这项工作是由弗里茨·蒂森基金会（Az.10.14.2.152）的支持。我们感谢组织包埋设施和CMMC的显微镜核心设施。我们是由实验室中心体和细胞骨架生物学的成员提供的讨论和技术支持表示感谢。我们感谢李明Gooi校对稿件。

1.脑类器官的生成（23天） <ol><li> 神经外胚层的启动（5天） 注:以下几点值得分化开始前予以考虑。重编程方法（lentiviral-，仙台病毒，附加体，或微小RNA-基于等 ）以获得人类iPSC理想地应为所有患者同一和控制iPSC系。各种重编程工具包，并根据公布的方案说明，请15，16，17，18。人类iPSC系的质量的关键是实现最佳的分化。监测菌落和细胞形态用显微镜和通过测试标记物的表达，如OCT3 / 4，Nanog的，或TRA-1-60验证多能性。 <ol><li>培养人无饲养条件下的iPSC在介质A上涂覆有Engelbreth-Holm的-群菜（EHS）矩阵。 注:生长人类iPSC无饲养和维持确定的培养条件，并避免对分化开始前除去小鼠胚胎饲养细胞（MEF中）的附加步骤的无血清。对于人类iPSC的最佳通道编号，以从通道15重新编程时开始分化范围到通道70的总额。传代时，分离的hiPSC菌落作为细胞聚集使用具有低机械应力的适当细胞剥离溶液和2毫升血清移液管的聚集体转移到新的培养皿中。避免解离为单细胞，因为这可能会诱导分化和凋亡在大多数iPSC系。据报道，长期，单细胞传代会增加基因组改变在人iPSC 19，20。避免细胞脱离程序，这需要离心步骤以除去剥离液，因为这降低iPSC的整体可行性。测试微生物污染，特别是支原体，定期，所有的文化，因为这可能会改变iPS细胞的质量和分化能力。 <ol><li>外套60毫米组织培养皿中以EHS矩阵按照制造商的说明书。 </li><li>解冻的1×10 6人类iPSC的等分试样。种子的iPSC在涂在EHS基质和含有5毫升培养基A（参见材料表 ）60毫米组织培养皿中。每日更换培养基和传代后5至7天当细胞达到〜80％汇合。 注意:在传代80％汇合使用标准方法，如集落21的不含酶的分离，至少一次在开始分化前的解冻的iPSC。简言之，移除的iPSC介质，具有预温37℃，Dulbecco氏改良的Eagle氏培养基一次洗涤细胞:营养混合物F12（DMEM / F12），并孵育的iPSC按照生产商的使用试剂A指令（见材料表）。不要为了避免解离为单细胞超过推荐的培养时间。人类iPSC应分离及浮动聚集，而不是单个细胞。然后可以将它们转移到新的EHS基质包被的培养皿;例如，iPSC集聚集来自一个60-mm皿可以被分配到4个新的60-mm培养皿。 </li><li>根据制造商的说明与支原体检测试剂盒检查支原体。只使用无支原体的iPSC，支原体可以改变iPS细胞的分化能力。 </li></ol></li><li>解离的iPSC（80％汇合），并使用试剂B.制备单细胞悬浮液<ol><li>用预热（37℃）的DMEM / F12洗涤的iPSC一次。 </li><li>添加预热的试剂B（ 例如，1毫升在60毫米培养皿）和孵育的iPSC在37℃5分钟和5％CO 2。 </li><li>上的侧部和底部的指尖轻弹20倍培养皿以分离的iPSC。检查细胞在显微镜下分离。 </li><li>用1毫升微量移液管将细胞悬浮液上下在培养皿5倍。 </li><li>加入3毫升培养基A稀释1mL的试剂B和收集细胞悬浮液在15mL的离心管中。 </li><li>轻轻降速的iPSC（500×g下），用于在室温下4分钟。 </li><li>重悬在1mL介质B的细胞沉淀和计数与血球细胞数目。 注意:请注意只使用介质B的再悬浮。避免使用介质A，因为它包含一个过于高浓度的bFGF，这可能会抑制分化。 </li></ol></li><li>稀释细胞悬浮液，以每毫升4.5×10 5个细胞在培养基B补充有10μMRho相关蛋白激酶抑制剂（Y-27632）。 </li><li>添加每孔100μL在非贴壁，V形底，96孔板。 注:确保细胞均匀分布在SUS通过取出100μL的部分之前每次摇动管退休金。使每孔应包含以获得神经球在尺寸和形状（圆形，限定的表面）均匀的等效细胞数量是重要的。 </li><li>与细胞在500×g下和室温下温和降速板3分钟，并在37℃和5％CO 2孵育。 </li><li>每日通过去除50微升和加入新鲜培养基B的50μL到每个孔中用于接下来5天改变介质。 </li></ol></li></ol> 2.嵌入神经球在EHS矩阵（4天） <ol><li> 200（V / V）的补充1，1:1:1:1:DMEM / F12和介质C（V / V），1的1:1混合物100（V / V）补充2无（重量/通过以下混合制备神经球介质○）维生素A，1:100 L-谷氨酰胺，0.05 mm，非必需氨基酸（MEM），100U / mL青霉素，100微克/ mL链霉素，1.6 g / L的胰岛素，和0.05mMβ巯基乙醇。 </li><li>收集神经球机智公顷使用〜2毫米尖端200μL微量预先切割用无菌剪刀。 </li><li>放置神经球大约5mm远离彼此上石蜡膜（3×3厘米2）在一个空的100mm培养皿，并小心地取出尽可能剩余的培养基。 </li><li>添加EHS矩阵一滴（7微升）到每个单个神经球。 </li><li>孵育EHS矩阵与用于神经球在培养箱中15分钟下降。 </li><li>通过与神经球中冲洗它们从石蜡膜仔细清洗的神经。要刷新，使用1mL的微量和含有10毫升神经球介质的新百毫米培养皿。 </li><li>孵育神经球在接下来的4天，添加在第2天2毫升的新鲜神经球培养基。 注:确保在孵化器的货架上是平的，这样的EHS基质埋入神经球不会对菜的一侧聚集在一起。 </li></ol> 3.在旋转悬挂类器官文化（14天） <ol><li> 200（V / V）的补充1，1:100（V / V）补充2 W / O 1:DMEM / F12和介质C（V / V），1的混合物通过以下混合制备脑类器官介质维生素A，1:100 L-谷氨酰胺，0.05毫MEM，100 U / ml的青霉素，100μg/ mL链霉素，1.6 g / L的胰岛素，0.5μMdorsomorphin，5μMSB431542，和0.05mMβ巯基乙醇。 </li><li>加入100毫升脑类器官培养基到每个转瓶中通过其侧臂并将它们放置在用于预热的培养箱中至少20分钟。 </li><li>在25rpm设置搅拌程序，根据制造商的说明。 注:EHS基质埋入神经球转移到转瓶中之前，确保它们都分开。如果两个或多个通过EHS矩阵连接，通过用手术刀切割所述连接矩阵将它们分开。 </li><li>小心转移EHS基质包埋神经球到含有100毫升类器官培养基的旋转烧瓶我们荷兰国际集团2毫升血清移液管。使用旋转瓶的侧臂的神经球转移到烧瓶中。 </li><li>放置在培养箱中的磁搅拌下平台旋转瓶在37℃和5％CO 2;这是化培养的第0天。 </li><li>改变介质每周一次（或更经常时，有一个颜色的变化）通过除去一半的培养基并添加新鲜培养基的相同的量。 注意:当取旋转烧瓶出培养箱，等待3-5分钟，以让类器官下沉到烧瓶的底部。通过将液体的表面上的玻璃移液管尖端（连接到泵）拆下介质;小心通过一个侧部开口/烧瓶的臂吸出培养基。这些操作必须在层流罩下进行。 </li></ol> 4.脑组织体分析<ol><li> 组织体固定 <ol><li>收集转瓶中培养无线的第14天的类器官TH切割1mL的微量（切割约5毫米）。把它们都在60毫米培养皿中并用5毫升温暖的DMEM / F12的一次洗3分钟。 </li><li>制备1.5mL管500μL温暖4％多聚甲醛的（PFA）。 注意:处理PFA固定时，戴上皮肤和眼睛的保护和工作安全罩下。 </li><li>将每个器官样分别在每个管和解决这些问题，在室温下至少30分钟。不要修复类器官的时间超过60分钟。要移动类器官，用接种环或其他任何设计出方便。 </li><li>除去PFA和洗涤固定类器官两次，每次10分钟，用1mL的PBS中。 </li><li>存储在1mL PBS的类器官在4℃至7天，直至进一步使用。 </li></ol></li><li> 嵌入类器官的冷冻切片 <ol><li>除去PBS并在每管脱水cryofreezing他们之前的类器官蒸馏水溶液加入1毫升30％蔗糖的;加入SUCRO后SE溶液，所述类器官应在表面上浮动。