细胞保持有丝分裂活跃，保留的Oct3 / 4的表达，并继续成为神经。使用这种方法，〜1.5×10 7] E旺通常恢复每个通道。二，生产的NPC通过掺入机械搅拌以防止分化过程中结块，表面上是增加养分无障碍聚集体的内部升高〜300％。在这个过程中，我们发现，用于ESC培养和神经元分化的血清是在维持胚胎干细胞源性的最关键部件。为了达到最佳的成功，我们建议悬浮适应的ES细胞向每批血清和储存血清在-20℃下通过到期日期，以支持ESC培养和神经元分化。 如同大多数突触活性培养物，DIV 14 + ESN信息极易受到pH的突然变化，甚至短暂暴露于大气中的CO 2浓度可导致神经毒性在24小时内。对于需要治疗的文化其次是孵化持久的O / N或更长的时间，神经元必须直接处理的实验我n中的孵化或转移至前实验的恒定的 CO 2室中。 多种技术确认神经和神经元的成熟，包括ICC，转录谱和全细胞膜片钳电。在基因表达和神经元形态学的时间变化是通过神经发生的发育阶段迅速进展一致，并且由格16，ESN信息表现兴奋和抑制微型突触后电流与紧急网络行为16。突触活动的证据表明，ESN信息可能会复制负责肉毒中毒和破伤风的临床表现的病理生理学。这是通过使用MIST证明中毒用的BoNT / A- / G或相比于载体处理的或未经处理的神经元的TeNT受损mEPSC频率由&gt; 95％的确认。 薄雾的BoNT / A的灵敏度的测量所指示的半数抑制浓度（IC 50）为0.013 PM（相当于0.5小鼠致死单位/ ml）和低于0.005 PM一个极限的检测，测定浴后加入20小时。基于这些值，雾大约两倍敏感和2 - 比MLA快4倍和30倍，比免疫系检测裂解SNAP-25的更敏感。 总的来说，这些数据表明，干细胞衍生的神经元网络的人群提供中毒的生理上相关的，基于细胞的模型。在具有改进的方法从悬浮液适于胚胎干细胞衍生的网络的ESN文化组合，利用雾的应减少需要进行动物试验和相关的缺点，费用和MLA的伦理问题，同时提供更迅速，敏感和特异的措施中毒。虽然全细胞膜片钳电是低通量的方法用于鉴定活性的神经毒素的存在下，它提供了一个分辨率和SPEED采用分子生物学方法是无法实现的。这些研究结果还提供了证据，证明活性依赖性方法的应用，以评估突触活动和网络行为可使得快速和特异性检测的神经毒素。这种较高通量的方法将使机理研究，治疗的筛选或诊断测定法的神经调节剂，包括碳纳米管各种各样可行的。

