我们感谢伊丽莎白Benevides校对文章。感谢Drs。 John Hussman和Gene Blatt进行了有益的讨论和评论。

神经祖细胞的产生<ol><li> 在补充有小分子的人iPSC培养基中，在γ-照射的小鼠胚胎饲养细胞（MEF）细胞层上，将来自成纤维细胞和PBMC的hiPSC保持在6孔板中（参见材料表）。 注:日常保养是以前报道的程序1，16修改后的版本。 <ol><li>在6孔组织培养级板的每个孔上以600μL/孔推荐的培养基（以下制造商的方案）将6×10 5个 MEF板铺板。 </li><li> 48小时后，加入hiPSCs，用300μL/孔hiPSC培养基取代MEFs培养基1 h。 </li><li>在37℃水浴中迅速解冻hiPSC，并逐渐缓慢加入1mL hiPSC培养基。将hiPSC和培养基转移到15 mL锥形管中。加入培养基使总体积达到5 mL。以129 xg离心4分钟，吸出超新星在1:1,000浓度的ROCK抑制剂的1mL hiPSC培养基中轻轻重新悬浮沉淀。 </li><li>板中的细胞悬浮液（通常在30万个细胞/孔接种）到MEF细胞层，并在37℃，5％CO 2，95％湿度下孵育。 </li><li>使用光学显微镜密切监测iPSC培养。 48小时后，去除具有不均匀边缘的培养物，在光学显微镜下生长成为囊状或显示为黄棕色，以最小化自发分化。 注意:7 - 10天后，应形成hiPSC菌落。殖民地应该圆形，边界清晰，细胞密度均匀，没有松散包装的不均匀细胞。 </li><li>手动选择圆形hiPSC菌落，以清除自发分化细胞的培养。将它们放置在新鲜的MEF层上来扩展菌落。展开1:3，每7天，当接近融合的文化和冻结1，16。 </li></ol></li><li>在扩增和冷冻细胞（用于备用）后，在板上生长hiPSC菌落直至达到50-75％汇合。 </li><li>电镀后7天，使用温和的酶处理（用300μL酶溶液5-10分钟）和温和研磨（用1,000μL移液管2-4次）收获并制备hiPSC用于神经前体细胞分化。 </li><li>以129 xg离心细胞悬浮液4分钟，吸出上清液，并将沉淀重悬于5 mL人iPSC培养基中。将细胞悬浮液转移到0.1％明胶包被的5cm细胞培养皿中，在37℃下1小时以消除MEF并使hiPSC产率最大化。将培养基（非贴壁细胞）转移到另外5厘米明胶包被的培养皿中。 </li><li>使用转移吸管将非贴壁细胞转移到15 mL离心管中。用3 mL培养基轻轻冲洗平板，并加入15 mL管中。 </li><li>将细胞悬浮液以129 xg离心4分钟，吸出超级细胞将其沉淀在培养基中。使用自动细胞计数器确定细胞计数。在96孔低粘附V底板中板9,000个细胞/孔。 </li><li>离心板163 xg 3分钟。在37℃，95％湿度和5％CO 2下孵育该板。使用移液管，每隔一天更换50％的培养基，取出一半的培养基，并用新鲜的化学成分分化培养基1（DM1; 图1 ; 材料表）替换14天。 </li></ol> 2.无血清胚胎体（SFEB）诱导（ 图2 ） <ol><li>在加入聚集体之前，将40μm细胞培养插入物置于6孔板中，并加入1 mL DM1培养基至少1 h。 </li><li> 在第14天，使用200μL宽嘴尖将20μL培养基的聚集体转移到40μm细胞培养物插入物上，并将培养基改变为DM2。 <br/&gt;注意:细胞培养插入物是位于孔底部稍微偏离并由悬浮在塑料框架上的聚四氟乙烯膜组成的专用插入物。该膜是生物相容的，可以有效地维持营养和氧气输送到放置在顶部的SFEB。它们通常与来自小鼠或大鼠7，17，18取海马脑切片培养物使用。 <ol><li>将4-6个聚集体转移到一个细胞培养插入物，并允许聚集体之间的足够的空间。使用精细的吸头（ 例如 200μL提示）尽可能多地除去多余的溶液。 注意:SFEB可以短暂的干扰SFEB，因为它们将作为从SFEB周围移除的溶液移动到细胞培养插入物上。 </li><li>在37℃，95％湿度和5％CO 2的 1mL DM2中维持培养物。使用移液器，更换75％每隔一天中间除去四分之三的培养基，并用四分之三的新鲜培养基代替。例如，移除750μL培养基，加入750μL新鲜培养基。 </li></ol></li><li>培养14天后，每隔一天切换至含有75％培养基的DM3培养基，并持续16天。 </li><li>为了维持超过30天的细胞培养插入物的SFEB，每隔一天更换培养基60天，90天和120天。 