Kristen Brennand is a New York Stem Cell Foundation - Robertson Investigator. The Brennand Laboratory is supported by a Brain and Behavior Young Investigator Grant, National Institute of Health (NIH) grant R01 MH101454 and the New York Stem Cell Foundation.

1. hiPSC分化为神经前体细胞<ol><li>发展和扩大在人胚胎干细胞人iPS细胞（HES）培养基（ 表1）共培养上的小鼠胚胎成纤维细胞（MEF）饲养层，直到大（但汇合）菌落准备神经分化经由胚状体（EB）的中间（ 图2）。常规hiPSC培养条件是公别处16,17描述简单地说，生长在HES媒体人iPS细胞在MEF饲养层，直到融合，然后通过酶胶原酶（1mg / ml的DMEM中），并扩大约为1:3，每7天。 </li><li>在无菌水中塑料表面制备聚-L-鸟氨酸/层粘连蛋白包被的板，涂层板用10μg/ ml的聚-L-鸟氨酸（50μg/ ml的玻璃表面）并在室温下孵育3-24小时。用无菌水，然后涂上5克/ ml的层粘连蛋白洗至少一次在无菌PBS在RT 3-24小时。 注意:如果包裹在塑料，板材可贮存长达六个月-20 C。 </li><li>在第1天，酶解除从板一样大菌落中的DMEM / F12利用1mg / ml的胶原酶IV汇合人iPS细胞（通常生长在MEF中，或人iPS细胞在限定培养基如TESR 18上生长的基质胶）。 注:继37ºC1-2小时的潜伏期，殖民地将会漂浮在菜。 </li><li>轻轻洗hiPSC菌落（通过沉降，离心分离不）1-2x用DMEM / F12，重悬在2毫升/孔的N 2 / B27培养基（ 表1），并转移到非粘附6孔培养皿（结合3-井成1- -嗯）19。 注:一夜之间，菌落形成浮动球团称为"胚体"（EBS）。预计大量的细胞死亡。 </li><li>第2天，倾斜板，并允许胚解决。小心地取出媒体和DMEM / F12洗一次，清除杂物。饲料用N2 / B27媒体辅以双SMAD抑制剂（0.1M LDN193189和10M SB431542）12 </SUP&gt;。 </li><li>关于天3-7，neuralization将发生在双SMAD抑制的上下文中检查胚是圆的，但不是囊。订阅的EB每隔一天用补充有0.1M LDN193189和10M SB431542 N 2 / B27培养基。 </li><li>在7-14天，下列的EB电镀聚L-鸟氨酸/层粘连蛋白涂层板，检查使用明显微镜神经花环开始在几天之内出现。（定性为圆簇神经上皮细胞apico - 基底极性的; 图1）。继续喂粘附的EB每隔一天用N 2 / B27培养基补充有0.1M LDN193189，10M SB431542和1μg/ ml的层粘连蛋白。 </li></ol>神经玫瑰花奖2丰收注:我们建议的神经花环使用神经玫瑰花选择试剂20或类似的选择试剂酶收获。虽然神经花环可以手动挑入6孔聚-L-鸟氨酸/层粘连蛋白包被的平板，THI方法论需要大量的训练来掌握，而且，依赖于用户的技术，可能需要第二轮采摘20天，以进一步丰富了筹备和消耗非神经细胞类型。 <ol><li>对于在第14天的酶的选择神经花环，从附着的EB抽吸媒体和加入1 ml，每孔神经花环选择试剂的一个6孔板。在37ºC1小时。 </li><li>随着P1000的Pipetman，轻轻地从每孔除去酶。加入1毫升每孔的DMEM / F12的洗。 </li><li>与P1000，收集1毫升DMEM / F12，并迅速排出DMEM / F12回入井，从而分离从板块的花环。