该方法的总的目标是在功能上评估突触活动中暴露于梭菌神经毒素的干细胞衍生的神经元网络培养物。这是一种在体外派生模型，功能复制负责肉毒中毒的临床表现的病理生理学和验证一个ESN作为一个合适的模型检测碳纳米管，治疗的筛选和机理研究的第一个示范。 BoNTs是由梭菌属的成员产生高致命性细菌的神经毒素。包括最近提出的BoNT / H，8抗原性不同的BoNT血清型已经描述（/ A / H）1,2。所有血清型被表达为150 kDa的肽是翻译后缺口，以产生一个100kDa的重链（HC）构成迪辰和一个50kDa的轻链（LC）通过二硫键3相连。慧聪介导结合突触前受体和耳鼻喉科RY毒素进入突触通过内吞作用的神经元。期间内体处理，所述HC经历结构重新组织，以在小泡膜的孔，便于LC的易位进入突触前细胞溶胶。该LC然后专门针对和突触前车厢切割可溶性ñ-ethylmaleimide敏感融合附着蛋白受体（SNARE）蛋白质:SNAP-25（肉毒毒素/ A，/ C，/ E），VAMP2（肉毒毒素/ B，/ D， /楼/ G）或突触（肉毒毒素/ C）1。任何这些SNARE蛋白的裂解防止突触胞吐机制的组件，从而阻断神经递质释放。有效的神经元定位和突触前定位的这种组合使得BoNTs最有效的毒药已知，估计人类致死剂量低至0.1 - 1纳克/千克1。 生化测定法可用于检测的CNT LC的具有高灵敏度的存在或活性。然而，这些方法不能我nterrogate结合，输入毒素的能力，激活和神经元内的功能，并且因此是活性的毒素的存在的间接和可能误导的措施。与此相反，中毒的功能测定，例如在小鼠致死性试验（MLA）综合评价CNT的成功协商摄取和激活的所有阶段的能力，提供了毒素活性的更相关的和具体的评估。而MLA是肉毒毒素检测的黄金标准，它是一种使用死亡作为终点的体内方法，因此具有有限的效用的研究平台。尝试开发适合机理研究和治疗筛查CNT中毒的细胞为基础的车型也遭遇显著的局限性。而初级神经元培养提供生理相关程度高，它们的使用是由许多因素复杂，包括高资源成本，相对低的产率，对多个神经子的存在类型和涉及动物使用广泛的监管和行政监督。作为替代原代神经元，神经细胞系（其可以被诱导以采用由化学刺激神经 ​​元样性质），如神经母细胞瘤和肾上腺嗜铬细胞已被用作体外模型中毒。因为这些细胞继续培养诱导之前，它们是高度可扩展的，因此，非常适合于中等通量的方法。然而，他们的相关性是有问题的，因为诱导的表型通常是异质的，都是不良敏感CNT和不能显示出临界神经行为，包括无法以形成组装成正常突触4前和突触后车厢。在没有生理上的完整突触前车厢，全系列的毒素:神经元相互作用不能被复制和中毒，因此功能性测试是不可能的5。没有吨奇怪的是，尝试进行了成果是在体内和初级神经元的研究6不一致的机理研究和药物筛选使用引起的神经细胞系BoNTs。 已经提出了干细胞衍生的中枢神经系统（CNS）中的神经元可提供下一代基于细胞的平台的BoNT研究相结合的原代神经元的关联性与培养的细胞系7-10的灵活性。具体地，小鼠的干细胞衍生的神经元（ESN信息）已被发现是向生理剂量的BoNT / A- / G高度敏感和复制许多体内反应中毒，包括差动不放过，活性增强的中毒，和血清型特异性效力5,8,11。这些行为表明，肉毒毒素的吸收，加工，贩运和活动的体内机制是保守的ESN信息。然而，抑制突触activit的y是肉毒中毒的署名表现，并得出结论认为ESN信息是一个合适的体外衍生的细胞系模型的CNT研究之前因此由碳纳米管表明突触活动的下列中毒损失是必不可少的。 为了测量中毒与突触活动碳纳米管的影响，全细胞膜片钳电生理学来定量在媒介物处理的CNT或处理DIV 21 + ESN信息突触电流。我们发现一个ESN与肉毒毒素/ A- / G或帐篷是中毒引起的范围内20小时在所有情况下的突触活动&gt; 95％的损失。进一步表征进行中毒后20小时用的BoNT / A表明对突触活性的剂量依赖性作用，具有检测低于0.005 PM期限及0.013 PM的IC 50值。这些结果表明，电生理检测中毒的灵敏度和时间帧都大大超过可比免疫系ANA改进的SNAP-25裂解和体内小鼠致死性测定裂解，证明中毒的突触活性的神经元培养物的功能测量可以便于更灵敏，特异和快速的方法来对的BoNT的细胞应答表征。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Table 1. ESC and ESN&lt;/strong&gt;</td></tr><tr><td>Knockout DMEM</td><td>Life Technologies</td><td>10829-018</td><td>Media: ESC culture medium&lt;br/&gt; Formulation and notes: 500 mL</td></tr><tr><td>100x MEM NEAA</td><td>Life Technologies</td><td>11140-050</td><td>Media: store at 4 &amp;deg;C for up to 1 month&lt;br/&gt; Formulation and notes: 6 mL</td></tr><tr><td>200 mM L-Alanyl-L-Glutamine</td><td>ATCC</td><td>30-2115</td><td>6 mL</td></tr><tr><td>ES qualified FBS</td><td>Applied Stem Cell</td><td>ASM-5007</td><td>90 mL</td></tr><tr><td>100x antibiotics</td><td>Sigma-Aldrich</td><td>A5955</td><td>3 mL</td></tr><tr><td>55 mM 2-mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td>1.1 mL</td></tr><tr><td>10&lt;sup&gt;7&lt;/sup&gt; Units/mL LIF</td><td>Millipore</td><td>ESG1107</td><td>60 &amp;mu;L</td></tr><tr><td>Knockout DMEM</td><td>Life Technologies</td><td>10829-018</td><td>Media: ESC differentiation medium&lt;br/&gt; Formulation and notes: 436.6 mL</td></tr><tr><td>100x MEM NEAA</td><td>Life Technologies</td><td>11140-050</td><td>Media: store at 4 &amp;deg;C for up to 1 month&lt;br/&gt; Formulation and notes: 5 mL</td></tr><tr><td>200 mM L-Alanyl-L-Glutamine</td><td>ATCC</td><td>30-2115</td><td>5 mL</td></tr><tr><td>ESC-qualified serum</td><td>Applied Stem Cell</td><td>ASM-5007</td><td>50 mL</td></tr><tr><td>100x antibiotics</td><td>Sigma-Aldrich</td><td>A5955</td><td>2.5 mL</td></tr><tr><td>55 mM 2-mercaptoethanol</td><td>Life Technologies</td><td>21985-023</td><td>0.9 mL</td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td>Media: NPC trypsinization medium&lt;br/&gt; Formulation and notes: 100 mL</td></tr><tr><td>0.5 M EDTA (18.3%)</td><td>Sigma-Aldrich</td><td>3690</td><td>Media: freeze in 5 mL aliquote&lt;br/&gt; Formulation