我们感谢K.戴瑟罗特和S.济的的ChR2和Synapsin1-tdTomato慢病毒结构分别C.柴上的iPSC代共享专业知识和协议，WY丰隆技术支持，吴作栋实验室分享试剂和专长的成员。雅培营养与卓越学术中心（ACE）从葛兰素史克公司（GSK）研究奖，以ELG根据其竞争研究计划（NRF 2008 NRF-CRP 002-082），以ELG这项工作是支持的，由国家研究基金会和新加坡GJA，并通过世界级协会（WCI）计划韩国国家研究基金会（NRF）受教育，科学和韩国的技术（MEST）部资助（NRF授权号:2009-003 WCI），以GJA

注:所有实验执行以下批准的机构动物护理和使用委员会在新加坡保健协议。 1.早期收获产后老鼠大脑<ol><li>解剖前，准备海马组织消化解决方案:每1ml 1X厄尔的平衡盐溶液（EBSS）（+ 1％HEPES）与脱氧核糖核酸酶I（DNA酶I）（250单位/毫升）和分散酶II（1 U / 10微升木瓜蛋白酶毫升）。使大约2.5毫升组织消化溶液和使用0.20微米的过滤器单元进行过滤。保持溶液温暖，在37℃，直到使用。 </li><li>通过把它们放在冰上5分钟麻醉产后第5天（P5）小鼠的幼崽。通过脚趾或尾巴捏冰和测试去除疼痛反射。喷雾用70％乙醇的幼崽。斩首幼仔和放置头在培养皿在冰上。 </li><li>做一个切口在沿中线皮肤，从头骨到鼻子的基础上，有一对小scissoRS或精细解剖钳。剥开皮肤，并通过头骨类似的切口。使头骨两侧各两个额外的水平削减一喙（位于前囟门）和一个尾（在拉姆达）。 </li><li>剥离打开颅骨沿切开线，以暴露底层的大脑。用一对镊子除去头骨覆嗅球，以及它们之间的任何中线骨。为此，同时保持头稳定有一对镊子的在非解剖手。 </li><li>缓解脑的腹侧部分和颅底的尾端之间的刮刀的扁平部分。保持平行锅铲大脑在颅骨的基础上，将其向头骨的喙端切断大脑和颅底之间的粘连。 <ol><li>舀脑用刮刀并将其放置在含有冰 - 冷的1×EBSS（+ 1％HEPES）的陪替氏培养皿中。继续组织淹没在任何时候。 </li></ol></li><li>对于所有显微解剖的步骤，在解剖显微镜下工作，并使用一对细解剖镊在每只手，一个用于切割组织，和其他用于保持组织稳定。 </li><li>做一个切只是后路大脑皮层把它从后脑分开。沿中线切开到大脑皮层分离成两个半球。从大脑半球去除脑膜。 </li><li>将一个半球内侧和插入钳入海马下面侧脑室。切去脑。 <ol><li>使切口在海马的上，下两端，靠近齿状/嗅皮层交界以隔离从皮质海马。收集含冰冷的1X EBSS（+ 1％HEPES）菜的海马。 </li></ol></li></ol> 2.海马组织的解离<ol><li>海马转移到含预热海马组织消化解决方案，一盘菜。切末吨他海马组织以尽可能细，并培育30分钟，在37℃。 </li><li>轻轻离解组织成单个细胞通过机械吸液和转移的细胞入15ml离心管中。 </li><li>离心，在200×g离心5分钟，并除去上清液。 </li><li>在20毫升重悬细胞沉淀0.5X Hank氏含有蔗糖（0.9 M）和离心机平衡盐溶液（HBSS）中，在200×g离心10分钟。 </li><li>在2ml鼻咽癌维持培养基（ 表1）的去除上清，重悬细胞沉淀，放在在1X EBSS溶液和离心机加入10ml 4％的牛血清白蛋白（BSA）的顶部，在200×g离心5分钟。 </li><li>除去上清液，加入小鼠NPC维持培养基（ 表1）的细胞沉淀。轻轻吸取上下5-10次，以确保细胞沉淀被分解成单个细胞。 </li><li>传输介质含有细胞进入预涂覆基质胶板，并添加表皮生长因子（EGF）和fibroblaST生长因子2（FGF2）（均20毫微克/毫升）的肝素（5毫克/毫升）。孵育板在37℃和5％的CO 2。 </li></ol>贴壁小鼠海马的NPC 3.维护<ol><li>准备至少一个小时之前，传代小鼠海马的NPC涂层板/瓶。添加足够的基质胶以覆盖各板或烧瓶中的表面面积，并在室温下孵育1小时。 </li><li>从90％汇合小鼠NPC培养物除去介质和用1×磷酸盐缓冲盐水（PBS）中洗一次，然后吸PBS中断。 </li><li>添加足够的细胞剥离溶液以覆盖所有孔/烧瓶中并孵育5分钟，在37℃。 </li><li>确保所有的细胞都被倒置亮视野显微镜下观察放大10倍分离。如果仍有附着的细胞，孵育培养容器用于进一步1-2分钟，并再次检查细胞。加的Dulbecco氏改良Eagle培养基/火腿的F-12营养混合物的量的两倍（DMEM / F-12）作为细胞分离溶液至孔/瓶一旦所有细胞脱离。 </li><li>转移的DMEM / F-12含有细胞入15ml离心管中并离心，在200×g离心5分钟。 </li><li>在5毫升的鼠标NPC维护中（ 表1）去除上清，重悬细胞沉淀。轻轻移液器上下打散细胞沉淀成单个细胞。 </li><li>板块第三细胞总数为补充EGF和FGF2新文化井/瓶加满油足够的培养基。孵育培养容器，在37℃和5％的CO 2。补充培养基每隔一天和分裂细胞1:3，每3至4天。避免鼠标NPC文化的过度生长，防止聚集和脱离。 </li></ol> 4.分化鼠标的NPC为星形胶质细胞<ol><li>制备聚-L-赖氨酸（PLL）/层粘连蛋白包被12毫米盖玻片至少提前一天开始的小鼠的NPC分化为星形胶质细胞前。添加COVerslips成24孔板并覆盖孔有足够的PLL（1毫克/毫升的硼酸盐缓冲液中）的整个表面区域。 <ol><li>过夜孵育板在4℃下。次日，除去PLL和用1×PBS洗涤一次。 </li></ol></li><li>加层粘连蛋白（1毫克/毫升在冰冷的DMEM / F-12），再覆盖每个的整个表面面积以及，然后在37℃下孵育至少4小时。 </li><li>从小鼠NPC文化取出介质，并与1X PBS洗一次，然后吸PBS关闭。 </li><li>添加足够的细胞剥离溶液以覆盖所有孔/烧瓶中并孵育5分钟，在37℃。 </li><li>确保所有细胞脱离再加入DMEM / F-12作为细胞分离溶液至孔/烧瓶的量的两倍。 </li><li>转移的DMEM / F-12含有细胞入15ml离心管中并离心，在200×g离心5分钟。 </li><li>除去上清，重悬细胞在5ml星形胶质细胞分化培养基（ 表1）。轻轻吸细胞悬索桥Ñ​​上下打散细胞沉淀成单个细胞。计数单​​细胞用血细胞计数器的数量。 </li><li>板的细胞以5×10 4个细胞/ cm 2在500μl培养基中于24孔板的每个孔中。孵育板在37℃和5％的CO 2。更换一半的培养基用新鲜培养基每隔一天。小鼠的NPC迅速分化为星形胶质细胞在2天内并在开始进一步的实验前分化为一个星期。 </li></ol> 5.收获胚胎鼠脑和皮质组织解离<ol><li>从星形细胞培养星形胶质细胞除去分化培养基（ 表1），并替换为初级神经元培养基（ 表1）之前开始收获胚胎大鼠的大脑中的至少4小时。 </li><li>解剖前，准备皮层组织消解液:10微升每1ml 1X EBSS（+ 1％HEPES）与12.1毫克/毫升的L-半胱氨酸木瓜蛋白酶。使约20.5毫升组织消化溶液和使用0.20微米的过滤器单元进行过滤。保持溶液温暖，在37℃，直到使用。 </li><li>安乐死的怀孕水坝用压缩气体二氧化碳窒息。测试通过脚趾或尾巴捏疼痛反射。 </li><li>打好动物腹侧上的吸收垫，并浸泡其用70％乙醇的腹部区域。捏用一对镊子的皮肤，使用一对手术剪的横向切口，足够宽以露出腹部。抓住用钳子腹膜和剖开腹腔。 </li><li>抬起角子宫与镊子和削减它免费沿子宫系膜。将解剖子宫在培养皿中在冰上。通过切割它们之间的子宫组织分隔每个胚胎。 <ol><li>使胚胎囊切开。轻轻壳胎儿出通过开口。斩首胎儿和放置头在培养皿中在冰上。 </li></ol></li><li>收获胎IC大鼠脑，遵循步骤1.3至1.5的描述。 <ol><li>解剖皮质组织，放置一个脑半球的横向朝上。从中间切开半球两个横向。丢弃一半更靠近大脑底部。修剪切除的组织去除脑。 </li></ol></li><li>传送皮质组织到包含预热皮质组织消化溶液一道菜。剁碎皮质组织以尽可能细，并培育30分钟，在37℃。 </li><li>传输来自组织的消化溶液皮质组织至含有10ml预热的初级神经元培养基（ 表1）在50ml的离心管中。通过反复吹打他们上下分离皮质组织中一个血清移液管，直到获得没有组织碎片均匀的溶液。 </li><li>通过一个70微米的细胞过滤通过所述组织液过滤出剩余的组织团块。分装组织溶液在15ml离心管，加入约3毫升每管。 </li><li>在1×PBS中添加800微升7.5％的BSA的每个管的底部，形成了两层，在底部的顶部和BSA的组织方案。 </li><li>离心，在200×g离心5分钟。除去上清液，从所述两个层的界面吸。避免扰乱细胞沉淀在试管的底部。 </li><li>重悬的每个细胞沉淀在1ml初级神经元培养基（ 表1）和将它们组合在一15ml离心管中。确定使用血球细胞密度。 </li></ol> 6.电穿孔和主电镀的皮层神经元注:基因转移可以使用几种方法，包括电穿孔，磷酸钙转染和病毒转导来实现。在这个协议中，我们将描述电穿孔传递的ChR2构造成原代皮层神经元。 <ol><li>离心机6×10 6大鼠皮层神经元吨200×g离心5分钟，去除上清。至少6×10 6个皮层神经元所需的电穿孔，以确保由存活的神经元神经元网络的形成。 </li><li>加100ul电的解决方案，以细胞沉淀和悬浮平缓。结合100微升细胞悬浮液与6微克的Plenti-突触-hChR2的（H134R）-EYFP-WPRE 4质粒并转移到认证的反应杯中。避免在传输过程中产生气泡。 </li><li>选择并根据制造商的说明电应用适当的程序。 </li><li>电穿孔后，加入500μlMEM的细胞/ DNA悬液的试管中。将样品转移到11.5毫升初级神经元中（ 表1），轻轻重悬。为整个24孔板的介质的总量是足够的。等份加入500μl培养基的24孔板的每个孔。孵育板在37℃和5％的CO 2。 </li></OL&gt; 7.生成诱导多能干细胞的注:此方法已被改编自冲田等[10] <ol><li>在DMEM培养人成纤维细胞在6孔板中补充有10％胎牛血清（FBS）。 </li><li>之前开始的成纤维细胞，与基质胶涂层6阱的重新编程孵育至少一个小时，在室温下进行。在80％汇合，由成纤维细胞培养中删除媒体和1X PBS洗一次。 </li><li>添加足够的0.25％胰蛋白酶-EDTA覆盖整个板孵育在37℃下进行10分钟。 </li><li>一旦细胞已经从井脱落，添加的DMEM的量的两倍有10％FBS作为胰蛋白酶以猝灭胰蛋白酶消化。轻轻移液管细胞悬液上下分手细胞。计数单​​细胞用血细胞计数器的数量。 </li><li>转印7×10 5个细胞入15ml离心管中，离心所述细胞悬浮液在200×g离心5分钟。 </li><li>除去上清，悬浮细胞沉淀在电的解决方案。根据制造商的说明电穿孔的成纤维细胞。重悬的细胞在100μl的电穿孔缓冲液，并添加1微克每附加型载体的构建。电穿孔细胞与1650 V设置，10毫秒和3次脉冲。 </li><li>转移细胞悬液到DMEM补充有10％FBS和板每一个6孔板的井5×10 4个细胞。孵育板在37℃和5％的CO 2。 </li><li> 48小时后，从转染的成纤维细胞培养中删除媒体和更换mTeSR中等。每天更换培养基直到诱导多能干细胞（IPSC）菌落准备隔离。的iPSC集落可以后28-35天观察到。 </li><li>当准备隔离，机械切割的的iPSC集落，并用10μMRho相关的蛋白激酶（ROCK）抑制剂（Y-27632）补种在mTeSR培养基中到Matrigel包被6孔板12。每天更换培养基。 </li></ol> 8.神经诱导和人类的NPC保养注意:此方法已描述由Li 等人 11 <ol><li>当IPSC的文化是在20％左右汇合，删除mTeSR中，替换神经诱导培养基（ 表1）。中补充每隔一天。 </li><li> 7天后，取出神经诱导培养基并替换为人类鼻咽癌维持培养基（ 表1）。