在4℃下过夜存储类器官中的蔗糖溶液;在第二天，类器官应该已经沉没到管的底部。 注意:可以类器官在4℃如有必要，可以保存长达3-5天在蔗糖溶液。 </li><li>填充400μL最佳切割温度（OCT）化合物的乙烯基标本模具，并使用接种环将一个类器官在模具的中心。标记与样品名称模具的边缘。 </li><li>冷冻在-80℃下的含类器官模直到冷冻切片。 </li><li>用PBS中0.1％的聚-L-赖氨酸溶液（PLL），在室温下5分钟，涂层玻璃cryoslides并让干燥载玻片3小时。在4℃保存幻灯片，使用前预热起来到室温温和。 注:PLL涂层是重要的一步，因为它会阻止该组织体片漂浮之遥。收集并在4℃下存储PLL溶液长达3个月。重新使用前，用0.22微米注射器过滤它，让它温暖至室温。 </li><li>部cryofrozen类器官成20-50微米厚的切片上PLL涂布的玻璃cryoslides 22。让与所述部分中的干燥载玻片在室温下1个小时。在-80℃直到进一步处理存储该部分。 </li></ol></li></ol> 5.类器官切片的免疫荧光染色注:有关组织体的一般特征，与巢蛋白，神经祖细胞标记，TUJ1，泛神经元标记染色，值得推荐。作为附加示例，免疫荧光染色与磷酸化波形蛋白（P-VIM），哪些标签有丝分裂顶端径向胶质细胞，和Arl13b，为纤毛，进行说明。为了测试细胞凋亡，使用末端脱氧核苷酸转移酶（TdT）的dUTP缺口末端标记（TUNEL）测定法。放置在塑料盒中载片温育期间，以保护它们免受灰尘，光，和干燥。 <ol><li>解冻载玻片在室温下30分钟。 </li><li>用PBS-甘氨酸200μL（在PBS中0.225克甘氨酸）3分钟洗两次滑动以淬灭PFA诱导的自体荧光。 </li><li>透化切片用/ 0.5％的Triton X100的200μL中在室温下10分钟的PBS溶液0.1％吐温。 </li><li>用3分钟PBS-甘氨酸溶液200微升洗两次。 </li><li>与/ 200μL0.5％鱼明胶的0.1％的Triton X100的PBS孵育它们在室温下或在4℃下过夜1个小时，以封闭非特异性的抗原结合。 注:如果需要TUNEL分析，进行测定按照制造商的方案。进行免疫，因为它可能与第二抗体干扰和以后使用时，荧光淬灭之前与TUNEL法启动。 </li><li>稀释的抗体在阻断以下列浓度溶液:巢蛋白，1:200; P-Vim的，1:500; TUJ1，1:200;Arl13b，1:20;抗体和第二抗体，1:1,000。 </li><li>与所述第一初级抗体的200μL（ 例如，巢蛋白）1-2小时，在室温下或在4℃下孵育过夜。 </li><li>洗3×3分钟用封闭溶液的200μL。 </li><li>稀第一（抗小鼠488）的第二抗体1:1000在封闭溶液中孵育在200μL滑动，在室温下1-2小时。从现在起，始终保护将幻灯片从光。 </li><li>洗3×3分钟用封闭溶液的200μL。 </li><li>与下一个初级抗体的200μL（ 例如，TUJ1）孵育在室温下1-2小时或过夜，在4℃。 </li><li>洗3×3分钟用封闭溶液的200μL。 </li><li>添加下一个第二抗体（抗 - 兔647）的200μL，在室温下1-2小时。 </li><li>洗3×3分钟。用封闭溶液的200μL。 </li><li>添加200μL的4&#39; ，6-二脒基-2-苯基吲哚（DAPI）在30nM的浓entration的PBS在室温下核染色15分钟。 </li><li>洗2×3分钟用封闭溶液的200μL。 </li><li>洗1×1分钟用蒸馏水200μL，并让部分干燥10-20分钟，直到没有明显的水滴是可见的了。 </li><li>安装与包埋剂的部分。将它们储存在4℃下避光长达数周。 </li><li>与微观分析图像的组织体，脑室区，原始的骨板，以及其他感兴趣的领域的概述进行。 </li><li>使用与63X油浸物镜和荧光滤光器的共聚焦显微镜，根据所使用的1，10中的荧光染料标记的第二抗体选择。 </li></ol>