and notes: 0.266 mL</td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T8802</td><td>50 mg</td></tr><tr><td>Polyethyleneimine (PEI)</td><td>Sigma-Aldrich</td><td>P3143</td><td>Media: Surface coating solutions&lt;br/&gt; Formulation and notes: 2.5 &amp;micro;g/mL in H&lt;sub&gt;2&lt;/sub&gt;0</td></tr><tr><td>Poly-D-lysine (PDL)</td><td>Sigma-Aldrich</td><td>P7280</td><td>Media: use within 1 wk&lt;br/&gt; Formulation and notes: 100 &amp;micro;g/mL in H&lt;sub&gt;2&lt;/sub&gt;0</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td>5 &amp;micro;g/mL in H&lt;sub&gt;2&lt;/sub&gt;0</td></tr><tr><td>DMEM/F12 + GlutaMAX</td><td>Life Technologies</td><td>10565-018</td><td>Media: NPC culture medium&lt;br/&gt; Formulation and notes: 492.5 mL</td></tr><tr><td>100x N2 vitamins</td><td>Life Technologies</td><td>17502-048</td><td>Media: store at 4 &amp;deg;C for up to 1 month&lt;br/&gt; Formulation and notes: 5 mL</td></tr><tr><td>100x antibiotics</td><td>Sigma-Aldrich</td><td>A5955</td><td>2.5 mL</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Neurobasal A</td><td>Life Technologies</td><td>10888-022</td><td>Media: ESN culture medium&lt;br/&gt; Formulation and notes: 482.5 mL</td></tr><tr><td>50x B27 vitamins</td><td>Life Technologies</td><td>17504-044</td><td>Media: store at 4 &amp;deg;C for up 1 month&lt;br/&gt; Formulation and notes: 10 mL</td></tr><tr><td>200 mM L-Alanyl-L-Glutamine</td><td>ATCC</td><td>30-2115</td><td>5 mL</td></tr><tr><td>100x antibiotics</td><td>Sigma-Aldrich</td><td>A5955</td><td>2.5 mL</td></tr><tr><td>5-fluoro-2&#039;-deoxyuridine (5FDU)</td><td>Sigma-Aldrich</td><td>F0503</td><td>Media: 2000x Mitotic inhibitors aliquot and store at -20 &amp;deg;C&lt;br/&gt; Formulation and notes: dd 150 mg 5FDU and 350 mg uridine to 10 mL Neurobasal A. Sterile filter, aliquot and freeze. Use 2000x in ESN culture medium.</td></tr><tr><td>Uridine</td><td>Sigma-Aldrich</td><td>U3003</td><td></td></tr><tr><td>TrypLE Express Trypsin</td><td>Life Technologies</td><td>12605-010</td><td>Media: Miscellaneous&lt;br/&gt; Formulation and notes: store at RT</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td>store at RT</td></tr><tr><td>soybean trypsin inhibitor (STI)</td><td>Sigma-Aldrich</td><td>T6414</td><td>store at -20 &amp;deg;C</td></tr><tr><td>ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td>store at RT</td></tr><tr><td>ascorbic acid</td><td>Sigma-Aldrich</td><td>A4403</td><td>Prepare 100 mM stock by dissolving 100 mg ascorbic acid in 5.7 mL of 50:50 DMSO/ethanol mix.</td></tr><tr><td>retinoic acid (RA)</td><td>Sigma-Aldrich</td><td>R2625</td><td>Resuspend 15 mg RA in 9 mL 50:50 DMSO/ethanol. Supplement with 1 mL of 100 mM ascorbic acid stock. Aliquot and stored at -80 &amp;deg;C. Stable for 6 mos.</td></tr><tr><td colspan="4">&lt;strong&gt;Table 2. MIST&lt;/strong&gt;</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>71386</td><td>Media: Extracellular Recording Buffer&lt;br/&gt; Final Concentration (mM): 58.44&lt;br/&gt; MW: 140</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>60135</td><td>Media: (ERB; pH = 7.3, 315 mO)&lt;br/&gt; Final Concentration (mM): 74.56&lt;br/&gt; MW: 3.5</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S3139</td><td>Media: Stable at 4 &amp;deg;C for at least 6 mo.