Name: protocol for the differentiation of human induced pluripotent stem cells into mixed cultures of neurons and glia for neurotoxicity testing
Uploaded: 2017-06-09T14:00:00.000+00:00
Duration: 9 min 2 s
Description: Watch this Scientific Journal Video about Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing at JoVE.com

作者要感谢Marc Peschanski博士（I-Stem，法国埃夫里）提供IMR90-hiPSC; Giovanna Lazzari博士和Silvia Colleoni博士（Avantea srl，Cremona，意大利）; Simone Haupt博士（德国波恩大学）; Tiziana Santini博士（意大利罗马技术研究所）提供关于免疫荧光染色评估的建议; Benedetta Accordi博士，Elena Rampazzo博士和Luca Persano博士（意大利帕多瓦大学）为RPPA分析和抗体验证做出了贡献。资金来源:这项工作得到欧盟资助项目"SCR＆To​​x"（拨款协议第266753号）的支持。

人诱导多能干细胞（hiPSC）扩增注意:在含有mTeSR1 5x补充剂的mTeSR1培养基（根据制造商的说明书制备;板〜100个菌落片段/ 60-mm培养皿）的存在下，hiPSC可以在合适的蛋白质混合基质上培养。当hiPSC菌落达到合适的大小（参见图2A中的菌落实例）时，如下所述（每周一次）传代细胞。 <ol><li> 用hESC合格的基底膜基质（以下称为"合格基质"）或任何其他合适的蛋白质底物涂层。 <ol><li>在冷的1.5 mL试管和冷的5 mL或10 mL移液管中，将合格的基质（见材料表）存放在-80°C的200μL等分试样中。 </li><li>在传代之前，在冰上解冻200μL合格的基质。 </li><li>在20 m稀释200μL合格基质L的DMEM / F12培养基（1:100稀释）。 </li><li>用该溶液（5mL /皿）涂上60mm培养皿。 </li><li>将包衣的培养皿在37℃孵育至少1小时。 </li></ol></li><li> HiPSC菌落片段冷冻保存和解冻 <ol><li>切割hiPSC菌落后（参见步骤1.3的hiPSC菌落切割程序），轻轻慢慢地将hiPSC菌落片段重悬于干细胞冷冻培养基，〜100个片段/250μL（参见材料表）。 </li><li>将菌落碎片等分于合适的小瓶中以进行冷冻保存（250μL/小瓶）。 </li><li>将小瓶放入装有2-丙醇的容器中，并将容器放置在-80℃至少2小时，最多2周。 </li><li>将小瓶转移到液氮罐的气相中。 </li><li>要重新开始培养，在37℃的水浴中解冻1个冷冻小瓶。 </li><li>在7 mL预热的完整hiPSC中轻轻收集hiPSC菌落片段培养基（参见材料表 ），使用1,2或5mL移液管在15mL管中。 </li><li>将hiPSC菌落碎片以130×g离心3分钟。 </li><li>取出上清液，并使用1,2或5 mL移液管轻轻将hiPSC菌落碎片重悬于1 mL完整的hiPSC培养基中。 </li><li>进一步稀释3或4 mL完整的hiPSC培养基中的细胞悬液。 </li><li>在合格的基质涂覆的60mm培养皿（〜100片/碟;如步骤1.1中所述包衣的盘子）上放置hiPSC菌落碎片。 </li><li>在37℃和5％CO 2下孵育hiPSC。 </li><li>每天进行一次中等的改变。 </li></ol></li><li> 传播hiPSC菌落 注意:未分化的hiPSC应该是圆形的，具有大的核仁和没有丰富的细胞质。未分化菌落的特征应该是平坦且紧密堆积的形态。只有未分化的菌落（约1毫米，n直径）应进行切割以进一步传送。 <ol><li>使用具有30G针或任何其他市售工具的1-mL注射器切割约200μm×200μm的正方形的干细胞集落（参见材料表）。在室温下在层流柜中以4倍放大率使用立体显微镜。 </li><li>使用200μL移液管，从培养皿表面分离菌落碎片，轻轻移取底下的培养基以提起碎片。 </li><li>将菌落碎片（〜100片）转移到装有4mL完整hiPSC培养基的合格基质DMEM / F12涂覆板（参见材料表;如步骤1.1所述包衣）。 </li><li>在37℃和5％CO 2下孵育新板。 </li><li>每天进行一次全面的介质变化，并使用4倍和10倍放大倍数的相差显微镜检查菌落的形态。 </li></ol></li></ol> HiPSC的差异混合神经元和胶质细胞注意:程序大约需要28天才能完成，主要步骤如图1 （上半部分）所述。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55702/55702fig1.jpg" /> 图1:神经元分化方案的示意图。 （上部）IMR90-hiPSC菌落可以切成片段形成胚状体（EB）。 体外培养 2天后，将EBs铺在层粘连蛋白或标准基质包被的培养皿上，并在神经上皮诱导培养基（NRI）培养基中培养，产生神经外胚层衍生物（玫瑰花结，这里染色巢蛋白（绿色）和β -III-微管蛋白（红色））。玫瑰果可以在层粘连蛋白或标准基质包被的培养皿上解离，收集，复制，并进一步分化成成熟神经元（NF200，红色）和胶质细胞（GFAP，绿色）细胞在神经元分化（ND）培养基存在下。 （下部）玫瑰花源衍生的NSCs（巢蛋白，红色）可以在存在神经诱导（NI）培养基的情况下扩增，冷冻保存，或在ND培养基存在下进一步分化，形成混合神经元（NF200，绿色）和胶质细胞GFAP，红色）培养。 <a href="http://ecsource.jove.com/files/ftp_upload/55702/55702fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol><li> 胚胎体（EB）的生成（第0→1天） 注意:此过程需要良好的手动技能和精度。 HiPSC菌落片段应具有相同的大小，以在后续步骤中获得均质的胚状体（EB）。应该丢弃形态上分化的菌落（具有大细胞质级分和小核仁）。 <ol><li>在切割未分化的hiPSC菌落（约1毫米直径）之前，先刷新hiPSC培养基（3mL / 60mm培养皿）在无菌条件下（如步骤1所述）参见图2A ）。 </li><li>使用具有30G针的1-mL注射器将未分化的菌落（ 如图2A和图2B所示 ）切割成约200μm×200μm的片段。在室温下在层流柜中以4倍放大率使用立体显微镜。 </li><li>使用200μL移液管，从培养皿表面分离菌落碎片，轻轻移液培养基，以提起碎片。 </li><li>使用1,2或5 mL移液管将所有分离的碎片和培养基转移到15 mL管中。 </li><li>用2 mL完整的hiPSC培养基冲洗盘子，以回收所有碎片。 </li><li>以112 xg离心1分钟。 </li><li>吸出上清液并轻轻地将片段重悬于5 mL完整的hiPSC EB培养基中（见材料表 ）。 </li><li>板公司在60mm超低附着培养皿（5mL / 60mm培养皿）中的Lony碎片。 </li><li>在37℃和5％CO 2下孵育培养皿过夜。 </li><li>第二天（第1天），使用1,2或5 mL移液管将EBs及其培养基收集在15 mL管中。 </li><li>以112 xg离心EB 1分钟。 </li><li>仔细吸取上清液，并使用1,2或5 mL移液管轻轻将EB悬浮于5 mL完整的hiPSC EB培养基中。 </li><li>将EB替换到新的60毫米超低附着培养皿（5毫升/ 60毫米培养皿）上。 </li><li>在37℃和5％CO 2下孵育培养皿过夜。 </li></ol></li><li> 在第1天，用基底膜基质（ 例如，基质胶，以下称为"标准基质"）或任何其它合适的蛋白质底物（ 例如层粘连蛋白）包衣。 <ol><li>在-80°C储存标准基质（参见材料表 ）使用冷的1.5毫升管和冷的5毫升或10毫升移液管的200μL等分试样。 </li><li>在冰上解冻200μL标准基质。 </li><li>在20mL DMEM / F12培养基（1:100稀释）中稀释200μL标准基质。 </li><li>用该溶液（5mL /皿）涂上60mm培养皿。 </li><li>将包衣的菜肴在37℃下孵育过夜。 注意:这些菜肴将用于平铺EB（约50个EB /碟）并产生神经上皮聚集体（玫瑰花结）;见步骤2.3。 </li></ol></li><li> 神经上皮聚集体（玫瑰花结）的产生（第2天→第7天） <ol><li>在第2天，从60 mm培养皿中取出标准基质涂层溶液（无需冲洗板），并填充5 mL /皿的完整神经上皮诱导培养基（NRI）;见材料表 。 </li><li>将漂浮的EB（从步骤2.1.14）转移到涂覆的盘（〜50个EB /皿）上，使用200μL移液管在立体mi以4倍放大倍率放置在层流柜中。 注意:选择均匀的中等大小的EB（直径约200-300μm）至关重要。神经外胚层分化过程中，太小的EBs可能不能很好地存活，而过大的EB往往会发生核心坏死。 </li><li>在37℃和5％CO 2下孵育。 </li><li>在第二天（第3天）之后，以10倍放大倍率在显微镜下检查菜肴，以确保所有的EB都被连接。 </li><li>使用完整的NRI培养基轻轻地进行中等变化。 </li><li>每天第二天将NRI培养基更换至第7天，当神经上皮聚集（玫瑰花结））应可见时。 </li><li>在第7天，如步骤2.2所述将标准基质（或层粘连蛋白）涂覆到任何所需的板或盘形式:96孔板（100μL/孔），24孔板（250μL/孔），12-孔板（500μL/孔），MEA芯片（用于电活动，1mL /单孔芯片）或60mm Petri dishes（4mL /皿）。 </li><li>在37℃和5％CO 2下将涂覆的板/皿孵育至少2小时。 </li></ol></li><li> 玫瑰花解离和神经元分化（第8〜28天） 注意:此过程需要良好的手动技能和精度。