收集到玫瑰花猎鹰管。 </li><li>吸取的另一个1毫升DMEM / F12，并迅速排出到同一以及分离剩余花环。 （不要磨碎，尽量不要分手花环聚集）。收集神经花环成相同的猎鹰管。 </li><li>重复步骤需要2.4。如果花环不容易分离，添加钼重新酶和重复上述过程（步骤2.1-2.4） </li><li>旋转细胞在300×g离心3分钟。吸洗，并重新暂停2毫升NPC媒体（ 表1）花环。转移的细胞（1:1）到聚-L-鸟氨酸/层粘连蛋白包被的6孔板中。 </li><li>从15-21天，神经花环将扩大;饲料花环每隔一天与NPC媒体。 </li><li>评价神经花环的质量，并确保扁平成纤维细胞样细胞不培养内扩大。 注意:如果不坚持花环，全国文化有时可以通过人工采摘打捞上来，只选择最好的采摘玫瑰花成的Accutase。分离15分钟，在37ºC。颗粒，洗净，板NPC媒体到24孔聚L-鸟氨酸/层粘连蛋白涂层板。 </li></ol> 3.扩大神经祖细胞注:hiPSC的NPC可以生长在任Matrigel-或聚-L-鸟氨酸/层粘连蛋白包被的板。我们通常使用Matrige升感光板，因为它们可以更快速地制备并以更低的成本。 <ol><li>为了制备基质胶包被的板，快速解冻，重悬基质胶的1mg的冷冻等分试样以24毫升冷的DMEM / F12，并立即分发两个六孔板2毫升至每个孔中。孵育至少1小时，在37ºC。 注:基质胶仅需要被吸入，而不是洗涤，在使用前立即。 </li><li>饲料的NPC每隔一天，并以极高的密度保持或他们就会自发地分化为神经元。 注:虽然更成熟的NPC一般分为1:每周4，极低的通道的NPC可以被分割1:2的次数，每两周一次。 </li><li>分裂，第一抽吸媒体和加入1毫升温的Accutase（1X）每6孔板井。在37ºC10-15分钟。 </li><li>分离的细胞轻轻转移到15毫升管含有DMEM / F12用尽可能少的机械应力成为可能。不要滴定细胞，而酶。颗粒细胞spinni在1000×g离心纳克5分钟。 </li><li>吸洗，并在〜1毫升NPC媒体重新挂起的NPC每原井的6孔。 注:期望6孔培养皿的汇合阱具有5-10百万个细胞;这可以通过用血细胞计数之前重新电镀计数细胞的10升等分来确认。 </li><li>下面再悬浮，重制版的NPC作为NPC的，神经元或神经球。以保持的NPC，板每一个6孔板的大约1-2百万个细胞。的神经球的形成效率可以实验和细胞系之间有所不同，但一般发生最好之间200000和百万细胞每一个非粘附6孔板的井接种;如果神经球的聚集发生，降低细胞接种的数量。分化为神经元，板约200000每一个6孔板的细胞中，与神经元的媒体替换鼻咽癌介质。 </li><li>免疫组化验证每个NPC建立行。一旦有足够的多孔材料一直EXPANDED，标签的NPC与抗体NESTIN和SOX2。验证NPC线也应该分化成神经元4-6周，并标有抗体III微管蛋白和MAP2AB。 <ol><li>固定的细胞在PBS中的4％多聚甲醛在4℃下10分钟。透的NPC在RT在1.0％的Triton的PBS中15分钟，然后在5％驴血清阻滞与0.1％的Triton在RT 30分钟。 </li><li>孵育在5％驴血清初级抗体用0.1％的Triton，于4℃过夜。 注意:我们推荐以下抗体:羊抗SOX2，1:200;小鼠抗人巢蛋白，1:200;兔抗βIII微管蛋白，1:500;小鼠抗βIII微管蛋白，1:500;小鼠抗MAP2AB，1:200。 （ 图2）。 </li><li>以下用PBS洗涤，孵育二级抗体的细胞与合适的共轭次级抗体山羊，小鼠或兔1:300，在5％驴血清和0.1％的Triton为1-2小时，在室温。