&lt;br/&gt; Final Concentration (mM): 119.98&lt;br/&gt; MW: 1.25</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>21115</td><td>Final Concentration (mM): 110.98&lt;br/&gt; MW: 2</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>63069</td><td>Final Concentration (mM): 95.21&lt;br/&gt; MW: 1</td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G8644</td><td>Final Concentration (mM): 180.16&lt;br/&gt; MW: 10</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H0887</td><td>Final Concentration (mM): 238.3&lt;br/&gt; MW: 10</td></tr><tr><td>K-gluconate</td><td>Sigma-Aldrich</td><td>P1847</td><td>Media: Intracellular Recording Buffer&lt;br/&gt; Final Concentration (mM): 234.5&lt;br/&gt; MW: 140</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>71386</td><td>Media: (IRB; pH = 7.3, 320 mO)&lt;br/&gt; Final Concentration (mM): 58.44&lt;br/&gt; MW: 5</td></tr><tr><td>Mg-ATP</td><td>Sigma-Aldrich</td><td>A9187</td><td>Media: Stable at 4 &amp;deg;C for at least 6 mo.&lt;br/&gt; Final Concentration (mM): 507.18&lt;br/&gt; MW: 2</td></tr><tr><td>Li-GTP</td><td>Sigma-Aldrich</td><td>G5884</td><td>Final Concentration (mM): 523.18&lt;br/&gt; MW: 0.5</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>21115</td><td>Media: Stable at 4 &amp;deg;C for at least 6 mo.&lt;br/&gt; Final Concentration (mM): 110.98&lt;br/&gt; MW: 0.1</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>63069</td><td>Media: Stable at 4 &amp;deg;C for at least 6 mo.&lt;br/&gt; Final Concentration (mM): 95.21&lt;br/&gt; MW: 1</td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td>380.35</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H0887</td><td>Media: Stable at 4 &amp;deg;C for at least 6 mo.&lt;br/&gt; Final Concentration (mM): 238.3&lt;br/&gt; MW: 10</td></tr><tr><td>bicuculline</td><td>Tocris</td><td>131</td><td>Media: Miscellaneous&lt;br/&gt; Final Concentration (mM): 0.01&lt;br/&gt; MW: 417.85</td></tr><tr><td>CNQX</td><td>Sigma-Aldrich</td><td>C239</td><td>Final Concentration (mM): 0.01&lt;br/&gt; MW: 276.12</td></tr><tr><td>Tetrodotoxin</td><td>Sigma-Aldrich</td><td>A8001</td><td>Final Concentration (mM): 0.005&lt;br/&gt; MW: 645.74</td></tr><tr><td>BoNT/A-/G</td><td>Metabiologics</td><td>N/A</td><td>Media:&lt;br/&gt; Final Concentration (mM): TBD&lt;br/&gt; MW: 150,000</td></tr><tr><td>Tetanus toxin</td><td>Sigma-Aldrich</td><td>T3194</td><td>Final Concentration (mM): TBD&lt;br/&gt; MW: 150,000</td></tr><tr><td>Sigmacote</td><td>Sigma-Aldrich</td><td>SL-2</td><td>Final Concentration (mM): N/A&lt;br/&gt; MW: N/A</td></tr><tr><td colspan="4">&lt;strong&gt;Table 3. Equipment and Software&lt;/strong&gt;</td></tr><tr><td>MiniAnalysis (version 6.0.7)</td><td>Synaptosoft, Inc.</td><td>N/A</td><td>Event detection software</td></tr><tr><td>Igor Pro (version 6.22A)</td><td>WaveMetrics</td><td>N/A</td><td>Software for visualization of recordings</td></tr><tr><td>Patchmaster</td><td>Heka</td><td>N/A</td><td>Data acquisition software</td></tr><tr><td>Olympus IX51 inverted microscope</td><td>Olympus</td><td>N/A</td><td></td></tr><tr><td>micromanipulator</td><td>Sutter Instrument</td><td>MPC-365</td><td></td></tr><tr><td>patch-clamp amplifier</td><td>Heka</td><td>ECB10USB</td><td></td></tr><tr><td>micropipette puller</td><td>Sutter Instrument</td><td>P-1000</td><td></td></tr><tr><td>borosilicate capillary tubes</td><td>Sutter Instrument</td><td>B150-86-10</td><td>Corning 7740</td></tr><tr><td>18 mm glass coverslips</td><td>Fisher</td><td>12-545-84</td><td></td></tr><tr><td>plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G</td><td></td></tr><tr><td>cell strainer (40 &amp;micro;m)</td><td>Fisher</td><td>08-771-1</td><td></td></tr><tr><td>Stovall Belly Dancer Shaker</td><td>Fisher</td><td>15-453-211</td><td></td></tr><tr><td>low adhesion dishes</td><td>Fisher</td><td>05-539-101</td><td>Corning 3262</td></tr><tr><td>bacterial dishes</td><td>VWR</td><td>25384-302</td><td>100 x 15 mm</td></tr></tbody></table>