为了避免收集中胚层和内胚层细胞，只应外分泌和收集外胚层类玫瑰花结构。 <ol><li>在第8天，在无菌条件下，在立体显微镜下以10倍放大倍数将玫瑰花状结构切成碎片。使用带有30G针头的1 mL注射器。请注意，当与针接触时，花环容易从盘中脱离。 </li><li>使用200μL移液管完成玫瑰花瓣碎片的分离。 </li><li>将盘子转移到层流罩下方，并使用1,2或5毫升移液管将玫瑰花瓣碎片及其培养基收集到15 mL锥形管中。用2mL的NRI培养基冲洗该培养皿以恢复所有片段。 </li><li>以112 xg的速度旋转玫瑰花瓣碎片2分钟。 </li><li>吸出上清液。 </li><li>轻轻地将沉淀物重悬于1毫升1x DPBS（不含钙和镁）中，并使用1,000μL移液管轻轻吸取玫瑰花瓣碎片，使其部分解离。 </li><li>加入4 mL完整的NRI培养基，并用台盼蓝和自动细胞计数器计数细胞（参见材料表 ） 注意:将20μL细胞悬浮液稀释成20μL台盼蓝。如果细胞不能进入单细胞悬浮液，则可以省略该步骤。如果莲座碎片看起来没有完全解离，则从约50个EB / 60-mm皿中衍生的玫瑰花粉碎片可以重新悬浮在50mL的完整NRI培养基中并铺板，如表1所示 。 </li><li>从培养皿，平板和/或MEA碎片吸出标准基质（或层粘连蛋白）涂层溶液（来自步骤2.3）。7）。不要让他们干</li><li>根据研究计划（约15,000个细胞/ cm 2 ; 参见表1的电镀体积适应症）将细胞在完整的NRI培养基中培养。 </li><li>在37℃和5％CO 2下孵育板过夜。 </li><li>在第10天，使用完整的神经元分化培养基（ND）进行总介质变化;见材料表。 </li><li>每周两次刷新完整的ND培养基，直到第28天。 </li><li>描述神经元/神经胶质细胞衍生物，如步骤5所述（ 参见表2的一般验收标准）。 </li></ol></li></ol> 3. HiPSC衍生的神经干细胞（NSC）扩增和分化成混合神经元和胶质细胞注意:从玫瑰花序片段衍生的神经干细胞可以按照下述步骤进行扩展和维护（ 图1 ，下图 ）。这允许增加the用于分化和化学测试的细胞数。 <ol><li>用60 mL标准基质DMEM / F12涂层溶液涂抹60 mm培养皿（或T-25烧瓶），并在37°C和5％CO 2下孵育至少2 h（如步骤2.2所述）。 </li><li>将圆锥形15 mL管中的步骤1-2（见步骤2.4。），以112 xg的速度旋转2分钟。 </li><li>将沉淀轻轻重悬于5 mL神经诱导培养基（NI）中;见材料表。 </li><li>将细胞转移到标准基质涂层的60mm培养皿（或T-25烧瓶）上。 </li><li>在NI培养基存在下培养玫瑰花结的神经干细胞，每隔一天刷新培养基直至细胞达到汇合。 </li><li>汇合时，如以下步骤所述通过NSCs。 注意:每周一次通过NSCs;考虑使用新鲜包装的菜肴，烧瓶或盘子，这取决于研究计划。 </li><li>取出完整的NI培养基和gentl用DPBS（不含钙和镁）冲洗NSCs。 </li><li>向装有细胞的60-mm培养皿（或T-25烧瓶）中加入预先温热至37℃的1.5mL的0.05％胰蛋白酶-EDTA，并置于培养箱中1分钟。 </li><li>轻轻敲打盘（或烧瓶）以分离细胞。 </li><li>加入预先温热至37℃的1.5 mL胰蛋白酶抑制剂，并将细胞转移至15 mL管中。 </li><li>用等体积的NI培养基（1.5mL）冲洗培养皿（或T-25烧瓶），并将体积收集在相同的15-mL管中。 </li><li>将细胞以130×g离心3分钟。 </li><li>去除上清液，并使用1,000μL移液管轻轻将细胞重悬于1 mL完整的NI培养基中。 </li><li>进一步稀释3或4 mL完整的NI培养基中的细胞悬浮液，并使用台盼蓝和自动细胞计数器计数细胞。 </li><li>将NSCs以约50,000个细胞/ cm 2的密度从步骤3.1将NSCs平板化到60-mm培养皿（或T-25烧瓶）上。 </li><li&gt;每隔一天用完整的NI培养基进行总体介质变化。 </li><li>表征细胞是否存在神经元/神经胶质细胞衍生物，如步骤5所述。 注意:NSCs可以在完整的ND培养基中分化成神经元和神经胶质的混合培养物（如步骤2.4.11-2.4.13所述），每周二次清洁完整的ND培养基21天。 </li></ol> 4. HiPSC衍生的NSC冷冻保存和解冻注意:传代后，按照这个程序，NSCs可以冷冻并重新融化。 <ol><li>将离心的NSCs（来自步骤3.12）以130×g离心3分钟。 注意:细胞应在步骤3.14进行计数。 </li><li>轻轻和缓慢地重新悬浮在3×10 6 / mL冷冻介质中的NSCs（参见材料表 ）。 </li><li>将细胞等分于合适的小瓶中用于冷冻保存（约0.5mL = 1.5×10 6 /小瓶）。 </li><li>将小瓶放入容器中装入2-丙醇，将容器放在-80°C至少2小时，最长可达2周。 </li><li>将小瓶转移到液氮罐的气相。 </li><li>重新开始细胞培养，在37℃的水浴中解冻1个冷冻小瓶。 </li><li>使用1000μL移液管在15 mL管中轻轻收集7 mL预热的完整NI培养基中的细胞。 </li><li>将细胞以130×g离心3分钟。 </li><li>取出上清液，用1000μL移液管轻轻将细胞重悬于1 mL完整的NI培养基中。 </li><li>进一步稀释3或4 mL完整的NI培养基中的细胞悬浮液，并使用台盼蓝和自动细胞计数器计数细胞（注意:20μL台盼蓝稀释20μL细胞悬浮液;解冻后的存活率应≥80 ％）。 </li><li>以约50,000个细胞/ cm 2的密度将NSCs放置在涂覆的60-mm培养皿（或T25烧瓶）中。 </li></ol> CharahiPSC衍生的神经元和神经胶质细胞的整合注意:分化后，神经元和神经胶质衍生物可用不同的技术进行表征，如以下部分所述的技术。 <ol><li> 定量实时PCR（qPCR）分析10 <ol><li>将hiPSC菌落碎片，EB和/或NSCs以130×g离心3分钟。 </li><li>将细胞沉淀重悬在100μL冷RNA溶解缓冲液中，提供在合适的RNA提取试剂盒中。 </li><li>或者，通过吸出培养基直接从平板收集神经元/神经胶质衍生物，并向孔中加入冷RNA裂解缓冲液以收集细胞。 </li><li>按照制造商的说明隔离RNA。 </li><li>逆转录500纳克的总RNA使用合适的RNA套件到cDNA逆转录。 </li><li>使用适当的主混合物和底漆，重复使用qPCR反应套（见材料表 ）。 </li><li>记录荧光发射实时:45个循环，引物在60℃退火。 </li><li>将GAPDH和β-肌动蛋白的相对RNA量归一化为参考基因，并使用未分化的hiPSC或未处理的细胞进行校准条件（ΔΔCt方法）。或者，使用另一种合适的方法。 </li></ol></li><li> 免疫细胞化学和高含量成像（HCI） 6,11 <ol><li>在室温下用冷的4％多聚甲醛固定hiPSC菌落，NSCs和/或神经元/胶质细胞衍生物15分钟。 </li><li>轻轻洗涤1X PBS中的细胞，并将板在4℃下储存长达1个月。 </li><li>准备进行染色时，在室温下将渗透缓冲液（含有0.1％triton-X-100和3％BSA的1×DPBS）中的细胞透化15分钟。 </li><li>去除渗透液在室温下将细胞在封闭缓冲液（3％BSA / 1X DPBS）中孵育15分钟以防止抗体的非特异性结合。 </li><li>取出封闭缓冲液，并在含有合适的一抗的封闭缓冲液中于4℃孵育细胞过夜（参见材料表 ）。 </li><li>用1x PBS洗涤细胞3次。 </li><li>在含有荧光染料缀合的二级抗体的封闭缓冲液中，在室温下孵育细胞45分钟（参见材料表 ），用DAPI染料重新染色细胞核。 </li><li>使用合适的高含量成像平台（如果有的话）量化平均荧光强度和细胞类型的相对百分比（参见材料表 ）。 注意:为了确定荧光背景的强度水平，单独用二次抗体孵育一些细胞/孔。流动细胞分析活（不固定），和可以进行分化的hiPSC以评估PSC特异性标记物如SSEA4 的表达 （参见材料表 ）。按照制造商的说明书，可以使用市售的BCIP / NBT试剂盒分析未分化的hiPSC菌落碱性磷酸酶活性（参见材料表 ）。另外，如参考文献12（测试抗体列表，参见材料表 ）所述，可以进行反相蛋白质阵列（RPPA）测定和分析。 </li></ol></li><li> 电生理测量13 <ol><li>在完整的ND培养基（〜1×10 5个细胞/单孔MEA芯片）中，将衍生自玫瑰花簇上的解离的玫瑰花束片段（7DIV）或衍生自包被的多电极阵列上的玫瑰花簇（MEA;参见材料表 ）。 </li><li>在完整的ND培养基中分化细胞3周，清爽e媒体每周两次。 </li><li>在分化结束时，用层流罩下的半透膜密封MEA碎片，以保持培养物无菌以进行重复测量。 </li><li>用一个接地参考电极替换一个电极，允许从剩余电极进行记录。 </li><li>使用MEA放大器记录平均燃烧速率（MFR;峰值/分钟数），将集成温度过程控制调整到37℃和5％CO 2 。 </li><li>使用阈值限制-4.7σ（σ表示基础噪声的标准偏差）从MEA原始数据中检测峰值。 </li><li>使用合适的软件处理后记录数据。 </li></ol></li></ol>

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作者没有什么可以披露的。