注意:SFEBs的直径将增长至约1,000μm，通常为100-150μm厚1 。 </li></ol> 3.确定SFEB组成<ol><li>使用200μL宽口吸头，轻轻移液培养基，从插入物中分离SFEB。要转移SFEB，请使用宽口吸头将其轻轻吸入吸头。将SFEB装入移液管末端后，轻轻转移至12孔板中，用300μL磷酸盐缓冲盐水洗涤（PBS）。 注意:SFEB将悬浮在12孔板中的溶液中。这对于允许固定剂，封闭溶液和抗体的完全渗透是重要的。如果使用多个SFEB进行染色，则可以在12孔板中每孔加入10个SFEB。这将节省解决方案和抗体。 </li><li>在室温下将SFEB固定在300μL/孔4％多聚甲醛中30-45分钟。用300μLPBS洗涤固定的SFEB两次，每次洗涤3-5分钟。 注意:处理多聚甲醛时，穿戴适当的个人防护装备。 注:为探讨以免疫标记来确定成熟度，细胞类型等。在这项研究中鸡TUJ1在1使用标记的神经元:500，为其他标记，请见表2和参考文献1，16。 </li><li>渗透并用PBS中的0.1％Triton-X100和10％正常驴血清（封闭溶液;300μ; L /孔）在室温下1小时。 </li><li>去除阻断溶液，并在封闭溶液中用300μL一抗处理SFEB。在4℃下孵育过夜。在含有0.1％Triton-X的300μLPBS中洗涤SFEB三次（每次洗涤3-5分钟）。 </li><li>在阻断溶液中制备1:1000的二抗。在室温下用二次抗体孵育SFEB 1小时。用300μLPBS洗涤SFEB，3次，每次3-5分钟。 注意:在这个阶段，SFEB可以准备成像。 </li><li>为了成像，使用宽孔移液管将SFEB移液到载玻片上。使用温和的吸力去除SFEB周围的多余溶液。 注意:对于类似的染色条件，可以将多个SFEB放置在同一张载玻片上。 </li><li>在SFEB上添加一滴安装介质，放置玻璃盖玻片，轻轻按下以确保没有气泡。允许安装介质硬化并进行成像和定量（步骤3.8）。 <br/&gt;注意:安装介质硬化后，SFEB可以进行成像。 </li><li>通过利用穿过SFEB关注区域（ROI）的多个区域，并使用Z堆栈通过在一个标准共焦显微镜每个ROI具有最大投影图像组合，如参考文献1中 ，16中所述进行细胞定量。 注:全SFEBs可以使用基于图像的拼接（详见参考文献1，16）进行量化。由于SFEB薄至约100μm，组织中光的散射最小，因此不需要双光子显微镜。这个协议与成纤维细胞衍生iPSC的较早的用途制备的SFEB命运映射揭示复杂多样的结构与相似CGE和前端脑命运1,16转录身份的interneurons的亚群。 </sup&gt; </li></ol>记录SFEB中的神经元活动<ol><li>根据制造商的说明准备12孔MEA板。 </li><li>在无菌条件下用无菌H 2 O洗涤MEA板3次5分钟以进行清洁。用75％乙醇洗涤5分钟，然后用100％乙醇洗涤，对板进行灭菌。 </li><li>烘烤板在烘箱中在50℃下倒置4-5小时以完成灭菌过程。 </li><li>向每个孔中加入500μL的0.2％聚乙烯亚胺溶液（PEI），并在室温下孵育1小时。吸PEI和洗涤各孔用无菌蒸馏水H 2 O.空气干燥4倍在引擎盖过夜。 </li><li>在L15培养基中制备20μL/ mL层粘连蛋白溶液，并将10μL层粘连蛋白加入每个孔的中心。 注意:请勿涂覆周围的参考电极和接地电极。 </li><li>向周围的水库中加入无菌dH 2 O以防止介质或层粘连蛋白蒸发。在37℃孵育板1小时。 </li><li>将SFEB（见第1部分和第2部分）通过轻轻地将其吸入广泛的吸液管尖端并转移到层粘连蛋白包被的MEA板上。向每个孔中加入200μLDM2培养基，并在37℃，95％湿度和5％CO 2下孵育过夜。 </li><li>在24小时后再加入200μL培养基，并在37℃，95％湿度，5％CO 2下回收30分钟，然后再读板。 </li><li>将MEA板置于读板器中（预热至37℃）以记录神经元活动。使用相关的MEA录制软件记录活动10分钟。详见参考资料19 。 </li><li>使用统计分析软件生成原始数据 - 连续流和神经元活动的栅格图19 。 注意:MEA录音是在SFEB电镀7天后进行的。对于这项研究，记录采取10分钟以检测网络爆发活动，con连续痕迹和光栅图。 SFEB可以在MEA板材上长期保持，并且可以使用环境条件在更长的时间内记录（ 图6 ）。 </li></ol>