functional evaluation of biological neurotoxins in networked cultures of stem cell-derived central nervous system neurons

在突触前靶向梭菌神经毒素（碳纳米管）的治疗和机理研究已经由需要产生正常突触和经历生理反应中毒可扩展的，基于细胞的模型的限制。在这里，我们描述一个简单的和可靠的方法，以有效地分化的鼠胚胎干细胞（ESCs）到突触活性，网络的神经元定义谱系。以下内容的8天分化方案，小鼠胚胎干细胞衍生的神经元（ESN信息）快速表达和划分neurotypic蛋白质，形成神经元的形态和发展的内在电响应。由分化后的18天（DIV 18），ESN信息显示出活性的谷氨酸和γ氨基丁酸（GABA）GABA能突触和特征是兴奋性紧急网络行为:抑制平衡。为了确定中毒碳纳米管是否功能拮抗突触神经传递，从而复制次E 在体内的病理生理，它负责肉毒中毒或破伤风的临床表现，全细胞膜片钳电被用来量化自发微小兴奋性突触后电流（mEPSCs）在暴露于破伤风神经毒素（帐幕）或肉毒毒素ESN信息（的BoNT）血清型/ A- / G。在所有的情况下，表现出ESN信息内20小时突触活动的近乎完全丧失。陶醉的神经元仍然可行，具体表现为静止不变的膜电位和内在的电反应。这种方法的灵敏度进一步表征，中毒突触活性的剂量依赖性影响，测定另外的BoNT / A的后20小时。中毒与0.005时肉毒毒素/ A导致了mEPSCs一个显著递减，与0.013时的中位数抑制浓度（IC 50）。平均剂量的比较表明，抑制突触功能的测量速度更快，更具体，比SNARE更敏感裂解测定法或鼠标致死测定。这些数据验证了使用突触耦合，干细胞衍生的神经元为CNT的高特异性和灵敏的检测。