为了显现细胞核，染色的细胞用0.5微克/毫升的DAPI（4&#39;，6-二脒基-2-苯基吲哚），然后装入带的Vectashield。 </li></ol></li></ol> 4.全国人大转导<ol><li>使用spinfection（细胞培养板离心中病毒的存在下，在1000×g离心1小时），以增加转染的细胞21的百分比。抽吸培养基并替换为相关过表达（或控制）慢病毒（或反转录病毒），滴定感染的所需复数（MOI）（通常为1-10），稀释在鼻咽癌介质。使用每一个6孔板的孔1.5ml体积（或其他类型的组织培养板中的适当缩放体积）。旋在1000×g离心37ºC1小时在平板离心机。 注意:理想情况下，在1-2天内分裂转导的NPC与慢病毒或逆转录病毒载体。是否使用商业或实验室制备的病毒，也可以是有帮助的通过连续稀释至适当滴度的病毒的批次提前。 （ 图3）。 </li><li>为了减少CEL细胞性死亡，在spinfection 8小时更换介质。 注:病毒整合和转基因的表达应该是在24小时内报告基因的荧光检测。 （如果病毒缺乏荧光报道，这是更难以评估;成功的转染可以最好地通过免疫印迹，免疫组织化学或定量PCR对表达产物进行验证。）全部表达可能需要3-7天;可能需要重复spinfections与其他病毒以提高转染的细胞的百分比。复发性spinfections每天可以发生，但不必如此频繁。理想情况下，如果使用的是荧光报道，&gt; 80％的细胞应被标记。 </li></ol> 5.神经球迁移实验注:神经球形式自发，以下的NPC的酶解（通过等同于在鼻咽癌膨胀中使用的方式 - 步骤3.3-3.6），如果细胞是在悬浮液中鼻咽癌培养基中培养。 <ol><li>简单地说，长大分离在鼻咽癌媒体48小时在非粘附板ð的NPC，以便产生神经球。 （ 图4）。 </li><li>为神经球迁移，采用冷鼻咽癌媒体，而不是培养基，在96孔板中，以神经球采摘前1-2小时制备的新鲜基质胶板。不要从油井吸基底膜。 </li><li>最初公布的胚胎干细胞衍生的神经球22，手工挑NPC来源的神经球在显微镜下，在全国人民代表大会媒体清洗一次，以去除细胞碎片之后。传送一个神经球到基质胶包被的96孔板的每个孔中。 </li><li>添加额外的0.5毫克的Matrigel，稀释于冷鼻咽癌媒体，向神经球在每个96孔板中。 </li><li>手动居中每个单独的神经球在使用吸管尖阱的中间。 注意:挑大小相似的神经球，以减少可变性的结果是很重要的。 </li><li>允许迁移发生48小时，然后修复细胞瓦特第i个4％多聚甲醛和染色与期望的免疫组织化学标记。 </li><li>如果放射状迁移要确定自己完全采用4倍显微镜物镜，照片神经球。排除任何神经球接触所述井的边缘或从分析的第二神经球。 注意:如果发生的时间超过48小时的迁移，采用了4倍物镜所得径向迁移可能会过大，以图像。 </li><li>测量从用NIH ImageJ的软件（ 图5）的每个神经球平均迁移。这可以使用任何一种下面描述的分析方法来完成: <ol><li>放射状迁移: <ol><li>手动追踪使用徒手选择工具然后使用测量（CTRL + M）函数来计算得到的形状的区域中的最远迁移细胞的边缘。前测量的面积确保区域在分析选项卡下设置测量窗口进行检查。使用面积测量与方程为一个圆（A =πR2）的面积的值来确定所述半径（外）。 </li><li>跟踪原始神经球的边缘，测量面积，并计算半径（内）以相同的方式。计算总径向迁移作为外半径和内半径之间的差异。 </li></ol></li><li>最大的细胞迁移: <ol><li>测量采用直线选择工具其次是测量（按Ctrl + M）功能从内神经球的边缘移动的五个细胞最远的距离。 </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