传代培养之前补充媒体每隔一天最多7天。 </li><li>之前传代培养物，外套板/瓶与基质胶在室温下至少一个小时。 </li><li>从融合的人类NPC文化取出介质，并与1X PBS洗一次，然后吸PBS关闭。 </li><li>添加足够的细胞剥离溶液以覆盖所有孔中，然后在37℃下孵育5分钟。 </li><li>确保所有的细胞已经脱落。加入DMEM量的两倍/与F-12作为细胞分离溶液至孔/瓶。 </li><li>转移的DMEM / F-12含有细胞入15ml离心管中并离心，在200×g离心5分钟。 </li><li>在5毫升人类NPC维护中（ 表1）取出上清和悬浮细胞。轻轻移液管细胞悬液上下分手细胞。 </li><li>板块第三细胞总数为基质胶包被的培养井/瓶加满油辅以10μMY-27632中型充足。孵育培养容器，在37℃和5％的CO 2。 <ol><li>分裂培养1:3每隔3至4天，补充培养基每隔一天。维护人类文化的NPC高密度，防止分化。 </li></ol></li></ol> 9.分化人类的NPC成神经元共培养<ol><li>开始分化为神经元才加的慢病毒载体Synapsin1-tdTomato人类NPC文化。制备星形细胞/皮层神经元共培养物提前一天区分人类的NPC面前。 </li><li>用1×PBS洗涤一次人鼻咽癌培养，并添加足够的细胞剥离溶液以覆盖所有孔/烧瓶然后孵育5分钟，在37℃。 </li><li>确保所有的细胞已经脱落。添加的DMEM / F-12的量的两倍的该细胞剥离溶液至孔/瓶。转移到一15ml离心管中。离心，在200×g离心5分钟。 </li><li>在5毫升的神经元分化培养基（ 表1）去除上清和悬浮细胞。轻轻移液管细胞悬液上下打散细胞沉淀成单个细胞。计数单​​细胞用血细胞计数器的数量。 </li><li>小心吸从星形胶质细胞/皮层神经元的文化媒介。板的人的NPC在5×10 3细胞/ cm 2在500μl神经分化培养基（ 表1）补充有10μM的Y-27632对于每个24孔。轻轻地加入含有人类的NPC中到每口井为了避免干扰星形胶质细胞和皮层神经元。 </li><li>孵育板在37℃和5％的CO 2。更换一半的培养基用新鲜培养基每两至三天，至少28天。 </li></ol>的源自iPSC的神经元10电录音<ol><li>确认人类iPS细胞是否表现得像成熟神经元。 <ol><li>发现细胞iPS细胞人用tdTomato的可视化区分使用一个堂堂正正的共聚焦显微镜配备了60X（0.9 NA）的水浸泡镜头。 </li><li>拉记录吸管4-6分枝灌注细胞在室温下的电阻在外部溶液（ 表1）不断通入95％O 2/5％的CO 2。填有内部溶液（ 表1）的记录微量。 </li><li>补丁tdTomato +细胞中全细胞模式和记录动作电位触发响应于电流注入（1秒持续时间，10 pA的增量）在CUR租夹。 </li></ol></li><li>确认是否表达的ChR2皮层细胞的动作电位是可靠的光刺激诱发。 <ol><li>查找共聚焦显微镜下表达的ChR2通过GFP可视化皮层细胞。通过下面的步骤来10.1.2 10.1.3执行上皮层细胞全细胞膜片钳记录。 </li><li>检查动作电位是通过光照射与汞弧光灯（100瓦）可靠地诱发。激发滤光器的波长是四十分之四百八十〇纳米。 </li></ol></li><li>执行光遗传学刺激。 <ol><li>补丁tdTomato +细胞在全细胞模式，并设置电压钳位在-70mV。滤除电流信号以2千赫和数字化在10千赫。监测串联电阻范围从10到录制过程中20男的一致性。 </li><li>由100瓦汞灯四十分之四百八nm激发滤光片刺激整个字段，持续30秒。 </li><li>记录突触后电流引起的光刺激修补细胞（物业服务公司）的ChR2表达突触前心病蒂卡尔神经元。检测和测量产品分成合同。设定的阈值振幅和电流范围为5个PA和20件，分别。手动检查这些事件放弃任何非PSC痕迹。 </li></ol></li></ol>