我们已经开发出一种分化方案，使经济生产大量高纯度的干细胞衍生的神经元的小鼠胚胎干细胞（ 图1A中详细说明）8,11。这种方法已被用于在一段年可再现区分私人生成和市售ESC行到限定谱系12-14的神经元。该协议的关键要素包括:（1）适应胚胎干细胞馈线无细胞悬浮培养; （2）适当的保养悬浮培养的;及（3）分化旋转的条件下。过渡到悬浮培养显着减少的时间和ESC维护成本，并省却了在微分之前除去饲养细胞。以下悬浮适应，的Oct3 / 4的表达是通过至少30代一致，表明悬浮适应不改变的多潜能标志物的表达（ 图1B-D- </stroNG&gt;）。胚胎干细胞是适合的神经元分化一旦细胞产量持续超过每通道1×10 7个细胞。这通常发生在暂停调整后，10通道或以前悬挂适应胚胎干细胞解冻后，5代。微分集料的机械转动也被发现是增加的神经元的产量的关键。增加了一个旋转振荡器中消除高聚集的形成，增加总存活率和产生在DIV 0（ 图1E）神经祖细胞（NPC的）的产量增加了〜300％。 一个典型的分化开始于3.5×10 6的胚胎干细胞在DIV -8，生产115×10 6的NPC在DIV 0，其中约60％将生存下来并成为神经元。剩余的40％包含非神经元细胞，并大大消除由血清剥夺DIV 0之间 - 1.少数持续胶质细胞可通过加入有丝分裂INH的去除从DIV ibitors 2 - 4或DIV 8 - 12镀层密度在DIV 0的关键;元太镀人口稀少将无法生存超过2周。内镀层的天，分化的神经元表现出神经元的形态和划分neurotypic蛋白质，如somatodendritic标记MAP2和轴突标记头（ 图2A）。通过DIV 14突触-1 +泪点可确定在axodendritic接口，暗示突触的形成。这是与现有突触标记蛋白的表达，以DIV 7 8,11一致。神经元形态继续成熟至DIV 21，在该时间培养物表现出精细的axodendritic乔木和大型突触泪点（ 图2A）。如果保持适当，ESN信息电镀后保持存活和活性为至少4周。 利用RNA测序纵向表达分析证实了神经规范中的形态和蛋白组学的证据ð催熟11,15。发育进展的代表性标志物表现出阶段特异性表达谱，其中的Oct3 / 4，巢蛋白，DCX，的NeuN和KCC2（ 图2B）。通过DIV 0，ESN信息表达神经元特异性结构蛋白，包括MAP2和Tau丰富的副本。与以前的研究结果，观察指标只有两个神经元的亚型:vGluT2表达中脑/后脑谷氨酸能神经元和GABA能中间8。相应地，谷氨酸和GABA能标记一系列广泛展出急剧增加的表达DIV 0和DIV 7之间，包括所需的神经递质释放突触前SNARE蛋白;需要突触后的反应神经递质受体;和所需的脚手架蛋白质系绳这些受体的突触后膜。神经元表现出成熟的内在的电气特性由DIV 14和自发微型兴奋性突触后电流通过DIV16 16。 的突触传递（雾）测定中测得的抑制来评价中毒对突触活动的影响。由酣和载体处理的DIV之间比较mEPSC频率24+ ESN信息，薄雾提供中毒基于突触活性（ 图3A）的功能性抑制进行定量和特定的测量。 MIST被用于测量的BoNT / A- / G或帐篷在ESN信息突触活动的影响，在浴加入每种毒素后20小时。毒素进行的浓度相当于加入10倍的EC 50值，如先前通过的SNARE蛋白切割位11免疫印迹分析来确定。所有的毒素减少mEPSC频率媒介物处理的对照的5％以内。突触率降低并不归属于细胞死亡或改变固有的反应，因为陶醉ESN信息是能够被修补，解雇反复动作电位响应于电流注入和表现出在静息膜电位（ 图3B，C）的无显著改变。 到雾的灵敏度比较，以现有的方法来检测碳纳米管，检测和半数抑制浓度的极限（IC 50）测定加入的BoNT / A的到ESN信息后的20小时。中毒由少至0.005 PM的BoNT / A产生了统计学上显著降低mEPSC频率，具有0.013 PM的IC 50值，并在0.5以上时（ 图4A）的突触活性的完全沉默。该IC 50值对应于约0.5小鼠致死单位/ ml，表明雾两倍敏感并在检测的BoNT / A的存在2至4倍，比所述MLA更快。的SNAP-25裂解免疫测量产生下午12时38分和下午12时05分的最低检出量的EC 50值，表明MIST大约30倍更敏感比免疫系检测裂解SNARE蛋白（ 图4B）的。 <img alt="图1" src="/files/ftp_upload/52361/52361fig1.jpg" /> 图1.