我们已经描述了通过该分化人iPS细胞的NPC成，神经细胞类型，其中hiPSC衍生的神经元的基因标记的显著分数是保守，并且可以作为一个代理可能造成疾病的发病机制8的发育途径的方法， 11。此外，正如我们已经详细介绍，NPC的是一个稳健的复制和转导容易神经元群，我们相信这可能是用于疾病易感性的分子和生化研究。 虽然我们已经详细方法分化和培养前脑图案化的NPC，他们可以很容易地适用于产生的NPC构图以其他细胞命运。例如，如果浮置的EB首先用0.5毫克/毫升DKK1，10毫SB431542，0.5毫克/ ml成头蛋白和​​1mM的环巴胺，海马的NPC将生成可以在鼻咽癌媒体进行扩展并分化在补充有20纳克神经介质/ ml的WNT3A 7 </suP&gt;。或者，通过用100ng / ml的Shh的C25II，2M嘌吗啡胺，100代替FGF2微克/毫升FGF8和3M CHIR99021，NPC的可交替地图案化以脑多巴胺能命运，能够产生富含FOXA2-和酪氨酸羟化酶阳性多巴胺能神经元群体的神经细胞12,23。鉴于大多数亚型特异性神经元分化协议通过巢蛋白进行，SOX2，positve中间，我们预测，类似的方法可以适用于GABAergic- 2,24和glutamatergic- 3,25,26特定的NPC人群。 有协议值得注意中的几个关键步骤。首先，如果过表达或敲除内源基因表达，最好是使用一个控制向量从相同的病毒主链表达的荧光蛋白，我们已经观察到荧光显着的差异时，记者从可变大小的矢量表示（由于为减少包装效率矢量大小）或发​​起人的差异（由于在促进效率的差异）。其次，我们建议在完成病毒转导前神经球的形成，正如我们所观察到的病毒载体的外显率较差进入密集神经球结构。第三，超越通道10的筹备增加通道数量，我们经常观察神经球试验大大受损迁移。事实上，给定的NPC的倾向，得到的星形胶质细胞通路的较大百分比，这是至关重要的，以配合在各实验鼻咽癌线的通过。 最后，要考虑所描述的技术的生物和技术的限制是重要的。虽然的NPC似乎是用眼睛形态相似，他们是一个异类人群包括能产生两种谷氨酸和GABA能神经元细胞。同时我们观察到的NPC线非常一致脑图案个人之间使用此其实现方法具ð1，这是只有在比较充分验证的高品质的NPC线-有好的和质量差的NPC线之间的巨大差异。此外，病毒转导的产量遗传异质细胞，每个病毒载体在一个独特的基因组位置的积分;因此，各个单元都将在基因组整合位点的位置和数量而异。当遗传操作针对的是精确而定义的位置，是否发生了锌指，TALEN和CRISPR为基础的战略总是发生在基因表达更一致的变化。 患者hiPSC衍生的NPC可以被用来研究在两者的神经系统疾病中发生的全基因表达的扰动，并在分子和/或细胞操纵候选基因或微RNA在任一健康的或患病的患者来源的神经细胞的上下文中的影响。以这种方式，所带来的后果操纵一个或多个关键风险等位基因的特点可以在不同遗传背景的情况下。