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No conflicts of interest declared.

光遗传学提供了激活神经元13的特定人群的时间和空间的精度。在我们的实验中，显微镜物镜的整个领域被照射30秒，以光刺激仅皮层神经元表达的ChR2。这使我们能够确定是否突触连接进行的联合培养的神经元内不同群体之间形成。我们的研究结果表明，光刺激增加的PSC频率在源自iPSC的神经元，这表明这些神经元接收从突触前皮质神经元表达的ChR2突触输入。这，反过来，确定，源自iPSC的神经元成功掺入与皮质神经元的电路。 有此共培养体系的产生几个关键步骤。确保了小鼠NPC来源星形细胞培养是健康的，具有最小的细胞死亡，在进行电镀之前是很重要其它类型的细胞。皮层神经元和人类的NPC以及附着到健康的星形胶质细胞和电生理记录在很大程度上取决于文化的条件。在这个协议中的其他关键步骤是电和皮层神经元电镀到星形胶质细胞的文化。作为大量细胞的电穿孔后死亡，​​它确保至少6000000皮层神经元是电穿孔在24孔板上镀星形细胞培养是很重要的。我们已观察到，当少于600万细胞电穿孔，即生存的神经元的数量并没有形成致密的神经网络，其中受影响的电生理记录。电穿孔的皮层神经元的数目也应该是一致的每个共培养实验，以允许不同研究之间的比较。对于涉及酶促消化的所有步骤，必须不离开细胞在消化溶液太久，因为它可能导致细胞死亡。 此方法可用于筛选从患者特异性的成纤维细胞衍生的，以形成与其他神经元突触联系的神经元的能力。来自于成纤维细胞，从患者的神经发育紊乱雷特综合症（RTT）患有神经元表现出突触传递的缺陷:RTT神经元表现出，大概是由于以下突触连接14一显著降低的PSC的频率和幅度相对于健康的神经元。我们的共同培养​​系统允许检查对神经元显示发育缺陷的药物的效果。这可以通过加入感兴趣化合物的患病的人的NPC播种期间以及连续暴露于化合物在整个神经元分化的时间期间进行。 虽然这种共培养系统也可以被用来作为一种模式用于药物测试，这种方法的限制是，调查每一个候选化合物的效果的方法可以是长期的，乏味的它不是一个高通量筛选方法。以下用候选化合物处理，源自iPSC的神经元必须单独修补以确定的PSCs各化合物的作用。此外，目前有从患者没有许多可用的成纤维细胞和iPS细胞。因此，这将是在不久的将来的兴趣有更多的患者的细胞，也以适应该协议用于高通量筛选。例如，通过标记源自iPSC的神经元的活动的指标，如离子或膜电位的基因编码的传感器，将有可能通过侧步进耗时电过程大幅提高吞吐率。作为产生该共培养体系，从重新编程的成纤维细胞，以进行电生理记录的整个过程中，需要90天以上，则建议无论是人类iPSC或NPC的扩大，重新编程和神经诱导步骤后分别并冷冻保存，以减少所需要的未来实验的时间。 在我们的实验中，我们在下面的synapsin1子皮层神经元表达的ChR2。因为这是促进泛神经元，我们大概是photostimulating既抑制和兴奋皮层神经元。如果未来的实验的目的是为了具体询问或者抑制或兴奋性的电路，更具体的启动子也可以使用。可替代地，皮质神经元可以从转基因（或病毒注射的小鼠），其中的ChR2表达被特异性地靶向神经元的基因定义的亚群来获得。例如，来自小鼠具有Cre重组收获皮质组织表达在含小清蛋白-的interneurons（PV的interneurons），其越过一个鼠标两侧装接loxP的ChR2将产生一种情况，唯一的皮层神经元表达的ChR2将光伏的interneurons 15。使用这样的ChR2表达在我们的interneurons共立方米的lture系统将使修补源自iPSC的神经元是否能够将与PV的interneurons电路的决心。 根据先前的研究9，皮层神经元的成熟与后体外 14天重叠树突。因此，如果光刺激不引起从修补源自iPSC的神经元的响应，它应表明，神经元不具有以与皮质神经元突触连接的能力。一个源自iPSC的神经元能够接收或者从其他源自iPSC的神经元或皮质神经元突触输入。如果采用传统的膜片钳记录，它不可能分辨其中所述突触输入是来自。我们的系统的优点是，从皮质突触前输入突触后的反应，可以选择性地诱发和鉴定。这里列出的程序提供了一个简单的方法来评估整合源自iPSC的神经元的能力成神经细胞定义网络。