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作者没有竞争的经济利益。

这里描述的方案提供了将hiPSC源区分为重现前额叶皮质早期发育阶段的3-D结构的条件。该过程产生可以询问电生理学的结构，同时也适用于显微镜。 SFEB的最终形态类似于器官型脑切片培养物的形态，并允许高质量的详细共聚焦成像。该方案可以成功地从成纤维细胞和外周血单核细胞衍生的hiPSC中产生SFEB。由外周血单个核细胞来源的hiPSCs制成的SFEB表现出与从成纤维细胞衍生的hiPSC产生的形态特征相...

我们以前报告了由患者衍生的人类多能干细胞（hiPSC）产生的3-D模型系统，其概括了早期皮层网络发展的某些方面1 。此3-D模型，无血清胚状体（SFEB），改善了以前的简单聚合的hiPSC模型2,3。工作的一个增长的身体被显露像我们SFEBs 3-D结构，神经发育的近似方面在体内和在比2维（2-D）观察到更早的时间点通常观察到的/单层的hiPSC模型4,5。初步研究集中在3-D体的自组织复杂性，而不表现出其生理复杂性2 。 本文描述的方案已用于源自成纤维细胞的未分化的hiPSC<html>外周血单核细胞（PBMCs）。这些细胞维持在γ照射的小鼠胚胎喂养器（MEFs）上。将这些hiPSC菌落手工清洗自发分化的细胞，酶促收获，并重新悬浮于含Rho-Kinase抑制剂Y-27632（ROCKi）的培养基中。将未分化的hiPSC在转移到96孔低粘附性V底板之前进行离解和离心。电镀后，使用双SMAD抑制（SB431542和LDN193189以及dickkopf 1（DKK-1））启动神经诱导，以驱动前额叶前脑神经元命运谱系6 。 14天后，将SFEB转移到6孔板中的细胞培养插入物中。一旦转移，圆形SFEB开始扩散和稀释，同时维持局部网络连接，如使用类似的细胞培养插入物1的海马器官切片培养物制备中常见的那样，ss ="xref"&gt; 7。 以这种格式使用基于SFEB的3-D平台适用于可以使用基于细胞的生理测定例如钙成像或电生理测定（例如单细胞记录或多电极阵列（MEA））来询问的皮层网络的有效生产） 1 。虽然3-D系统具有早期皮层发育的标志，但其他研究已经表明，这些3-D体可能需要更长的孵育时间，以允许人体组织发育的本质上较慢的速度8 。该SFEB协议成功地从未分化的hiPSC产生3-D SFEB，其捕获皮质早期发育的方面。 SFEBs在神经系统疾病中模拟网络畸变的潜力是该系统的优势。源自患者组织的hiPSC可以生长成受屁股的神经系统的细胞与细胞生物学有关的ays以及伴随的基因表达。人类iPSC被用来确定大群体的个体的遗传谱与不同神经障碍具有复杂的病因，如孤独症谱系障碍（ASD），精神分裂症9，Rett综合症10，和阿尔茨海默氏病11，12。直到最近，iPSC模型通常是单层制剂，虽然精通评估分子相互作用，但在破译体内发现的复杂细胞相互作用方面是不够的。动物模型已经成为重建整个器官平台的默认替代品。这些动物模型受到研究结果差的翻译困扰，复制由大型遗传筛选研究确定的人类遗传图谱的能力有限。因此，iPSC开发的3-D系统增加了人类所需的复杂层次疾病建模13，14。用于3D的hiPSC平台下一个步骤是，以适应使用基于细胞的测定筛选15高通量的大规模要求。