暂停适应一个ESN保持稳定的有丝分裂，并表示多能性的标志。对ESC维修和分化（A）示意图。视黄酸（RA）或白血病抑制因子（LIF）的存在或不存在的标志是+或 - 。 在体外 （DIV）和经典的发育阶段（DS）初级神经元培养天之间的比较提供17。 （B）的增殖率R1，D3和C57BL / 6J ES细胞系转变为悬浮培养稳定后，由五个段落。 （C）流式细胞仪检测数据显示测量超过25代悬浮培养的Oct3 / 4的表达没有实质性的变化，在R1，D3和C57BL / 6J ES细胞系（N = 6个）。 （D）的常规传代对于通路5和30（黑线）之间测得的悬浮液，适于R 1 ESC线期间的实际细胞产量。如果没有细胞传代期间被丢弃的理论累积产量也列（红线）。 （E）的DIV 0聚集在静（左）或旋转条件（右）所产生明视场图像。旋转条件下生产的球形聚集体，而不附聚和增大鼻咽癌产生3倍（P &lt;0.001，采用学生t检验确定）11。 *表示P &lt;0.05。这个数字已经被修改哈伯德等 11 <a href="https://www.jove.com/files/ftp_upload/52361/52361fig1large.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/52361/52361fig2.jpg" /> 图2.形态，公关神经元规范和成熟oteomic和转录证据（A）中从DIV 7免疫细胞化学- DIV 49表明神经元树枝状和突触泪点中的axodendritic界面的外观。轴突标记头（绿），树突标以MAP2（红色），突触前室标以突触（白色），并且细胞核用DAPI染色（蓝色）。 （B）代表的基因证实发育阶段特异性表达和指定的神经元亚型的纵向表达谱。所有的基因转录表达为每单元成绩单大致数量。 <a href="https://www.jove.com/files/ftp_upload/52361/52361fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52361/52361fig3.jpg" /> 图3.下一个ESN。去抑制突触活动的时候接触到肉毒毒素/ AG与帐篷（A）从一个ESN代表收集的痕迹除了洗澡后20小时〜10×EC的肉毒毒素/ A 50值- / G帐篷或汽车。每个神经毒素超过95％相比，降低控制突触活动。 （B）中的神经元酣仍能够触发重复的AP响应于去极化电流注入（这表现为的BoNT / A，但在所有的酣培养观察）和（C）显示出在负静息膜电位（℃无变化; N&gt; 18每处理）。 <a href="https://www.jove.com/files/ftp_upload/52361/52361fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52361/52361fig4.jpg" /> 图4.确定MIST的肉毒毒素/灵敏度的处理ESN信息。（A）的浴室除了肉毒毒素/ A后突触活动20小时MIST测量。代表性的电压钳痕迹段展出mEPS​​C频率以下的BoNT / A曝光（左侧）的降低。 mEPSC频率的定量（条形图，右侧中，n = 20对于对照; N = 11 - 22为每个剂量）证实了剂量依赖性降低mEPSC频率。半数抑制浓度是用最小二乘拟合使用四参数可变斜率（R 2&gt; 0.91）的非线性回归来确定。由雾ESN信息注检出限至少为0.005时。 （B）中的BoNT / ESN信息的结果中的SNAP-25的裂解甲中毒作为可视化通过凝胶迁移率改变（在左侧的代表性免疫印迹用SNAP-25裂解在右侧的条形图显示量化; N = 4） 。使用SNARE蛋白裂解（SNAP-25）作为一个端点注意降低的敏感性（EC 50下午12点36分） 主场迎战 </eM&gt; MIST（0.013 PM IC 50）。 *表示P &lt;0.05; ***表示P &lt;0.001。 <a href="https://www.jove.com/files/ftp_upload/52361/52361fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons at JoVE.com