这项工作描述了一个稳健和相对快速的方案，用于将IMR90-hiPSC分化为后有丝分裂神经元和神经胶质细胞。以前公布的基于hESC和hiPSC的神经元分化方案通常产生高百分比的神经前体25,26和大量神经元靶细胞27,28,29,30,31,32,33。类似地，本文描述的分化方案适用于产生GABA能，谷氨酸能和多巴胺能神经元细胞的不均匀培养物，以及神经胶质和离散比例的巢蛋白+细胞。谷氨酸能（〜35-42％）和GABA能（〜15-20％）神经细胞的存在表明这种文化具有前脑，皮质样特征，并且离散数量的多巴胺能神经元（〜13-20％）的存在也可能表明中脑特异性。此外，适度比例的巢蛋白+细胞的持久性可能适用于神经发生的研究和化学物质对神经干细胞的可能作用，其主要限于前脑34的海马区和脑室下区（SVZ）。进一步的免疫细胞化学和基因表达分析将有助于更好地定义分化细胞衍生物的区域特异性。 本文件描述的分化方案中的两个最关键的步骤是:（i）将hiPSC菌落切割成均质片段（其对于产生具有均匀大小的EB是至关重要的）和（ii）切割神经外胚层结构（玫瑰花结）用于NSC分化，这需要大量的手工技能并且精确地避免收集可能减少分化后获得的神经元和神经胶质细胞的比例的中胚层和内胚层细胞。 在扩展期间（作为未分化的集落或NSCs）和在所有分化步骤期间表征细胞的表型是至关重要的。特别是神经元/神经胶质细胞衍生物的基因和蛋白质表达谱应显示神经元相关信号通路的上调和激活，而多能性标记物的表达应降低。 EB和神经外胚层衍生物（玫瑰花结）的产生可能是手工挑战性的并且易于变异。因此，我们开发了用于扩增玫瑰花源衍生的NSCs的方案，并进一步分化为神经元/神经胶质细胞。 这种差异化方案的可能局限性主要是（i）d的百分比相对较低分化胶质细胞衍生物和（ii）缺乏成熟的神经元网络功能（如缺少突发所示）。此外，星形胶质细胞的特定亚群可以作为初级祖细胞或NSCs发挥作用。虽然在这种分化的细胞培养物中没有观察到巢蛋白/ GFAP双阳性细胞（数据未显示），但假设这些混合培养物中的GFAP +细胞是星形细胞祖细胞和星形胶质细胞。通过延长分化时间是可行的，星形胶质细胞的数量可能会增加，并且其形态可能会变得更加成熟，正如张氏组36,37的先前作品已经指出的那样。 在新的毒性试验范式中，生物通路的化学诱导扰动知识在评估化学逆境时是至关重要的。因此， 体外测试系统应该能够根据不良结局通路（AOP）的概念，将不良反应与信号通路的紊乱相关联。作为概念证明，鱼藤酮可用于评估Nrf2途径的激活，其涉及针对氧化或亲电应激的细胞防御38 ，氧化应激是与各种AOP相关的重要且常见的关键事件发育和成人神经毒性39 。 鱼藤酮应引起Nrf2通路的激活，Nrf2蛋白核转位可以表现，Nrf2靶酶（包括NQO1和SRXN1）的表达增加。已经发现，鱼藤酮诱导GFAP蛋白水平的剂量依赖性增加，表明星形胶质细胞活化40,41 。鱼藤酮也减少多巴胺能（TH + ）细胞的数量，这与previ一致显示鱼藤酮依赖性多巴胺能细胞死亡的体外和体内研究，因为这种类型的神经元对氧化应激特别敏感21,22,23 。 总之，这种hiPSC衍生的神经元和神经胶质细胞培养模型是评估导致产生Nrf2途径激活的氧化应激的化学物质的神经毒性作用的有价值的工具。由于该分化方案允许产生神经元细胞（GABAergic，dopaminergic，谷氨酸能神经元）和星形胶质细胞的混合培养物，所以它可能被证明适用于研究生理和病理学条件下的神经元和神经胶质之间的串扰，例如神经变性疾病例如帕金森病）。此外，大量NSCs的存在可能有助于评估化学物质对神经进展的可能影响已知它是化学诱导突变或病毒感染的主要靶标的参与者42 。

美国国家研究委员会报告1设想了一种新的毒性测试范例，其中监管毒性测试将从依赖于在动物中观察到的表型变化的方法转变为侧重于使用人类细胞的机械性体外测定的方法。多能干细胞（PSC）衍生物可代表癌细胞模型的替代品，因为所获得的细胞可能更接近人类组织的生理条件，并提供更多的相关工具来研究化学诱导的不良反应。最有希望进行毒性测试的两种主要类型的PSC培养物是人胚胎干细胞（hESCs）和人诱导多能干细胞（hiPSCs），目前广泛应用于基础研究和再生医学领域2,3 。现在可以利用这一专门知识来开发一类新的毒素gical 体外测试旨在确定涉及体内不良反应发展的扰动生理途径。然而，基于hESC的监管安全评估的测试方法将不可能被所有欧盟成员国和全世界的国家接受，因为可能存在道德问题，以及规范使用胚胎衍生细胞的各种国家立法政策。 hiPSCs具有类似于hESCs 4,5的特征 ，并且对于体外方法具有巨大潜力，用于鉴定治疗靶标以及用于安全性评估。此外，hiPSC技术减轻了有限供体池的限制以及与胚胎细胞相关的伦理问题。 hiPSC的一个主要挑战是证明这些细胞可以重复产生显着范围的毒理相关的细胞衍生物，具有人体组织典型的特征和反应。所选标记的预定水平通常用于表征分化过程后的细胞群体，并提供对分化过程的稳定性的洞察。 以前的作品评估了hiPSC产生神经元和神经胶质细胞混合培养物的适用性，并评估了鱼藤酮（线粒体呼吸复合物I的抑制剂）对Nrf2通路的激活的影响，Nrf2途径是抗氧化防御机制的关键调节剂许多细胞类型6,7 。 这项工作描述了用于将hiPSC分化成混合神经元和胶质细胞培养物的方案，提供了在神经元/胶质细胞分化时被激活的信号通路（基因和蛋白质水平）的细节。此外，这项工作显示了代表性的结果，证明了这一点hiPSC衍生的神经元和神经胶质细胞模型可用于评估用鱼藤酮进行急性（24小时）治疗诱导的Nrf2信号传导激活，允许评估氧化应激诱导。 IMR90成纤维细胞通过使用pMIG载体6的2种转录因子（Oct4和Sox2）的病毒转导，在I-Stem（法国）上重编程为hiPSC。也可以应用类似的hiPSC模型。下文描述的方案总结了hiPSC分化为神经干细胞（NSCs）的所有阶段，并进一步分为有丝分裂后神经元和神经胶质细胞的混合培养物（步骤1和2，还可参见EURL ECVAM DBALM网站，详细描述协议） 8 。 用于分离，扩增，低温保存和进一步将NSCs分化为混合神经元和神经胶质细胞的方案详见步骤3和4（也参见EURL ECVAM DBALM我们bsite详细说明此协议） 9 。步骤5描述了在承诺和分化的几个阶段期间可以进行的评估细胞表型鉴定的分析。

<table><tbody><tr><td>&lt;strong&gt;&lt;u&gt;Complete hiPSC medium:&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>mTeSR1 Basal Medium</td><td>Stem Cell Technologies</td><td>05851</td><td>(Step 1.2.6). Complete mTeSR1 is stable when stored at 2 - 8 &amp;deg;C for up to 2 weeks. 5x Supplements can be dispensed into working aliquots and stored at -20 &amp;deg;C. Use frozen aliquots within 3 months.</td></tr><tr><td>mTeSR1 5x Supplements</td><td>Stem Cell Technologies</td><td>05852</td><td></td></tr><tr><td>Matrigel hESC-qualified Matrix</td><td>Corning</td><td>354277</td><td>1:100 (Step 1.1). Thaw Matrigel on ice, prepare 200 &amp;mu;L aliquots and store them in -80 &amp;deg;C. For coating, dilute 200 &amp;mu;L aliquot in 20 mL of DMEM/F12 medium.</td></tr><tr><td>CryoStem Freezing Medium</td><td>Stemgent</td><td>01-0013-50</td><td>Freeze ~ 100 fragments/250 &amp;mu;L/vial (Step 1.2.1)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;&lt;strong&gt;hiPSC EB medium:&lt;/strong&gt;&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Knockout DMEM</td><td>Thermo-Fisher</td><td>10829-018</td><td>(Step 2.1.7)</td></tr><tr><td>Knockout Serum Replacement (KOSR)</td><td>Thermo-Fisher</td><td>10828-028</td><td>20% final concentration (Step 2.1.7)</td></tr><tr><td>Non-Essential Amino Acids</td><td>Thermo-Fisher</td><td>11140-035</td><td>(Step 2.1.7)</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo-Fisher</td><td>15140-122</td><td>50 U/mL final concentration (Step 2.1.7)</td></tr><tr><td>L-Glutamine 200 mM Solution</td><td>Thermo-Fisher</td><td>25030-081</td><td>2 mM final concentration (Step 2.1.7)</td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Thermo-Fisher</td><td>31350-010</td><td>50 &amp;micro;M final concentration (Step 2.1.7)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Complete neuroepithelial induction medium (NRI):&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo-Fisher</td><td>3133-038</td><td>(Step 2.3.1)</td></tr><tr><td>Non-Essential