<table border="0" cellpadding="0" cellspacing="0" width="1173">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:573px;">SFEB Neuronal Differentiation Cell culture Media. Reagents. Components&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:164px;"></td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">STEMdiff Neural Induction Medium (hiPSC Media)</td>
			<td>STEMCELL Technologies</td>
			<td>0-5835</td>
			<td>250ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">PluriQ ES-DMEM Medium (MEF Media)</td>
			<td>GlobalStem</td>
			<td>GSM-2001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DM1 Media Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">D-MEM/F-12 (1X), Glutamax liquid, 1:1</td>
			<td>Invitrogen</td>
			<td align="right">10565018</td>
			<td>385ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Knockout Serum Replacement</td>
			<td>Invitrogen</td>
			<td align="right">10828028</td>
			<td>20% 100ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td align="right">15140122</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glutamax 200mM</td>
			<td>Invitrogen</td>
			<td align="right">35050061</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MEM Non-Essential Amino Acids Solution 10 mM (100X), liquid</td>
			<td>Invitrogen</td>
			<td align="right">11140050</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">2-Mercaptoethanol (1,000X), liquid</td>
			<td>Invitrogen</td>
			<td align="right">21985023</td>
			<td>900ul</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DM2 Media Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">D-MEM/F-12 (1X), Glutamax liquid, 1:1</td>
			<td>Invitrogen</td>
			<td align="right">10565018</td>
			<td>500ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glutamax 200mM</td>
			<td>Invitrogen</td>
			<td align="right">35050061</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td align="right">15140122</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">N-2 Supplement (100X), liquid</td>
			<td>Invitrogen</td>
			<td align="right">17502048</td>
			<td>10ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DM3 Media Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">NEUROBASAL Medium (1X), liquid</td>
			<td>Invitrogen</td>
			<td align="right">21103049</td>
			<td>500ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">B-27 Supplement Minus Vitamin A (50X), liquid</td>
			<td>Invitrogen</td>
			<td align="right">12587010</td>
			<td>10ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glutamax 200mM</td>
			<td>Invitrogen</td>
			<td align="right">35050061</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td align="right">15140122</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Small Molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thiazovivin</td>
			<td>Stemgent</td>
			<td>04-0017</td>
			<td>2uM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">SB431542</td>
			<td>Stemgent</td>
			<td>04-0010-10</td>
			<td>1:1000 (10uM)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dorsomorphin</td>
			<td>Stemgent</td>
			<td>04-0024</td>
			<td>1uM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">LDN-193189</td>
			<td>Stemgent</td>
			<td>04-0074-10</td>
			<td>250nM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Y27632 (ROCKi)</td>
			<td>Stemgent</td>
			<td>04-0012-10</td>
			<td>10uM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Recombinant Protiens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DKK-1</td>
			<td>Peprotech</td>
			<td>120-30</td>
			<td>200ng/ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Components/Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Cell Culture inserts 0.4uM, 30mm Diameter</td>
			<td>Millicell</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mouse Embryonic Fibroblasts</td>
			<td>GlobalStem</td>
			<td>GSC-6301G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">96 well V bottom w/Lids</td>
			<td>Evergreen</td>
			<td>222-8031-01V</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">StemPro Accutase Cell Dissociation Reagent</td>
			<td>ThermoFisher</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">TritonX-100</td>
			<td>ThermoFisher</td>
			<td align="right">85111</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher</td>
			<td align="right">10010023</td>
			<td>500 mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Normal Donkey Serum</td>
			<td>Jackson Labs</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Leibovitz&#39;s L-15 Medium</td>
			<td>ThermoFisher</td>
			<td align="right">11415114</td>
			<td>500 mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DRAQ5 (Nuclear Marker)&#160;</td>
			<td>ThermoFisher</td>
			<td>65-0880-96</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MEA Plates</td>
			<td>Axion Biosystems</td>
			<td>M768-GL1-30Pt200</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">6 well flat bottom</td>
			<td>Falcon</td>
			<td align="right">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nestin</td>
			<td>Millipore</td>
			<td>MAB5326</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brn-2</td>
			<td>Protein tech</td>
			<td>14596-1-AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VGLUT1&#160;</td>
			<td>Synaptic Systems&#160;</td>
			<td>135 303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pax6</td>
			<td>abcam</td>
			<td>ab5790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calretinin&#160;</td>
			<td>abcam</td>
			<td>ab702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calbindin</td>
			<td>abcam</td>
			<td>ab11426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoupTFII</td>
			<td>R&#38;D Systems</td>
			<td>PPH714700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nkx 2.1</td>
			<td>abcam</td>
			<td>ab12650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tuj1</td>
			<td>abcam</td>
			<td>ab41489</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reelin</td>
			<td>Millipore</td>
			<td>MAB5364</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tbr1</td>
			<td>Millipore</td>
			<td>MAB2261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NFH</td>
			<td>Dako</td>
			<td>M0762</td>
			<td></td>
		</tr>
	</tbody>
</table>