近年来，我们的神经元和神经元电路功能的理解已经彻底改变了由该控制神经元兴奋性的几个光遗传学工具。一种这样的工具是光激活的阳离子通道，channelrhodopsin-2（的ChR2）:神经元表达的ChR2火动作电位响应于光刺激1-3。由于神经元通常不怕光，这种非凡的能力，开辟了新的途径，神经元4的基因特定人群的活动的确切时间和空间的控制，并大大加快脑功能的研究。的ChR2已经应用到干细胞研究用于不同的目的，从监测神经元发育5至调节神经网络6的活性。 在这里，我们使用的ChR2增加神经元共培养系统的效用。共培养系统使不同细胞类型之间的相互作用的评价。共培养的神经元和神经胶质的，神经系统的主要细胞类型，通常被用来研究这些不同细胞类型之间的信令，以及评估该信令对生理反应的意义，损害的繁殖和检查这潜在地参与了这一信令7的分子机制。先前的研究显示，人类iPSC非常迅速分化成神经元的大鼠皮质初级含有培养的神经元，星形胶质细胞，少突胶质细胞，小胶质细胞和8的存在。 在这个协议中，我们证明了利用的ChR2在共培养体系审问从人类诱导多能干细胞（iPS细胞）衍生的大鼠皮层神经元和神经元之间的突触连接的方法。我们描述的步骤来剖析和收获脑组织9以及维持和分化小鼠神经祖细胞（NPC的）为星形胶质细胞和人类的NPC诠释Ò神经元来创建共培养体系。建立这一共培养体系中，我们使用由Okita 等人描述的一体化的无重编程方法。10，以产生人类iPSC。这些iPS细胞然后有效地分化成的NPC在化学成分确定的条件下，使用小分子抑制剂如由Li 等人 11 在该共培养，神经元的不同种群可以通过在皮层神经元的检测的ChR2表达的源自iPSC的神经元和标记通过荧光蛋白，串联二聚体番茄（tdTomato）识别。组合电生理记录从源自iPSC的神经元与皮质神经元的光遗传学光刺激允许这两种类型的神经元，以形成彼此电路的能力的评估。具体来说，的ChR2表达皮层神经元光刺激唤起已经形成突触电路源自iPSC的神经元突触反应与光激励皮层神经元网络。我们表明，增加的突触后电流在源自iPSC的神经元确实检测到这样的条件下进行。该协议允许突触电路的评估在各种实验条件，包括不同的神经系统疾病的模型通过使用来自患者的成纤维细胞衍生的iPSC概括。