神经元由我们1 体外分化自人类诱导多能干细胞（人iPS细胞）和其他2,3的基因表达的研究表明，神经元hiPSC类似于胎儿，而不是成人脑组织。目前，hiPSC为基础的模型可能更适合于易感性的研究，而不是最近的功能，神经系统疾病。我们以前曾报道，精神分裂症hiPSC衍生的神经元的基因标记的显著馏分保守精神分裂症hiPSC源性神经祖细胞（NPC的），这表明的NPC可能是一种有用的细胞类型用于研究分子途径促成精神分裂症1 。我们和其他人已经报道异常迁移，增加的氧化应激和活性氧，灵敏度到亚阈值的环境压力和在精神分裂症受损线粒体功能hiPSC的NPC 1,4-6，以及减小神经Çonnectivity和精神分裂症hiPSC神经元突触5,7-10功能。如果分子因素在精神分裂症hiPSC的NPC有助于异常迁移和/或氧化胁迫也背后精神分裂症hiPSC衍生的神经元的减少神经连接性，NPC的可能是一个强大的和高度复制神经元群，用以研究负责疾病的机制。此外，由于可以迅速地产生大量的细胞，并且不需要等待数周或数个月的神经元成熟，鼻咽癌基测定法适用于较大的患者群的研究，并且更适合于高通量筛选。我们相信，hiPSC的NPC可以作为可能造成疾病的发病机制的发育途径的代理，如已被证明在疾病等不同精神分裂症1和亨廷顿氏病11。 从人iPS细胞，在最初的神经分化的NPCduction由双SMAD抑制（0.1mM的LDN193189和10毫SB431542）12完成。通过拮抗骨形态发生蛋白和TGF与这些小分子信令，内胚层和中胚层规范被阻塞，加速神经元分化，并导致的可见神经花环形成电镀的一个星期内。神经形成发生在这一过程的早期，在神经花环形成，随后立即期间大概。在没有其他线索，这些原始神经细胞承担前端脑样的命运13。紧随其后的神经花环形成，并在整个NPC不断扩大，前脑的NPC与FGF2 8,14培养。它们具有双重谱系的潜力，并且可以分化为70-80％III微管蛋白阳性的神经元和20％-30％胶质纤维酸性蛋白（GFAP）阳性的星形胶质细胞（ 图1）的神经种群。大多数脑hiPSC神经元是VGLUT1阳性等等都是推测谷氨酸，尽管神经元的大约30％是GAD67阳性（GABA能）8。 NPC的例行传代在体外的十倍以上，同时保持一致分化型材，和没有积累核型异常。组报道传代的NPC超过40倍15，然而，我们发现，超过10通道，NPC的显示增加倾向星形胶质细胞分化。 NPC的良好耐受多次冷冻 - 解冻，并且可以转换通过简单地在培养非粘附板成长为神经球。的NPC用病毒载体转导的效率，从而使遗传扰动的分子和细胞后果快速评估，并易于扩展，以产生足够的材料用于生化研究。另外，因为病毒载体容许鲁棒过度表达和/或与疾病相关的基因敲除，在对照或患者来源neur人的细胞，可以使用这个平台来测试对这些操作的遗传背景的影响。虽然不适合突触或基于活动的检测需要成熟的神经元，NPC的可能是来自患者的神经细胞的许多简单的分子或生化分析一个实际的选择。