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

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, ...

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Nanyang Technological University

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Neuroscience

9.3K 次观看.  United States Army Medical Research Institute of Chemical Defense. 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.

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

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 ...

科研

发育生物学

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

作为一种工具来描绘分子途径参与神经毒性和神经保护神经元细胞在线实时阻抗为基础的细胞分析仪

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

神经科学

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

神经石生长分析与神经毒性评估与人类神经原生细胞衍生神经元

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

闭环神经机器人实验来检验神经网络的计算性能

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

<meta charset="utf8"></head><body>用于研究秀丽隐杆线虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

用于研究 秀丽隐杆线 虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

A Scalable Method to Study Neuronal Survival in Primary Neuronal Culture with Single-cell and Real-Time Resolution

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to study the process of neuronal survival upon cytotoxic stress. Most methods are based on biochemical approaches that do not allow single-cell resolution or involve complex and costly methodologies. Presented here is a versatile, inexpensive, and effective experimental paradigm to study neuronal survival. This method takes advantage of sparse fluorescent labeling of the neurons followed by live imaging and automated quantification. To this aim, the neurons are electroporated to express fluorescent markers and co-cultured with non-electroporated neurons to easily regulate cell density and increase survival. 
Sparse labeling by electroporation allows a simple and robust automated quantification. In addition, fluorescent labeling can be combined with the co-expression of a gene of interest to study specific molecular pathways. Here, we present a model of stroke as a neurotoxic model, namely, the oxygen-glucose deprivation (OGD) assay, which was performed in an affordable and robust homemade hypoxic chamber. Finally, two different workflows are described using IN Cell Analyzer 2200 or the open-source ImageJ for image analysis for semi-automatic data processing. This workflow can be easily adapted to different experimental models of toxicity and scaled up for high-throughput screening. In conclusion, the described protocol provides an approachable, affordable, and effective in vitro model of neurotoxicity, which can be suitable for testing the roles of specific genes and pathways in live imaging and for high-throughput drug screening.

<meta charset="utf8"></head><body>一种以单细胞和实时分辨率研究原代神经元培养中神经元存活的可扩展方法

Here, we present a simple, potent, and versatile methodology to investigate neuronal survival upon cytotoxic stress in primary cortical neurons with cellular resolution in real time or in fixed material.

一种以单细胞和实时分辨率研究原代神经元培养中神经元存活的可扩展方法

Neuronal loss is at the core of many neuropathologies, including stroke, Alzheimer's disease, and Parkinson's disease. Different methods were developed to ...

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

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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

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

作为一种工具来描绘分子途径参与神经毒性和神经保护神经元细胞在线实时阻抗为基础的细胞分析仪

神经石生长分析与神经毒性评估与人类神经原生细胞衍生神经元

闭环神经机器人实验来检验神经网络的计算性能

用于研究秀丽隐杆线虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

用于研究秀丽隐杆线虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

一种以单细胞和实时分辨率研究原代神经元培养中神经元存活的可扩展方法

一种以单细胞和实时分辨率研究原代神经元培养中神经元存活的可扩展方法

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

摘要

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此视频中的章节

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

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

作为一种工具来描绘分子途径参与神经毒性和神经保护神经元细胞在线实时阻抗为基础的细胞分析仪

神经石生长分析与神经毒性评估与人类神经原生细胞衍生神经元

闭环神经机器人实验来检验神经网络的计算性能

用于研究 秀丽隐杆线 虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

用于研究 秀丽隐杆线 虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

一种以单细胞和实时分辨率研究原代神经元培养中神经元存活的可扩展方法

一种以单细胞和实时分辨率研究原代神经元培养中神经元存活的可扩展方法

用于研究秀丽隐杆线虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法

用于研究秀丽隐杆线虫兴奋性中毒性坏死模型中神经退行性的活体动物成像和细胞分选方法