Amino Acids</td><td>Thermo-Fisher</td><td>11140-035</td><td>(Step 2.3.1)</td></tr><tr><td>N2 Supplement</td><td>Thermo-Fisher</td><td>17502-048</td><td>(Step 2.3.1)</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo-Fisher</td><td>15140-122</td><td>50 U/mL final concentration (Step 2.3.1)</td></tr><tr><td>Heparin Grade I-A, &amp;ge;180 USP units/mg</td><td>Sigma-Aldrich</td><td>H3149-100KU</td><td>2 &amp;micro;g/mL final concentration (Step 2.3.1)</td></tr><tr><td>bFGF</td><td>Thermo-Fisher</td><td>13256-029</td><td>20 ng/mL final concentration added before use (Step 2.3.1)</td></tr><tr><td>Matrigel Basement Membrane Matrix</td><td>Corning</td><td>354234</td><td>1:100 (Step 2.2). Thaw Matrigel on ice, prepare 200 &amp;mu;L aliquots and store them in -80 &amp;deg;C. For coating, dilute 200 &amp;mu;L aliquot in 20 mL of cold DMEM/F12 medium.</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td>1:100 (Step 2.2). Dilute in PBS 1x.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Complete Neuronal Differentiation medium (ND):&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Neurobasal Medium</td><td>Thermo-Fisher</td><td>21103049</td><td>(Step 2.4.11)</td></tr><tr><td>B-27 Supplements (50x)</td><td>Thermo-Fisher</td><td>17504044</td><td>(Step 2.4.11)</td></tr><tr><td>N2 Supplement</td><td>Thermo-Fisher</td><td>17502-048</td><td>(Step 2.4.11)</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo-Fisher</td><td>15140-122</td><td>50 U/mL final concentration (Step 2.4.11)</td></tr><tr><td>GDNF</td><td>Thermo-Fisher</td><td>PHC7045</td><td>1 ng/mL final concentration. Added before use. (Step 2.4.11)</td></tr><tr><td>BDNF</td><td>Thermo-Fisher</td><td>PHC7074</td><td>2.5 ng/mL final concentration. Added before use. (Step 2.4.11)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Neural induction medium (NI):&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo-Fisher</td><td>3133-038</td><td>(Step 3.3)</td></tr><tr><td>Non-Essential Amino Acids</td><td>Thermo-Fisher</td><td>11140-035</td><td>(Step 3.3)</td></tr><tr><td>N2 Supplement</td><td>Thermo-Fisher</td><td>17502-048</td><td>(Step 3.3)</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo-Fisher</td><td>15140-122</td><td>50 U/mL final concentration (Step 3.3)</td></tr><tr><td>Heparin Grade I-A, &amp;ge;180 USP units/mg</td><td>Sigma-Aldrich</td><td>H3149-100KU</td><td>2 &amp;micro;g/mL final concentration (Step 3.3)</td></tr><tr><td>B-27 Supplement (50x), minus vitamin A</td><td>Thermo-Fisher</td><td>12587010</td><td>(Step 3.3)</td></tr><tr><td>L-Glutamine 200 mM Solution</td><td>Thermo-Fisher</td><td>25030-081</td><td>2 mM final concentration (Step 3.3)</td></tr><tr><td>bFGF</td><td>Thermo-Fisher</td><td>13256-029</td><td>10 ng/mL final concentration. Added before use (Step 3.3)</td></tr><tr><td>EGF</td><td>Thermo-Fisher</td><td>PHG6045</td><td>10 ng/mL final concentration. Added before use (Step 3.3)</td></tr><tr><td>BDNF</td><td>Thermo-Fisher</td><td>PHC7074</td><td>2.5 ng/mL final concentration. Added before use (Step 3.3)</td></tr><tr><td>Defined Trypsin Inhibitor (DTI)</td><td>Thermo-Fisher</td><td>R007-100</td><td>Pre-warm at 37 &amp;deg;C. Add an equal amount of DTI to Trypsin-EDTA (Step 3.10)</td></tr><tr><td>Trypsin-EDTA (0.5%), no phenol red</td><td>Thermo-Fisher</td><td>15400054</td><td>1:10. Dilute Trypsin-EDTA in PBS 1x (without calcium and magnesium), pre-warm the solution at 37 &amp;deg;C (Step 3.8)</td></tr><tr><td>CryoStor cell cryopreservation medium</td><td>Sigma-Aldrich</td><td>C2874-100ML</td><td>(Step 4.2)</td></tr><tr><td>Trypan Blue (0.4%)</td><td>Sigma-Aldrich</td><td>T8154-100ML</td><td>multiple manufacturers/suppliers</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;TaqMan Probesets and reagents for gene expression analysis:&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RNAqueous-Micro kit</td><td>Thermo-Fisher</td><td>AM1931</td><td>(Step 5.1.6)</td></tr><tr><td>High Capacity cDNA Reverse Transcription