<ol>
	<li>Lancaster, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501, 373-379 (2013).</li><li>Eiraku, M., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryonic+stem+cell+culture+for+generation+of+three-dimensional+retinal+and+cortical+tissues.">Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.</a> Nat Protoc. 7, 69-79 (2012).</li><li>Nakano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10, 771-785 (2012).</li><li>Gabriel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CPAP+promotes+timely+cilium+disassembly+to+maintain+neural+progenitor+pool.">CPAP promotes timely cilium disassembly to maintain neural progenitor pool.</a> EMBO J. 35 (8), 803-819 (2016).</li><li>Al-Dosari, M. S., Shaheen, R., Colak, D., Alkuraya, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+CENPJ+mutation+causes+Seckel+syndrome.">Novel CENPJ mutation causes Seckel syndrome.</a> J Med Genet. 47, 411-414 (2010).</li><li>Wollnik, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+mechanism+for+microcephaly.">A common mechanism for microcephaly.</a> Nat Genet. 42, 923-924 (2010).</li><li>Thornton, G. K., Woods, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+microcephaly:+do+all+roads+lead+to+Rome?.">Primary microcephaly: do all roads lead to Rome?.</a> Trends Genet. 25, 501-510 (2009).</li><li>Barbelanne, M., Tsang, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+cellular+basis+of+autosomal+recessive+primary+microcephaly.">Molecular and cellular basis of autosomal recessive primary microcephaly.</a> Biomed Res Int. 2014, 547986 (2014).</li><li>Homem, C. C., Repic, M., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+control+in+neural+stem+and+progenitor+cells.">Proliferation control in neural stem and progenitor cells.</a> Nat. Rev. Neurosci. 16, 647-659 (2015).</li><li>Gabriel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CPAP+promotes+timely+cilium+disassembly+to+maintain+neural+progenitor+pool.">CPAP promotes timely cilium disassembly to maintain neural progenitor pool.</a> EMBO J. 35, 803-819 (2016).</li><li>Mak, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecules+greatly+improve+conversion+of+human-induced+pluripotent+stem+cells+to+the+neuronal+lineage.">Small molecules greatly improve conversion of human-induced pluripotent stem cells to the neuronal lineage.</a> Stem Cells Int. , (2012).</li><li>Morizane, A., Doi, D., Kikuchi, T., Nishimura, K., Takahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+bone+morphogenic+protein+and+activin/nodal+signals+promote+highly+efficient+neural+induction+from+human+pluripotent+stem+cells.">Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells.</a> J. Neurosci. Res. 89, 117-126 (2011).</li><li>Shi, Y., Kirwan, P., Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+pluripotent+stem+cells+to+cerebral+cortex+neurons+and+neural+networks.">Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks.</a> Nat Protoc. 7, 1836-1846 (2012).</li><li>Zhou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+induction+of+neural+conversion+in+human+ESCs+and+human+induced+pluripotent+stem+cells+with+a+single+chemical+inhibitor+of+transforming+growth+factor+beta+superfamily+receptors.">High-efficiency induction of neural conversion in human ESCs and human induced pluripotent stem cells with a single chemical inhibitor of transforming growth factor beta superfamily receptors.</a> Stem Cells. 28, 1741-1750 (2010).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Okita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+more+efficient+method+to+generate+integration-free+human+iPS+cells.">A more efficient method to generate integration-free human iPS cells.</a> Nat Methods. 8, 409-412 (2011).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells+free+of+vector+and+transgene+sequences.">Human induced pluripotent stem cells free of vector and transgene sequences.</a> Science. 324, 797-801 (2009).</li><li>Lieu, P. T., Fontes, A., Vemuri, M. C., Macarthur, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+with+CytoTune,+a+non-integrating+Sendai+virus.">Generation of induced pluripotent stem cells with CytoTune, a non-integrating Sendai virus.</a> Methods Mol. Biol. 997, 45-56 (2013).</li><li>Bai, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+analysis+of+genome+alterations+induced+by+single-cell+passaging+in+human+embryonic+stem+cells.">Temporal analysis of genome alterations induced by single-cell passaging in human embryonic stem cells.</a> Stem Cells Dev. 24, 653-662 (2015).</li><li>Garitaonandia, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+risk+of+genetic+and+epigenetic+instability+in+human+embryonic+stem+cells+associated+with+specific+culture+conditions.">Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.</a> PLoS One. 10, e0118307 (2015).