developing hipsc derived serum free embryoid bodies for the interrogation of 3-d stem cell cultures using physiologically relevant assays

虽然已经使用hiPSC开发了许多体外疾病模型，但一个限制是这些二维（2-D）系统可能不代表携带疑似疾病变异的受影响个体的基础细胞结构和功能复杂性。传统的二维模型仍然是体内结构的不完整表现，并没有充分地捕捉大脑的复杂性。因此，现在需要更多的基于3-D hiPSC的模型，其可以更好地概括在体内系统中看到的细胞相互作用和功能。 在这里，我们报告了一种基于无血清胚状体（SFEB）从未分化的hiPSC开发3-D系统的方案。这种3-D模型反映了开发的腹侧新皮层的各个方面，并且允许研究活体神经细胞和完整组织的整合功能，例如迁移，连通性，通讯和垫uration。具体来说，我们证明使用我们的方案的SFEB可以使用生理相关和高含量的基于细胞的测定法进行询问，如钙成像和多电极阵列（MEA）记录，而不进行冷冻切片。在MEA记录的情况下，我们证明SFEB在长期培养期间增加了穗活动和网络爆发活动。该SFEB协议提供了一个强大和可扩展的系统，用于研究三维模型中网络形成的研究，捕捉早期皮质发育的方面。

使用我们的技术生长的SFEB产生具有形态学特征的组织，其类似于具有广泛的Tuj1阳性神经元以及神经祖细胞的早期发展的皮质脑室下区域（ 图3A ）。在SFEB的外层和内层观察到许多发育中的皮质玫瑰花结（ 图3B ）。 SFEB的外缘类似于含有神经元神经元的显影皮层。 Brn2的表达支持这一表达，Brn2是最外侧皮质层中神经祖细胞的标志...

Watch this Scientific Journal Video about Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays at JoVE.com

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

在这里，我们报告了一种由称为无血清胚状体（SFEB）的人诱导多能干细胞（hiPSCs）开发三维（3-D）系统的方案。这种3-D模型可以像器官切片培养物一样用于模拟人类皮质发育和生理学询问发展中的神经回路。

开发HiPSC衍生的无血清胚胎体用于使用生理相关测定的3-D干细胞培养物的询问

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

developing-hipsc-derived-serum-free-embryoid-bodies-for-interrogation

Research

JoVE Journal

Neuroscience

8.8K 次观看.  The Hussman Institute for Autism. 在这里，我们报告了一种由称为无血清胚状体（SFEB）的人诱导多能干细胞（hiPSCs）开发三维（3-D）系统的方案。这种3-D模型可以像器官切片培养物一样用于模拟人类皮质发育和生理学询问发展中的神经回路。 

视频: Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

科研

神经科学

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

神经母细胞含量:从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

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

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

生物神经毒素在茎的网络文化功能评估细胞衍生的中枢神经系统神经元

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

使用克隆测定神经干细胞的计数。

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.