Kits</td><td>Thermo-Fisher</td><td>4368814</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Thermo-Fisher</td><td>4369016</td><td></td></tr><tr><td>GFAP</td><td>Thermo-Fisher</td><td>Hs00909233_m1</td><td></td></tr><tr><td>MAP2</td><td>Thermo-Fisher</td><td>Hs00258900_m1</td><td></td></tr><tr><td>NQO1</td><td>Thermo-Fisher</td><td>Hs02512143_s1</td><td></td></tr><tr><td>SRXN1</td><td>Thermo-Fisher</td><td>Hs00607800_m1</td><td></td></tr><tr><td>HMOX1</td><td>Thermo-Fisher</td><td>Hs01110250_m1</td><td></td></tr><tr><td>GSR</td><td>Thermo-Fisher</td><td>Hs00167317_m1</td><td></td></tr><tr><td>PAX6</td><td>Thermo-Fisher</td><td>Hs01088112_m1</td><td></td></tr><tr><td>NES</td><td>Thermo-Fisher</td><td>Hs00707120_s1</td><td></td></tr><tr><td>GRIA1</td><td>Thermo-Fisher</td><td>Hs00181348_m1</td><td></td></tr><tr><td>GAP43</td><td>Thermo-Fisher</td><td>Hs00967138_m1</td><td></td></tr><tr><td>GABRA3</td><td>Thermo-Fisher</td><td>Hs00968132_m1</td><td></td></tr><tr><td>GABRA1</td><td>Thermo-Fisher</td><td>Hs00168058_m1</td><td></td></tr><tr><td>NR4A2</td><td>Thermo-Fisher</td><td>Hs00428691_m1</td><td></td></tr><tr><td>TH</td><td>Thermo-Fisher</td><td>Hs00165941_m1</td><td></td></tr><tr><td>GAPDH</td><td>Thermo-Fisher</td><td>Hs02758991_g1</td><td></td></tr><tr><td>ACTB</td><td>Thermo-Fisher</td><td>Hs99999903_m1</td><td></td></tr><tr><td>MAPT</td><td>Thermo-Fisher</td><td>Hs00902194_m1</td><td></td></tr><tr><td>SYP</td><td>Thermo-Fisher</td><td>Hs00300531_m1</td><td></td></tr><tr><td>NANOG</td><td>Thermo-Fisher</td><td>Hs04260366_g1</td><td></td></tr><tr><td>POU5F1 (OCT4)</td><td>Thermo-Fisher</td><td>Hs04195369_s1</td><td></td></tr><tr><td>SOX1</td><td>Thermo-Fisher</td><td>Hs01057642_s1</td><td></td></tr><tr><td>AFP</td><td>Thermo-Fisher</td><td>Hs00173490_m1</td><td></td></tr><tr><td>KRT18</td><td>Thermo-Fisher</td><td>Hs01941416_g1</td><td></td></tr><tr><td>NPPA</td><td>Thermo-Fisher</td><td>Hs00383230_g1</td><td></td></tr><tr><td>T</td><td>Thermo-Fisher</td><td>Hs00610080_m1</td><td></td></tr><tr><td>NCAM1</td><td>Thermo-Fisher</td><td>Hs00941821_m1</td><td></td></tr><tr><td>NR4A1</td><td>Thermo-Fisher</td><td>Hs00374226_m1</td><td></td></tr><tr><td>PHOX2A</td><td>Thermo-Fisher</td><td>Hs00605931_mH</td><td></td></tr><tr><td>PHOX2B</td><td>Thermo-Fisher</td><td>Hs00243679_m1</td><td></td></tr><tr><td>NARG2</td><td>Thermo-Fisher</td><td>Hs00973298_g1</td><td></td></tr><tr><td>SLC18A3</td><td>Thermo-Fisher</td><td>Hs00268179_s1</td><td></td></tr><tr><td>SLC5A7</td><td>Thermo-Fisher</td><td>Hs00222367_m1</td><td></td></tr><tr><td>ISL1</td><td>Thermo-Fisher</td><td>Hs00158126_m1</td><td></td></tr><tr><td>LHX3</td><td>Thermo-Fisher</td><td>Hs01033412_m1</td><td></td></tr><tr><td>TaqMan Human Protein Kinase Array</td><td>Thermo-Fisher</td><td>4418721</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Antibodies and reagents for immunostaining:&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B-III-tubulin (Tuj1)</td><td>Covance</td><td>MMS-435P</td><td>1:500 (Step 5.2.5). Other antibodies may also be used.</td></tr><tr><td>MAP2</td><td>Sigma Aldrich</td><td>M4403</td><td>1:500</td></tr><tr><td>NF200</td><td>Sigma Aldrich</td><td>N4142</td><td>1:1000</td></tr><tr><td>GFAP</td><td>Acris Antibodies GmbH</td><td>AP02002SU-N</td><td>1:500</td></tr><tr><td>Nestin</td><td>Sigma-Aldrich</td><td>N5413</td><td>1:200</td></tr><tr><td>synaptophysin (SYN)</td><td>Abcam</td><td>AB14692</td><td>1:200</td></tr><tr><td>Tau</td><td>Thermo-Fisher</td><td>MA5-12808</td><td>1:100</td></tr><tr><td>Nrf2</td><td>Abcam</td><td>AB62352</td><td>1:200</td></tr><tr><td>Keap1</td><td>Abcam</td><td>AB66620</td><td>1:200</td></tr><tr><td>sulfiredoxin1 (SRXN1)</td><td>Abcam</td><td>AB92298</td><td>1:200</td></tr><tr><td>NAD(P)H quinone oxidoreductase 1 (NQO1)</td><td>Abcam</td><td>AB2346</td><td>1:200</td></tr><tr><td>OCT4</td><td>Millipore</td><td>MAB4401</td><td>1:100</td></tr><tr><td>SSEA3</td><td>Millipore</td><td>MAB4303</td><td>1:100</td></tr><tr><td>Tra1-60</td><td>Millipore</td><td>MAB4360</td><td>1:250</td></tr><tr><td>Tyrosine hydroxylase (TH)</td><td>Millipore</td><td>AB152</td><td>1:200</td></tr><tr><td>Gamma-aminobutyric acid (GABA)</td><td>Sigma-Aldrich</td><td>A0310</td><td>1:100</td></tr><tr><td>Vesicular glutamate transporter 1 (VGlut1)</td><td>Abcam</td><td>AB72311</td><td>1:500</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>4% (4% formaldehyde can also be used)</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo-Fisher</td><td>14190144</td><td></td></tr><tr><td>Triton-X-100 Solution</td><td>Sigma-Aldrich</td><td>93443-100ML</td><td>0.1%</td></tr><tr><td>BSA 35%</td><td>Sigma-Aldrich</td><td>A7979-50ML</td><td>3.5%</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Cross Adsorbed Secondary Antibody, DyLight 594 conjugate</td><td>Thermo-Fisher</td><td>SA5-10040</td><td>1:500. (Step 5.2.7) Other fluorochrome-conjugated secondary antibodies may also be used. In this case, appropriate dilutions should be tested by the enduser.