</li><li>Ohnuma, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-free+passage+of+human+pluripotent+stem+cells+by+controlling+divalent+cations.">Enzyme-free passage of human pluripotent stem cells by controlling divalent cations.</a> Sci. Rep. 4, 4646 (2014).</li><li>Currle, D. S., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+freezing+and+cryosectioning+E12.5+mouse+brain.">Flash freezing and cryosectioning E12.5 mouse brain.</a> J Vis Exp. , (2007).</li><li>Yingling, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroepithelial+stem+cell+proliferation+requires+LIS1+for+precise+spindle+orientation+and+symmetric+division.">Neuroepithelial stem cell proliferation requires LIS1 for precise spindle orientation and symmetric division.</a> Cell. 132, 474-486 (2008).</li><li>Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+step+for+the+cell,+a+giant+leap+for+mankind:+a+hypothesis+of+neocortical+expansion+during+evolution.">A small step for the cell, a giant leap for mankind: a hypothesis of neocortical expansion during evolution.</a> Trends Neurosci. 18, 383-388 (1995).</li><li>Hu, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+of+human+induced+pluripotent+stem+cells+follows+developmental+principles+but+with+variable+potency.">Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 4335-4340 (2010).</li><li>Wilson, P. G., Stice, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+differentiation+of+neural+rosettes+derived+from+human+embryonic+stem+cells.">Development and differentiation of neural rosettes derived from human embryonic stem cells.</a> Stem Cell Reviews. 2, 67-77 (2006).</li><li>Dhara, S. K., Stice, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+of+human+embryonic+stem+cells.">Neural differentiation of human embryonic stem cells.</a> J. Cell Biochem. 105, 633-640 (2008).</li><li>Christie, V. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+evaluation+of+synthetic+retinoid+derivatives+as+inducers+of+stem+cell+differentiation.">Synthesis and evaluation of synthetic retinoid derivatives as inducers of stem cell differentiation.</a> Org Biomol Chem. 6, 3497-3507 (2008).</li><li>Kim, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+enhancement+of+neural+differentiation+from+human+ES+and+iPS+cells+regardless+of+their+innate+difference+in+differentiation+propensity.">Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity.</a> Stem Cell Reviews. 6, 270-281 (2010).</li><li>Gabriel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Zika+Virus+Isolates+Induce+Premature+Differentiation+of+Neural+Progenitors+in+Human+Brain+Organoids.">Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids.</a> Cell stem cell. , (2017).</li><li>Wu, J., Hunt, S. D., Xue, H., Liu, Y., Darabi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+Characterization+of+a+MYF5+Reporter+Human+iPS+Cell+Line+Using+CRISPR/Cas9+Mediated+Homologous+Recombination.">Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.</a> Sci. Rep. 6, 18759 (2016).</li><li>Li, H. L., Gee, P., Ishida, K., Hotta, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genomic+correction+methods+in+human+iPS+cells+using+CRISPR-Cas9+system.">Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.</a> Methods. 101, 27-35 (2016).</li><li>Grobarczyk, B., Franco, B., Hanon, K., Malgrange, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Isogenic+Human+iPS+Cell+Line+Precisely+Corrected+by+Genome+Editing+Using+the+CRISPR/Cas9+System.">Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System.</a> Stem Cell Reviews. 11, 774-787 (2015).</li><li>Camp, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cerebral+organoids+recapitulate+gene+expression+programs+of+fetal+neocortex+development.">Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.</a> Proc. Natl. Acad. Sci. U. S. A. 112, 15672-15677 (2015).</li><li>Qian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-Region-Specific+Organoids+Using+Mini-bioreactors+for+Modeling+ZIKV+Exposure.">Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.</a> Cell. 165, 1238-1254 (2016).</li><li>Jo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbrain-like+Organoids+from+Human+Pluripotent+Stem+Cells+Contain+Functional+Dopaminergic+and+Neuromelanin-Producing+Neurons.">Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons.</a> Cell Stem Cell. 19, 248-257 (2016).</li><li>Mariani, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FOXG1-Dependent+Dysregulation+of+GABA/Glutamate+Neuron+Differentiation+in+Autism+Spectrum+Disorders.">FOXG1-Dependent Dysregulation of GABA/Glutamate Neuron Differentiation in Autism Spectrum Disorders.</a> Cell. 162, 375-390 (2015).</li><li>D'Avanzo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+in+3D+culture:+challenges+and+perspectives.">Alzheimer's in 3D culture: challenges and perspectives.</a> Bioessays. 37, 1139-1148 (2015).</li></ol>