<table><tbody><tr><td>DMEM/F12</td><td>Life Technologies</td><td>#11330</td><td>for HES media</td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>#10565</td><td>for neural media</td></tr><tr><td>KO-Serum Replacement</td><td>Life Technologies</td><td>#10828</td><td>Needs to be lot tested</td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>#35050</td><td></td></tr><tr><td>NEAA</td><td>Life Technologies</td><td>&amp;nbsp;#11140</td><td></td></tr><tr><td>N2</td><td>Life Technologies</td><td>&amp;nbsp;#17502-048</td><td>Needs to be lot tested</td></tr><tr><td>B27-RA</td><td>Life Technologies</td><td>&amp;nbsp;#12587-010</td><td>Needs to be lot tested</td></tr><tr><td>FGF2</td><td>Life Technologies</td><td>#13256-029</td><td>Resuspend in PBS + 1% BSA</td></tr><tr><td>LDN193189</td><td>Stemgent</td><td>#04-0074</td><td></td></tr><tr><td>SB431542</td><td>Stemgent</td><td>#04-0010</td><td></td></tr><tr><td>BDNF</td><td>Peprotech</td><td>#450-02</td><td>Resuspend in PBS + 0.1% BSA</td></tr><tr><td>GDNF&amp;nbsp;</td><td>Peprotech</td><td>&amp;nbsp;#450-10</td><td>Resuspend in PBS + 0.1% BSA</td></tr><tr><td>Dibutyryl cyclic-AMP</td><td>Sigma</td><td>&amp;nbsp;#D0627</td><td>Resuspend in PBS + 0.1% BSA</td></tr><tr><td>L-ascorbic acid</td><td>Sigma</td><td>#A0278</td><td>Resuspend in H&lt;sub&gt;2&lt;/sub&gt;0</td></tr><tr><td>STEMdiff Neural Rosette Selection Reagent</td><td>Stemcell Technologies</td><td>&amp;nbsp;#05832</td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT-104</td><td></td></tr><tr><td>Collagenase IV</td><td>Life Technologies</td><td>#17104019</td><td></td></tr><tr><td>CF1 mEFs</td><td>Millipore</td><td>#PMEF-CF</td><td></td></tr><tr><td>Poly-L-Ornithine</td><td>Sigma</td><td>P3655</td><td></td></tr><tr><td>Laminin, Natural Mouse 1 mg</td><td>Life Technologies</td><td>#23017-015</td><td></td></tr><tr><td>BD Matrigel</td><td>BD</td><td>#354230</td><td>Resuspend on ice in cold DMEM at 10 mg/ml, use 1 mg per two 6-well plate equivalent</td></tr><tr><td>Tissue culture treated 6-well plates</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Ultra low attachment 6-well plates</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>goat anti-Sox2&amp;nbsp;</td><td>Santa Cruz</td><td>sc&amp;shy;17320</td><td>use at 1:200</td></tr><tr><td>mouse anti-human Nestin</td><td>Millipore</td><td>MAB5326</td><td>use at 1:200</td></tr><tr><td>rabbit anti-&amp;beta;III-tubulin</td><td>Covance</td><td>PRB&amp;shy;435P</td><td>use at 1:500</td></tr><tr><td>mouse anti-&amp;beta;III-tubulin</td><td>Covance</td><td>MMS&amp;shy;435P</td><td>use at 1:500</td></tr><tr><td>mouse anti-MAP2AB</td><td>Sigma</td><td>M1406</td><td>use at 1:200</td></tr><tr><td>Plate centrifuge</td><td>Beckman Coulter</td><td>Beckman Coulter Allegra X-14 (with SX4750 plate carrier)</td><td></td></tr></tbody></table>