</td></tr><tr><td>Donkey anti-Mouse IgG (H+L) Cross Adsorbed Secondary Antibody, DyLight 488 conjugate</td><td>Thermo-Fisher</td><td>SA5-10166</td><td>1:500</td></tr><tr><td>Donkey anti-Goat IgG (H+L) Cross Adsorbed Secondary Antibody, DyLight 488 conjugate</td><td>Thermo-Fisher</td><td>SA5-10086</td><td>1:500</td></tr><tr><td>DAPI Solution (1 mg/mL)</td><td>Thermo-Fisher</td><td>62248</td><td>1:1000 (Step 5.2.7)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Antibodies for Reverse Phase Protein Array (RPPA):&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4E-BP1 (S65)</td><td>Abcam</td><td>AB81297</td><td>1:250 (Note after step 5.2.8)</td></tr><tr><td>Akt (T308)</td><td>Cell Signaling</td><td>9275</td><td>1:100</td></tr><tr><td>Akt (S473)</td><td>Cell Signaling</td><td>9271</td><td>1:100</td></tr><tr><td>AMPKalpha (T172)</td><td>Cell Signaling</td><td>2531</td><td>1:100</td></tr><tr><td>AMPKbeta1 (S108)</td><td>Cell Signaling</td><td>4181</td><td>1:100</td></tr><tr><td>ATF-2 (T71)</td><td>Cell Signaling</td><td>9221</td><td>1:100</td></tr><tr><td>c-Jun (S63)</td><td>Cell Signaling</td><td>9261</td><td>1:200</td></tr><tr><td>c-Jun (S73)</td><td>Cell Signaling</td><td>9164</td><td>1:200</td></tr><tr><td>c-Kit (Y719)</td><td>Cell Signaling</td><td>3391</td><td>1:250</td></tr><tr><td>CREB (S133)</td><td>Cell Signaling</td><td>9191</td><td>1:100</td></tr><tr><td>EGFR (Y1068)</td><td>Cell Signaling</td><td>2234</td><td>1:50</td></tr><tr><td>ErbB2/HER2 (Y1248)</td><td>Cell Signaling</td><td>2247</td><td>1:100</td></tr><tr><td>ERK 1/2, p44/42 (T202/Y204)</td><td>Cell Signaling</td><td>9101</td><td>1:2000</td></tr><tr><td>GSK-3alpha (S21)</td><td>Cell Signaling</td><td>9337</td><td>1:50</td></tr><tr><td>Jak1 (Y1022/1023)</td><td>Cell Signaling</td><td>3331</td><td>1:100</td></tr><tr><td>Lck (Y505)</td><td>Cell Signaling</td><td>2751</td><td>1:500</td></tr><tr><td>LKB1 (S428)</td><td>Cell Signaling</td><td>3051</td><td>1:100</td></tr><tr><td>mTOR (S2448)</td><td>Cell Signaling</td><td>5536</td><td>1:100</td></tr><tr><td>NFkB p65 (S536)</td><td>Cell Signaling</td><td>3031</td><td>1:50</td></tr><tr><td>p70 S6 Kinase (T389)</td><td>Cell Signaling</td><td>9205</td><td>1:200</td></tr><tr><td>PDK1 (S241)</td><td>Cell Signaling</td><td>3061</td><td>1:100</td></tr><tr><td>PKA C (T197)</td><td>Cell Signaling</td><td>4781</td><td>1:250</td></tr><tr><td>PRAS40 (T246)</td><td>BioSource</td><td>44-1100</td><td>1:2000</td></tr><tr><td>PTEN (S380)</td><td>Cell Signaling</td><td>9551</td><td>1:500</td></tr><tr><td>Smad1 (S463/465), Smad5 (S463/465), Smad8 (S426/428)</td><td>Cell Signaling</td><td>9511</td><td>1:500</td></tr><tr><td>Src (Y527)</td><td>Cell Signaling</td><td>2105</td><td>1:500</td></tr><tr><td>Src Family (Y416)</td><td>Cell Signaling</td><td>2101</td><td>1:200</td></tr><tr><td>Stat1 (Y701)</td><td>Cell Signaling</td><td>9171</td><td>1:200</td></tr><tr><td>Stat3 (S727)</td><td>Cell Signaling</td><td>9134</td><td>1:200</td></tr><tr><td>Zap-70 (Y319)</td><td>Enogene</td><td>E011159</td><td>1:100</td></tr><tr><td>&amp;beta;Catenin (S33/37/T41)</td><td>Cell Signaling</td><td>9561</td><td>1:250</td></tr><tr><td>CREB</td><td>Upstate Biotechnologies</td><td>06-863</td><td>1:100</td></tr><tr><td>Fos B</td><td>Cell Signaling</td><td>2251</td><td>1:200</td></tr><tr><td>GRB2</td><td>Cell Signaling</td><td>3972</td><td>1:2000</td></tr><tr><td>HSP70</td><td>Stressgen</td><td>SPA-810</td><td>1:100</td></tr><tr><td>c-Jun</td><td>Cell Signaling</td><td>9165</td><td>1:100</td></tr><tr><td>Kip1/p27</td><td>BD</td><td>610241</td><td>1:100</td></tr><tr><td>Lck</td><td>Cell Signaling</td><td>2984</td><td>1:250</td></tr><tr><td>Mcl-1</td><td>Cell Signaling</td><td>4572</td><td>1:80</td></tr><tr><td>Musashi</td><td>Cell Signaling</td><td>2154</td><td>1:100</td></tr><tr><td>NOTCH1</td><td>Cell Signaling</td><td>3439</td><td>1:100</td></tr><tr><td>PTEN</td><td>Cell Signaling</td><td>9552</td><td>1:500</td></tr><tr><td>SGK1</td><td>Abnova</td><td>PAB4590</td><td>1:250</td></tr><tr><td>Zap-70</td><td>Cell Signaling</td><td>2705</td><td>1:250</td></tr><tr><td>&amp;beta;-Catenin</td><td>Abcam</td><td>AB32572</td><td>1:1000</td></tr><tr><td>Dll4</td><td>Abcam</td><td>AB7280</td><td>1:500</td></tr><tr><td>Shh</td><td>Abcam</td><td>AB53281</td><td>1:250</td></tr><tr><td>HIF-1&amp;alpha;</td><td>BD</td><td>610958</td><td>1:50</td></tr><tr><td>NUMB PAN-ISO</td><td>Upstate Biotechnologies</td><td>07-207</td><td>1:400</td></tr><tr><td>NUMB-L</td><td>Chemicon</td><td>AB15145</td><td>1:750</td></tr><tr><td>Cyclin B</td><td>BD</td><td>610220</td><td>1:75</td></tr><tr><td>c-Myc</td><td>Calbiochem</td><td>OP-10</td><td>1:100</td></tr><tr><td>BCIP/NBT Kit</td><td>Thermo-Fisher</td><td>002209</td><td>(Note after step 5.2.8). Kit used to measure alkaline phosphatase activity, similar kits can be used.