The authors declare that they have no competing financial interests.

MCPH是一个复杂的人类神经发育障碍，不能在动物模型中在体内或在简单的人细胞培养物在体外方法中重现。 MCPH的临床表现开始在第一孕期出现，早期的神经开始时。因此，3D脑组织体代表了可靠的实验系统，MCPH发展模式。此外，三维人体脑组织体是一种理想的方法，因为i）它们允许与各种遗传背景的患者样本的光谱的适配，ⅱ）它们显示包含不同的神经细胞类型组织的组织，并?...

人类神经发育障碍，如小头畸形，只能研究甚少在动物模型中由于人类大脑有较长的皮质表面，独特的功能，从非人类动物不同。 该方面使得人脑发育一个复杂的过程，不能被充分研究在2D， 体外细胞培养系统。新兴的3D培养技术允许从诱导的多能干细胞（iPS细胞）的组织样类器官的生成。在3D悬浮培养多能干细胞的体外分化允许各种细胞类型的及时和特定区域的方式形成的，从而产生了一个有组织的，分层组织1，2，3。多亏了3D先锋文化技术和揭秘的器官形成的复杂性，从干细胞开始实验室，我们开发大脑产生对类器官描绘人类大脑发育的早期事件和小头畸形体外 1，2，3模型的鲁棒性的方法。值得注意的是，我们适于通过兰开斯特等人开发的原始方法。以产生脑类器官1。这种方法是根据我们的实验要求进行修改。 从加布里埃尔等人的一项研究的目的。是分析大脑发育过程中的神经干细胞维持的细胞和分子机制。为了做到这一点，一种机械的研究是通过分析在从一个小头畸形患者4衍生的三维大脑类器官的神经祖细胞（NPC）进行。这个病人在CPAP，中心体生物发生所需要5保守中心体蛋白进行的突变。一个被广泛接受的低论点是，小头畸形是NPC池的耗尽的结果，这可能是由于无论是细胞死亡或过早分化1，6，7，8，9。 通过分析头畸形脑类器官的心室区（VZS），已显示的NPC的显著数进行不对称细胞分裂，不同于来自健康供体4衍生的脑类器官。 microcephalic脑组织体的广泛微观和生化分析揭示了及时纤毛拆卸4 CPAP意想不到的作用。具体地，突变的CPAP与延迟纤毛拆卸和延迟细胞周期重新条目相关联，从而导致的NPC 4的过早分化。这些结果表明，在小头畸形和日纤毛作用神经和大脑的大小控制在10 EIR参与。 该协议的第一部分是一个三步骤的方法的描述，以产生均匀的脑类器官。正如前面提到的，原来的兰开斯特协议进行调整和修改，以适应我们的宗旨1。首先，人类iPSC在上Engelbreth-Holm的-群（EHS）矩阵定义的无饲养条件下培养。这一步骤避免了馈线依赖性多能干细胞培养物的变体。在这个协议中，神经分化的诱导以形成神经上皮直接从iPSC的开始。通过跳过胚状体（EB）形成步骤中，以更受控和定向的方式的神经分化前进。该方法限制了其他生殖细胞层，例如中胚层和内胚层的自发和无向的形成。通过施加这个协议中，含有神经花环神经球可以在第5天收获为EHS矩阵的嵌入和固定的悬浮培养。用于我们的协议的第三步骤中的类器官培养基补充有dorsomorphin和SB431542。 Dorsomorphin是骨形态发生蛋白（BMP）的小分子抑制剂，和SB431542抑制TGFβ/激活素/ Nodal信号通路。这些因素的组合可以促进神经分化比更有效地单独11，12，13，14视黄酸。 总之，这些修饰使再现的生成脑组织体，并在保持类器官最小变化。重要的是，施加这种方法来鲁棒地产生从患者的iPSC，其携带突变影响中心体和细胞周期动力学的基因microcephalic脑类器官。 此协议的第二部分发出指令，以制备BR艾因类器官的分析和畸形细胞缺陷的解释。这包括固定，冷冻切片，免疫荧光染色和共聚焦显微镜分析。该协议会为读者提供了预期结果的详细说明和解释为指导。