an optogenetic approach for assessing formation of neuronal connections in a co-culture system

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

这里，描述参与生成一个共培养的星形胶质细胞组成的多个步骤的协议，皮层神经元和神经元的人示出。人类iPSC通过使用游离型的成纤维细胞重新编程得到向量10和指定到神经谱系用的小分子抑制剂鸡尾酒，使得其维持在高密度（ 图1A）的NPC 11。需要几个步骤生成共培养:分化鼠标的NPC为星形胶质细胞，大鼠皮层神经元表达的ChR2电镀，播种人类的NPC标记tdTomato（ 图1B）。在分化的第2天，胶质纤维酸性蛋白（GFAP）+星形细胞可以容易地在我们的培养物（ 图1C）的观察。小鼠的NPC被允许电镀皮层神经元表达的ChR2到星形胶质细胞单层（ 图1D）前区分至少一个星期。经过3到4周人鼻咽癌的分化，tdTomato +神经元培养物（ 图1E）进行检测。 此共培养体系允许响应于光刺激（ 图2A）相一致的检测，从个体的源自iPSC的神经元突触后电流。当通过光激发，在皮质神经元表达的ChR2（ 图2B）中产生动作电位。源自iPSC的神经元也可激发（ 图2C），显示出增加的动作电位发放作为去极化电流脉冲的幅度增加（ 图2D）。因此，这些细胞表现出成熟的神经元的特性。在没有光线，源自iPSC的神经元接收到的突触输入（ 图2E）。这些外来突触后电流是主要由AMPA受体介导的，因为通过GABA受体的电流会向外NMDA受体不进行电流吨-70 mV的控股潜力。至少一些这些输入分别从突触前皮质神经元表达的ChR2，因为光刺激大大增加突触后电流（ 图2E）的频率。这种增加的突触输入的时间过程示于图2F:皮质神经元的光刺激物持续整个30秒长光闪烁。同时，物业服务公司的频率时的ChR2表达皮层神经元光激励（ 图2G）升高，其幅度并没有受到影响（ 图2H）。总之，这些结果表明，源自iPSC的神经元表现出成熟的神经元特性和可形成具有突触前皮质神经元突触连接。 <img alt="图1" src="/files/ftp_upload/52408/52408fig1highres.jpg" /> 图1:示意图对共培养的产生系统（A）的时间轴重新编程人成纤维细胞进入的iPSC和神经诱导到的NPC。（B）的时间轴为人类的NPC成上皮层神经元和星形细胞培养成熟的神经元的终末分化。（C）的小鼠的NPC迅速分化成GFAP +胶质细胞2后1周天体外 。（D）中后，皮质神经元表达的ChR2人类的NPC的分化后接种到GFAP +胶质细胞。（E）的在4〜5周，电生理记录了上tdTomato +神经元进行。刻度条表示100μm左右。 <img alt="图2" src="/files/ftp_upload/52408/52408fig2highres.jpg" /> 图2:功能性突触连接光遗传学分析。 （A）的源自iPSC的细胞中具有标记tdTomato（红色）共培养与皮质神经元表达的光活化通道的ChR2（绿色）。从源自iPSC的神经元的录音显示突触后电流（PSC）的反应，这可能来自任一皮层神经元或其它源自iPSC的神经元。（B）的照明（蓝色条）诱发的一系列的皮层神经元表达的ChR2动作电位。（ C）的源自iPSC的细胞是通过电流注入诱发。（D）烧成VS源自iPSC的细胞中的电流的曲线。所述的ChR2表达皮层神经元（E）的光刺激（蓝色条）增加的PSCs的源自iPSC的神经元的频率（F）的时间的增加，由光刺激所产生的PSC频率的过程。蓝条表示光刺激的时间。 （G，H）当PSC频率增加了光刺激（G）中 ，PSC振幅不受影响（H）。 *表示P &lt;0.05相比，控制用学生氏t检验。

Watch this Scientific Journal Video about An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System at JoVE.com

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

13.4K 次观看.  Duke-NUS Graduate Medical School. A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2. 

视频: An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

在小鸡视网膜有效的基因转移的原代细胞培养研究:<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化和共同培养小鼠的卵巢和卵巢卵泡

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

平行板灌注系统的体外模型研究了细菌对移植组织的粘附性

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

一种优化的O9-1/水凝胶系统，用于研究神经嵴细胞中的机械信号

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

使用光遗传TAEL/C120系统在斑马鱼胚胎中的光诱导GFP表达

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...