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;u&gt;Antibodies for Flow Cytometry:&lt;/u&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SSEA1 Antibody, Pacific Blue conjugate</td><td>Thermo-Fisher</td><td>MHCD1528</td><td>1:100 (Note after step 5.2.8)</td></tr><tr><td>SSEA4 Antibody (MC813-70), Alexa Fluor 647</td><td>Thermo-Fisher</td><td>SSEA421</td><td>1:100</td></tr><tr><td>Name</td><td>Company</td><td>Catalog number</td><td>Comments</td></tr><tr><td>Specific instruments, tools and softwares:</td><td></td><td></td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Thermo-Fisher</td><td>C10227</td><td>Neubauer chamber or other suitable glass hemocytometer can be used.</td></tr><tr><td>MEA1060-Inv-BC</td><td>Multichannel Systems</td><td>MEA1060-Inv-BC</td><td>(Step 5.3)</td></tr><tr><td>MEA1060-BC control software</td><td>Multichannel Systems</td><td>MEA1060-BC</td><td>(Step 5.3)</td></tr><tr><td>NeuroExplorer</td><td>Multichannel Systems</td><td>NeuroExplorer (NE)</td><td>(Step 5.3) For post-processing of MEA data</td></tr><tr><td>Multielectrode arrays (MEA)</td><td>Multichannel Systems</td><td>60MEA100/10iR-Ti-gr</td><td>(Step 5.3) Single-well MEA chip</td></tr><tr><td>ArrayScan XTI High Content Platform</td><td>Thermo-Fisher</td><td>ASN00002P</td><td>(Step 5.2.8) Mean fluorescence can be quantified by using specific ArrayScan algorithms (e.g., Cytotoxicity V.4 and NucTrans V.4 bioapplications). It is recommended to take minimum 20 pictures/well, and have 7-8 internal replicates per condition</td></tr><tr><td>7900HT Fast Real-Time PCR System</td><td>Thermo-Fisher</td><td>4351405</td><td>(Step 5.1.6)</td></tr><tr><td>BD ULTRA-FINE Needle Insulin Syringe (with 30G needle)</td><td>BD</td><td>328280</td><td>(Steps 1.3.1, 2.1.2, and 2.4.1)</td></tr><tr><td>StemPro EZPassage Disposable Stem Cell Passaging Tool</td><td>ThermoFisher</td><td>23181010</td><td>This colony cutting tool can be used as an alternative to the use of 30G needle 1 mL syringes (Step 1.3.1)</td></tr><tr><td>Ultra-Low attachment Petri dish (60 mm)</td><td>Corning</td><td>10010582</td><td>(Step 2.1.8) Also other brands can be used.</td></tr><tr><td>Mr. Frosty Freezing container</td><td>Sigma-Aldrich</td><td>C1562-1EA</td><td></td></tr></tbody></table>

protocol for the differentiation of human induced pluripotent stem cells into mixed cultures of neurons and glia for neurotoxicity testing

人类多能干细胞可以分化成可应用于基于人体的体外毒性测定的各种细胞类型。一个主要优点是体细胞重编程产生人诱导多能干细胞（hiPSCs）避免了与使用人类胚胎干细胞（hESCs）相关的伦理和立法问题。 HiPSC可以扩展和有效地分化成不同类型的神经元和神经胶质细胞，用作毒性测试的测试系统，特别是评估涉及神经毒性的不同途径。这项工作描述了将hiPSC分化为神经元和神经胶质细胞的混合培养物的方案。定义由神经元分化调节和/或激活的信号通路。该信息对于将新型毒素测试范例应用于细胞模型至关重要，其中化学物质基于其对pe的能力进行评估rturb生物学途径。作为概念证明，鱼藤酮是线粒体呼吸复合物I的抑制剂，用于评估Nrf2信号通路的活化，Nrf2信号通路是抵抗氧化应激的抗氧化反应元件（ARE）驱动的细胞防御机制的关键调节剂。

未分化hiPSC的表征为了评估hiPSC的表型，应进行菌落/细胞形态分析，PSC特异性标记的测定，以及基因表达和碱性磷酸酶活性的检测。未分化的hiPSC应该是圆形的，具有大的核仁和没有丰富的细胞质。大多数菌落的特征应该是平坦且紧密堆积的形态，指示未分化的表型（ 图2A和图2B ）。另外，超过80％的菌落对于碱性磷酸酶活性染色是阳性的（ 图2C ）。 大约80％的细胞应该是经典的多能性相关标志物，如Oct4，SSEA3，SSEA4和Tra1-60（ 图2D-H ），如免疫细胞化学和流式细胞术所示，而巢蛋白+和β-III-微管蛋白+细胞的百分比应显着低（分别为约8％和3％ 如图2H所示）。这些结果应该能够超过段落。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55702/55702fig2.jpg" /> 图2.未分化的IMR90-hiPSC的表征。 （A和B）未分化的IMR90-hiPSC菌落的代表性的对比度图像（10X和20倍放大）。 （C）碱性磷酸酶染色菌落的代表性图像（放大倍数4倍）。 （DF）代表性的免疫细胞化学图像（D） Oct4（红色）， （E） SSEA3（绿色）和（F） Tra1-60（绿色）。 （G）通过流式细胞术分析SSEA1（CD15）和SSEA4染色的代表性点图。 （H）条形图显示了Oct4 + （〜75-80％），Tra1-60 + （〜75-80％），SSEA3 + （〜75-80％），巢蛋白+ （〜10-15％ ）和β-III-微管蛋白+ （〜3-7％）细胞，用DAPI复染并用HCl定量，平均3至5个生物重复±SEM（图6从参考文献6修改）。 <a href="http://ecsource.jove.com/files/ftp_upload/55702/55702fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 通过EB形成评估多能性 HiPSC是多能的，这意味着它们在合适的条件下表达三个与胚层相关的基因。为了评估hiPSC多能性，可以应用常见的基于自发EB形成的方法，其诱导形成三个胚层14 。胚胎层特异性基因的分析必须表明内胚层（甲胎蛋白（AFP）和细胞角蛋白18（KRT18）），外胚层（巢蛋白，SRY盒1（Sox1））和配对盒6（Pax6）的时间依赖性增加）和中胚层（利钠肽A（NPPA）和Brachyury-T）相关基因表达（ 图3A和图3B ）;见材料表。