<table border="0" cellpadding="0" cellspacing="0" width="670">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-mouse 488</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">A-11001</td>
			<td style="width:212px;">Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti-rabbit 647</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:119px;">A-21245</td>
			<td style="width:212px;">Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Arl13b</td>
			<td style="width:123px;">proteintech</td>
			<td style="width:119px;">17711-1-AP</td>
			<td style="width:212px;">ARL13B rabbit polyclonal antibody&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CELLSPIN system</td>
			<td style="width:123px;">IBS Integra Bioscience</td>
			<td style="width:119px;">183001</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DAPI</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">32670</td>
			<td style="width:212px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM/F-12</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">31331093</td>
			<td style="width:212px;">Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dorsomorphin</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">P5499</td>
			<td style="width:212px;">Compound C; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Embedding medium</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A9011, 0100</td>
			<td style="width:212px;">Mowiol; embedding medium; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Engelbreth-Holm-Swarm (EHS) matrix</td>
			<td style="width:123px;">Corning</td>
			<td style="width:119px;">354277</td>
			<td style="width:212px;">Matrigel hESC-qualified matrix; important: hESC qualified</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fish gelatin&#160;</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">G7765-250ML</td>
			<td style="width:212px;">Gelatin from cold water fish skin; multiple suppliers; autoclave after adding to PBS to dissolve and sterilize, store at 4&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glycine</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A1067,1000</td>
			<td style="width:212px;">Glycine for molecular biology; multiple suppliers&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inoculation loop with needle, disposable (1 &#181;l)</td>
			<td style="width:123px;">Sigma Aldrich, US</td>
			<td style="width:119px;">BR452201-1000EA</td>
			<td style="width:212px;">multiple suppliers&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insulin</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">I3536-100MG</td>
			<td style="width:212px;">multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-glutamine</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">25030081</td>
			<td style="width:212px;">L-glutamine (200 mM)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Medium A</td>
			<td style="width:123px;">Stem cell technologies</td>
			<td style="width:119px;">#05850</td>
			<td style="width:212px;">mTeSR1 (hiPSC medium)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Medium B</td>
			<td style="width:123px;">Stem cell technologies</td>
			<td style="width:119px;">#05835</td>
			<td style="width:212px;">Neural induction medium (NIM); neural differentiation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Medium C</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">21103049</td>
			<td style="width:212px;">Neural Basal Medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MEM</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">11140035</td>
			<td style="width:212px;">MEM non-essential amino acids solution (100x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MycoAlert Mycoplasma Detection Kit</td>
			<td style="width:123px;">Lonza, Switzerland</td>
			<td style="width:119px;">#LT07-218</td>
			<td style="width:212px;">Mycoplasma detection kit; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nestin</td>
			<td style="width:123px;">Novus biologicals</td>
			<td style="width:119px;">NBP1-92717</td>
			<td style="width:212px;">Nestin mouse monoclonal antibody (4D11)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Paraformaldehyde (PFA)</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A3813, 0500</td>
			<td style="width:212px;">4% in PBS, store solution at -20&#176;C; caution: wear skin and eye protection and work under hood&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS tablets</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">18912014</td>
			<td style="width:212px;">See manufacturer&#180;s instructions; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin (10.000 U/ml)</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">15140122</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly-L-lysine solution (PLL)</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">P8920-100ML</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pVim</td>
			<td style="width:123px;">MBL</td>
			<td style="width:119px;">D076-3S</td>
			<td style="width:212px;">Phospho-Vimentin (Ser55) mAb</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Reagent A&#160;</td>
			<td style="width:123px;">Stem cell technologies</td>
			<td style="width:119px;"># 05872</td>
			<td style="width:212px;">Note to Protocol 1.1.1.2; ReLSR (Enzyme-free human ES and iPS cell selection and passaging reagent); please follow manufactorer&#180;s protocol; alternative products from muliple suppliers available</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Reagent B&#160;</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">A6964-100ML</td>
			<td style="width:212px;">Accutase solution is an enzymatic solution for single cell dissociation; multiple suppliers; protocol 1.1.2 &#34;enzymatic cell dissociation solution&#8221;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Research Cryostat Leica CM3050 S</td>
			<td style="width:123px;">Leica biosystems</td>
			<td style="width:119px;">CM3050 S</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SB431542</td>
			<td style="width:123px;">Selleckchem.com</td>
			<td style="width:119px;">S1067</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spinner flask 250 ml</td>
			<td style="width:123px;">IBS Integra Bioscience</td>
			<td style="width:119px;">182026</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A4734, 1000</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Superfrost ultra plus microscope slides</td>
			<td style="width:123px;">Thermo scientific, US</td>
			<td style="width:119px;">J3800AMNZ</td>
			<td style="width:212px;">Slides should be labeled with a &#34;+&#34; and positively charged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Supplement 1</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">17502048</td>
			<td style="width:212px;">N-2 supplement (100x)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Supplement 2 w/o Vitamin A</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">12587010</td>
			<td style="width:212px;">B-27 supplement (50x), minus vitamin A; multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue-Tek Cryomold</td>
			<td style="width:123px;">Sakura, NL</td>
			<td style="width:119px;">4565</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:123px;">Sakura, NL</td>
			<td style="width:119px;">4583</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A1388,0500</td>
			<td style="width:212px;">Multiple suppliers multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TUJ1</td>
			<td style="width:123px;">Sigma-Aldrich, US</td>
			<td style="width:119px;">T2200</td>
			<td style="width:212px;">&#946;-Tubulin III (rabbit polyclonal)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TUNEL assay</td>
			<td style="width:123px;">Promega, US</td>
			<td style="width:119px;">G3250</td>
			<td style="width:212px;">DeadEnd Fluorometric TUNEL system; multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween 20 for molecular biology</td>
			<td style="width:123px;">AppliChem</td>
			<td style="width:119px;">A4974,0500</td>
			<td style="width:212px;">Multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">waterproof sheet</td>
			<td style="width:123px;">BEMIS company, inc.</td>
			<td style="width:119px;">PM996</td>
			<td style="width:212px;">Parafilm &#8220;M&#8221;; multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Y-27632&#160;</td>
			<td style="width:123px;">Selleckchem.com</td>
			<td style="width:119px;">S1049</td>
			<td style="width:212px;">ROCK-inhibitor (Y-27632 2HCL); multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#946;-mercaptoethanol</td>
			<td style="width:123px;">Gibco, US</td>
			<td style="width:119px;">31350010</td>
			<td style="width:212px;">2-mercaptoethanol (50 mM); multiple suppliers</td>
		</tr>
	</tbody>
</table>