a guide to generating and using hipsc derived npcs for the study of neurological diseases

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long period of disease progression prior to the onset of symptoms. Because fibroblasts from patients and healthy controls can be efficiently reprogrammed into human induced pluripotent stem cells (hiPSCs), and subsequently differentiated into neural progenitor cells (NPCs) and neurons for the study of these diseases, it is now possible to recapitulate the developmental events that occurred prior to symptom onset in patients. We present a method by which to efficiently differentiate hiPSCs into NPCs, which in addition to being capable of further differentiation into functional neurons, can also be robustly passaged, freeze-thawed or transitioned to grow as neurospheres, enabling rapid genetic screening to identify the molecular factors that impact cellular phenotypes including replication, migration, oxidative stress and/or apoptosis. Patient derived hiPSC NPCs are a unique platform, ideally suited for the empirical testing of the cellular or molecular consequences of manipulating gene expression.

神经花环可以形态被识别，使用明场显微镜，由它们的特征外观圆形簇神经上皮细胞apico -基底极性（ 图1）的。虽然筹备通常培养在非常高的细胞密度，紧接传代，略呈锥体形体躯和双极突起结构是可见的（ 图1D）。验证的NPC表达巢蛋白和Sox2在大多数细胞中，虽然III微管蛋白染色也是可见在所有鼻咽癌群体，这表明的NPC的一些比例都在不断的培养物（ 图1F）内引发神经元分化。神经干细胞，如SOX2和PAX6，和前脑的神经元祖细胞，如TBR2的标记物，也都表示在筹备，而脑标记如LMX1A和FOXA2不是（ 图1G-1）。内的显著的比例大而扁平的成纤维细胞样细胞<html>一个NPC人口表示质量差的NPC已经产生，这是不太可能产生以下的神经元分化（ 图2）大量的神经元。 在成功转导有高滴度慢病毒或逆转录病毒载体，大于80％的细胞应被标记由荧光报道包含在载体（ 图3）。神经球代应该产生相对均匀的大小的神经球（ 图4），这与常规饲养，可以为大约一周中培养保持健康的群体。鲁棒发生在健康的神经球（ 图5）神经球迁移和hiPSC NPC的一个神经球迁移测定1的过程中经历快速分化。 <img alt="图1" src="/files/ftp_upload/52495/52495fig1highres.jpg" />帐篷"&gt; 图1.患者的具体人iPS细胞，筹备和神经元，对hiPSC神经分化明场图像，从人iPS细胞（A），为胚体（B），神经花环（C）的NPC（D）和神经元（E） 。100X，比例尺为100μm 的FI hiPSC NPC都正对一些前脑的神经干细胞和神经祖细胞标记物，包括人大标记巢蛋白（绿色）和神经元标记III微管蛋白（红色）（F）神经干细胞转录因子SOX2（绿色）和PAX6（红色）（G）;以及前脑祖标记TBR2（红色）（H）;但不是脑标记LMX1A（绿色）和FOXA2（红色）（Ⅰ）。 DAPI染色细胞核（蓝色）。100X，比例尺为100μm。 学家的NPC可以分化为70-80％III微管蛋白阳性的神经元（绿色）和20-30％的胶质纤维酸性蛋白（GFAP）阳性的星形胶质细胞（红色）。 200X，比例尺为100μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52495/52495fig1large.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/52495/52495fig2highres.jpg" /> 图2.验证已NPC线，高品质（左上）hiPSC的NPC快递NESTIN（红色）和SOX2（绿色）。在大多数细胞（细胞核用DAPI染色（蓝色）），而低质量（左下）的NPC有SOX2阴性和巢蛋白阴性细胞（右）的补丁。从优质的NPC分化4周龄的神经元表达MAP2AB（绿色）和III微管蛋白（红色）（右上），但那些从低质量的NPC分化不（右下图）。 100X，比例尺为100μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52495/52495fig2large.jpg" target="_blank">请点击此处查看大图已经</a>rsion这个数字。 <img alt="图3" src="/files/ftp_upload/52495/52495fig3highres.jpg" /> 的NPC。明场（顶） 的图3.病毒转导和GFP荧光（底部）hiPSC的NPC的前（左）和病毒spinfection后的图像。 100X，比例尺为100μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52495/52495fig3large.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/52495/52495fig4highres.jpg" /> 图4.神经球形成贴壁的NPC（左）和新形成的神经球（右）明场图像。 100X，比例尺为100μm。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52495/52495fig4large.jpg" target="_blank">请点击此处查看LAR</a>蒙古包版本这个数字。 <img alt="图5" src="/files/ftp_upload/52495/52495fig5highres.jpg" /> 图5.神经球迁移。 AB，神经球前明视图像（A）后（B）48小时在基底膜迁移。 100X，比例尺为100μm。CG。截屏图像演示了使用分析ImageJ的NIH软件桡神经移植的方法。点击ImageJ的工具栏上的"写意的选择"（C）。跟踪周围神经球（D）中的最远的迁移细胞的边缘。确保地区设置测量窗口（E）被选中。使用测量功能（按Ctrl + M）（F）。跟踪（G）的原神经球的边缘，并使用测量功能（按Ctrl + M）。测量就可以导出到Excel进行分析。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52495/52495fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 媒体 </td><td> 组件 </td></tr><tr><td> HES媒体 </td><td> DMEM / F12（Life Technologies公司＃11330） </td></tr><tr><td></td><td> 20％的KO-血清替代（Life Technologies公司＃10828） </td></tr><tr><td></td><td> 1个谷氨酰胺（Life Technologies公司＃35050） </td></tr><tr><td></td><td> 1X NEAA（Life Technologies公司＃11140） </td></tr><tr><td></td><td> 1×2-巯基乙醇（55mm的1000倍）（Life Technologies公司＃21985-023） </td></tr><tr><td></td><td> 20ng的/ ml的FGF2（Life Technologies公司13256-029） </td></tr><tr><td> N2 / B27媒体 </td><td> DMEM / F12（Life Technologies公司＃10565） </td></tr><tr><td></td><td> 1X N2（Life Technologies公司＃17502-048） </td></tr><tr><td></td><td> 1X B27-RA（Life Technologies公司＃12587-010） </td></tr><tr><td> NPC媒体 </td><td> DMEM / F12（Life Technologies公司＃10565） </td></tr><tr><td></td><td> 1X N2（Life Technologies公司＃17502-048） </td></tr><tr><td></td><td> 1X B27-RA（Life Technologies公司＃12587-010） </td></tr><tr><td></td><td> 20毫微克/毫升的FGF2（Life Technologies公司） </td></tr><tr><td></td><td> 1mg / ml的天然小鼠层粘连蛋白（Life Technologies公司＃23017-015） </td></tr><tr><td> 神经介质 </td><td> DMEM / F12（Life Technologies公司＃10565） </td></tr><tr><td></td><td> 1X N2（Life Technologies公司＃17502-048） </td></tr><tr><td></td><td> 1X B27-RA（Life Technologies公司＃12587-010） </td></tr><tr><td></td><td> 1mg / ml的天然小鼠层粘连蛋白（Life Technologies公司） </td></tr><tr><td></td><td> 20毫微克/毫升的BDNF（派普泰克公司＃450-02） </td></tr><tr><td></td><td> 20纳克/毫升GDNF（Peptrotech＃450-10） </td></tr><tr><td></td><td> 500微克/毫升丁酰环-AMP（西格玛＃D0627） </td></tr><tr><td></td><td> 200nM的L-抗坏血酸（Sigma公司＃A0278） </td></tr></tbody></table> 表1.媒体的食谱。

Watch this Scientific Journal Video about A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases at JoVE.com

A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

指南生成和使用hiPSC派生的NPC的神经系统疾病的研究

Post-mortem studies of neurological diseases are not ideal for identifying the underlying causes of disease initiation, as many diseases include a long ...

a-guide-to-generating-using-hipsc-derived-npcs-for-study-neurological

Nanyang Technological University

Research

JoVE Journal

Medicine

18.2K 次观看.  Icahn School of Medicine at Mount Sinai. This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

视频: A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

科研

发育生物学

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

人脑神经前体细胞神经发育表型的快速检测 (npc)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

神经干/祖细胞在小鼠EAE的慢性全身性注射

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

免疫与感染

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

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 ...

Differentiation of Porcine Induced Pluripotent Stem Cells (piPSCs) into Neural Progenitor Cells (NPCs)

iPSC-derived neurons are attractive in vitro models to study neurogenesis and early phenotypic changes in mental illness, mainly when most animal models used in pre-clinical research, such as rodents, are not able to meet the criteria to translate the findings to the clinic. Non-human primates, canines, and porcine are considered more adequate models for biomedical research and drug development purposes, mainly due to their physiological, genetic, and anatomical similarities to humans. The swine model has gained particular interest in translational neuroscience, enabling safety and allotransplantation testing. Herein the generation of porcine iPSCs is described along with its further differentiation into neural progenitor cells (NPCs). The generated cells expressed NPC markers Nestin and GFAP, confirmed by RT-qPCR, and were positive for Nestin, b-Tubulin III, and Vimentin by immunofluorescence. These results show the evidence for the generation of NPC-like cells after in vitro induction with chemical inhibitors from a large animal model, an interesting and adequate model for regenerative and translational medicine research.

Differentiation of Porcine Induced Pluripotent Stem Cells piPSCs into Neural Progenitor Cells NPCs

This protocol describes a method for chemical differentiation and culture of neural progenitor cells derived from porcine induced pluripotent stem cells (piPSCs).

iPSC-derived neurons are attractive in vitro models to study neurogenesis and early phenotypic changes in mental illness, mainly when most animal models ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

无转基因生成血液衍生神经元细胞

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

使用从纤维细胞直接转换为神经元祖先细胞和分化为星形细胞的神经系统疾病体外建模

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first advent, protocols have been continuously optimized and are now widely used in research and drug development. However, the very long duration of these conventional differentiation and maturation protocols and the increasing demand for high-quality hiPSCs and their neural derivatives raise the need for the adoption, optimization, and standardization of these protocols to large-scale production. This work presents a fast and efficient protocol for the differentiation of genetically modified, doxycycline-inducible neurogenin 2 (iNGN2)-expressing hiPSCs into neurons using a benchtop three-dimensional (3D) suspension bioreactor.
In brief, single-cell suspensions of iNGN2-hiPSCs were allowed to form aggregates within 24 h, and neuronal lineage commitment was induced by the addition of doxycycline. Aggregates were dissociated after 2 days of induction and cells were either cryopreserved or replated for terminal maturation. The generated iNGN2 neurons expressed classical neuronal markers early on and formed complex neuritic networks within 1 week after replating, indicating an increasing maturity of neuronal cultures. In summary, a detailed step-by-step protocol for the fast generation of hiPSC-derived neurons in a 3D environment is provided that holds great potential as a starting point for disease modeling, phenotypic high-throughput drug screenings, and large-scale toxicity testing.

This article describes a protocol for the generation of human induced pluripotent stem cell-derived neurons in a benchtop 3D suspension bioreactor.

在三维悬浮生物反应器中产生人神经原蛋白2诱导神经元

The derivation of neuronal lineage cells from human induced pluripotent stem cells (hiPSCs) marked a milestone in brain research. Since their first ...