generation of ipsc-derived human brain organoids to model early neurodevelopmental disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

脑类器官的生成需要至少三周连续培养（ 图1A）的。为了实现可重复的结果，我们建议，研究人员记录了每一步，更重要的，避免了关于培养基成分，时间点和细胞处理任何改变。在这里，我们给出了如何在关键的里程碑，是为了在实验结束时获得足够的质量达到类器官评估总结。神经球在96孔板的形成应该是从第4天的神经球能够识别在每个孔的底部（ <strong...

Watch this Scientific Journal Video about Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders at JoVE.com

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

源自iPSC的人脑组织体的一代新型早期神经发育障碍

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

generation-ipsc-derived-human-brain-organoids-to-model-early

Research

JoVE Journal

Developmental Biology

19.6K 次观看.  University of Cologne. Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described. 

视频: Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

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 ...

科研

发育生物学

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

This article details the methods that are used to expand human fetal brain neural stem cells in culture, as well as how to differentiate them into various neuronal subtypes and astrocytes, with an emphasis on the use of neural stem cells to study Zika virus infection.

人胎脑神经干细胞 Zika 病毒感染的免疫细胞化学分析

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent endothelial progenitors (EPs), which can differentiate into the cell types necessary to assemble mature, functional vasculature, have been derived from both embryonic and induced pluripotent stem cells. However, these cells have not been rigorously evaluated in three-dimensional environments, and a quantitative measure of their vasculogenic potential remains elusive. Here, the generation and isolation of iPSC-EPs via fluorescent-activated cell sorting are first outlined, followed by a description of the encapsulation and culture of iPSC-EPs in collagen hydrogels. This extracellular matrix (ECM)-mimicking microenvironment encourages a robust vasculogenic response; vascular networks form after a week of culture. The creation of a computational pipeline that utilizes open-source software to quantify this vasculogenic response is delineated. This pipeline is specifically designed to preserve the 3D architecture of the capillary plexus to robustly identify the number of branches, branching points, and the total network length with minimal user input.

Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells&#8217; vasculogenic potential are described.

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

小鼠肝外胆管有机体的产生

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

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

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

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

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

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

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

多细胞人类原发性子宫内膜器官的生成

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

生成基于人类 iPSC 的血脑屏障芯片

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...