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/55702/55702fig3.jpg" /> 图3.通过EB形成评估多能性。 （A） EBs在第1天的代表性的对比图像（B）条形图显示中胚层（NPPA和brachyury），外胚层（Pax6，Sox1和巢蛋白）和内胚层（AFP和KRT18）的qPCR分析<html> ）基因，标准化为参考基因，β-肌动蛋白和GAPDH，并校准至未分化细胞（第0天）。这是ΔΔCt方法，平均5次独立分析±SEM * p &lt;0.05，** p &lt;0.01，*** p &lt;0.001;图表从参考文献6中修改。 <a href="http://ecsource.jove.com/files/ftp_upload/55702/55702fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 诱导神经元和胶质细胞分化 IMR90-iPSC可以按照图1和方案部分中总结的步骤分化为有丝分裂后神经元和神经胶质细胞的混合培养物。在完整的NRI培养基存在下，将EB铺在标准的基质或层粘连蛋白包被的培养皿上5-8天后，玫瑰花样结构应开始变得可见（="xfig"&gt;图4A）。 Rosette的特征在于存在nestin +细胞（神经元前体，约90％），几乎没有β-III-微管蛋白+细胞（致死的神经元细胞，约5-10％），后者通常主要位于玫瑰花结（ 图4B ，第12天玫瑰花结）。 在完整的ND培养基存在下，玫瑰花素解离并复制到层粘连蛋白或标准基质包被的培养皿或平板上，细胞开始分化成神经元和神经胶质的混合培养物，逐渐形成由神经突束连接的神经元细胞体簇（ 图4C和图4D ）。当分析通过扩大玫瑰花源衍生的NSCs并将其分化为神经元和神经胶质获得的神经元群体时，应该获得类似的结果。从花莲扩展的NSCs应为n雌激素+ （ 图4E ，插图显示巢蛋白+细胞）。 分化21天后，细胞应为β-III-微管蛋白阳性; NF200;头;和MAP2，树突的晚期标记（ 图4D ，4F和图4H），其中至少10-15％的细胞对胶质纤维酸性蛋白（GFAP）呈阳性，是星形胶质标记（ 图4G和图4H） 。此外，约20-30％的细胞应该在分化后保留巢蛋白的表达（ 图4H ）。重要的是要考虑每种细胞类型（ 即神经元，星形胶质细胞，巢蛋白+细胞）的百分比可能随传代而变化，并且可能观察到用户依赖的变异性。 通过分析特定的神经元亚群，GABA能神经元代表发送约15-20％的总细胞，多巴胺能神经元〜13-20％，谷氨酸能神经元约35-42％，如γ-氨基丁酸（GABA），酪氨酸羟化酶（TH）和囊泡谷氨酸的免疫染色转运蛋白1（VGlut1）（ 见图4H中的代表性定量）。分化的诱导也可以通过分析多能性相关标志物（ 例如 Oct4，Tra1-60和SSEA3）进行评估，其在分化细胞中相对于未分化的hiPSC应显着下调（未显示参见参考文献6）。这也可以通过qPCR的基因表达分析来证实，这应该表明Oct4和Nanog的降低以及神经元基因如神经细胞粘附分子1（NCAM1）和微管相关蛋白2（MAP2）的上调。突触前基因突触泡蛋白（synaptophysin，SYP）;和突触后基因，微管相关蛋白质tau（MAPT），如&lt;<html> strong class ="xfig"&gt;图4I。此外，多巴胺能（TH和NR4A1），去甲肾上腺素能（PHOX2A和PHOX2B），谷氨酸能（NARG2，GRIA1和GAP43），GABA能（GABRA1和GABRA3），运动神经元（ISL1和LHX3）和胆碱能（SLC5A7和SLC18A13）导致与未分化细胞相比上调的神经元细胞（ 图4J ）。 通过MEA分析自发电活动是评估分化hiPSC中神经元网络功能的有价值的读数。在分化期结束时，神经元衍生物通常以至少60尖峰/分钟的平均燃烧速率（MFR）表征（参见图4K中的代表性光栅图 ）。但是，没有观察到爆发。 55702fig4.jpg"/&gt; 图4.IMR90-hiPSC分化为神经元和胶质细胞的混合培养物。 （A和B） 7 DIV （A）和12 DIV （B）后玫瑰花的代表性图像，染色巢蛋白（绿色）和β-III-微管蛋白（红色））。 （C和D） 22 DIV （C）和28 DIV （D）后分化细胞的代表性图像，用于β-III-微管蛋白（红色）和NF200（绿色）染色）。 （E）源自玫瑰花簇解离和扩增的神经干细胞的代表性图像（插图显示巢蛋白+细胞，红色）。 （F和G）从神经干细胞分化的神经元细胞（ F ，染色的NF200（红色）和Tau（绿色））和神经胶质细胞（ G ，染色的GFAP（红色））的代表性图像（21DIV后）。 （H）巢蛋白，MAP2，GFAP，γ-氨基丁酸（GABA），囊泡型谷氨酸转运蛋白1（VGlut1）和酪氨酸羟化酶（TH）阳性细胞，比较IMR90-hiPSC衍生物和从IMR90-hiPSC衍生的NSCs分化的细胞（从参考文献7中修改的图）。 （I和J）显示多能性基因（Oct4和Nanog）和神经元基因（NCAM1，MAP2，SYP和MAPT） （I）和多巴胺能（TH和NR4A1），去甲肾上腺素能（PHOX2A和PHOX2B）的qPCR分析的条形图，谷氨酸能（NARG2，GRIA1和GAP43），GABA能（GABRA1和GABRA3），运动神经元（ISL1和LHX3）和胆碱能（SLC5A7和SLC18A13）相关基因（J） 。所有分析标准化为参考基因β-肌动蛋白和GAPDH，并校准至未分化细胞（绿色条）。这是ΔΔCt方法，平均5次独立分析±SEM * p &lt;0.05，** p &lt;0.01，*** p &lt;0.001。 I和J中的图从参考文献6修改。 （K） IMR90-NSC衍生神经的代表性光栅图（记录进行至少600秒;垂直条代表单峰）。 <a href="http://ecsource.jove.com/files/ftp_upload/55702/55702fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 在分化的IMR90-hiPSC中上调了神经元标志物的特异性标记在新的毒性测试范例中，必须定义暴露于给定毒物之后在细胞内发生的分子和细胞事件。因此，与正在研究的细胞模型中哪些信号通路被激活和/或上调有关。 用于分析蛋白激酶基因表达的市售阵列可用于比较未分化的hiPSC与分化的细胞。 Differen相关的IMR90-hiPSCs经历了参与神经营养因子受体控制的基因的上调，轴突指导调节，神经突增生调节，神经胶质来源的神经营养因子（GDNF）受体，骨形态发生蛋白（BMP）/ TGF-β途径和血小板 - 衍生生长因子（PDGF）受体（ 图5A和图5B ）。 RPPA的分析显示分化的IMR90-hiPSC中特异性神经元特征的上调。特别地，Erk / CREB，Akt / PDK1 / mTOR和Notch1信号通路在分化时被激活（ 图5C ）。 <img alt="图5" class="xfigimg" src="/files/ftp_upload/55702/55702fig5.jpg" /> 图5.分化神经元和神经胶质细胞显示神经元相关途径的激活。 （一个和B）条形图报告神经元相关激酶（A）和其他激酶相关基因（B）的qPCR分析。将基因表达数据归一化为参考基因18S和GAPDH（在阵列中提供）并校准至未分化细胞。对于这些分析，当基因的表达高于未分化细胞（2- ΔΔCt≥2 ）时，基因被认为是显着上调的;平均3次独立分析±SEM）。 （C）条形图显示通过RPPA分析，比较分化（红色条）和未分化细胞（绿色条）的绝对蛋白质定量。属于同一信号通路级联的蛋白质聚类如下:CREB ​​/ Erk / Akt，PDK1 / mTOR / Erk / Akt，Notch1，Shh和Wnt，SAPK / JNK和BMP / SMAD。 4次独立分析的平均值±SEM。 * p &lt;0,05，** p &lt;0.01，*** p &lt;0.001;图修改from参考6. <a href="http://ecsource.jove.com/files/ftp_upload/55702/55702fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> IMR90-hiPSC衍生的神经元/胶质细胞培养物可用于评估鱼藤酮的作用已知线粒体呼吸链复合物I的抑制剂鱼藤酮酮通过触发Nrf2途径的激活而引起氧化应激。在静止条件下，Nrf2通过Keap1（Kelch样ECH相关蛋白1）锚定在细胞质中，Nrf2阻遏物促进Nrf2泛素化和蛋白水解15 。诱导氧化应激后，Nrf2转位入细胞核并激活Nrf2-ARE靶基因的表达。 IMR90-hiPSC衍生神经元d胶质细胞可用于通过将细胞暴露于不同浓度的鱼藤酮（ 例如 ，1,10和100nM）24小时来评估鱼藤酮对Nrf2活化的影响。这些浓度是根据以前的研究17,18建立的 。 在这些浓度和暴露时间下，鱼藤酮不会导致细胞毒性，如活体DAPI +细胞核的定量所示（ 图6A ）。鱼藤酮诱导Nrf2核易位，特别是在将细胞暴露于100nM鱼藤酮后（ 图6B和图6E ）。在相同的浓度下，观察到细胞质Keap1的显着减少（ 图6C ），以及NAD（P）H醌氧化还原酶1（NQO1）和磺脲毒素1（SRXN1）的增加，两个Nrf2靶zymes 19,20 （ 图6D ）。 <img alt="图6" class="xfigimg" src="/files/ftp_upload/55702/55702fig6.jpg" /> 图6.鱼藤酮对Nrf2核易位，Keap1，SRXN1和NQO1蛋白水平的影响。 （A）在用 1,10,100nM鱼藤酮处理24小时后对活的DAPI +细胞（ 即非固定核）进行定量，并对未处理的细胞进行标准化（对照，Ctr）。 （B）暴露于鱼藤酮24小时后的Nrf2蛋白核易位（ 即核/胞质比），通过使用HCI分析测量荧光强度进行评估。 （C）通过HCl分析评估鱼藤酮处理后细胞质Keap1蛋白水平的定量。 （D） NAD（P）H醌氧化还原酶1（NQO1）和Sulfi的定量用鱼藤酮处理24小时后，通过免疫荧光和HCl进行氧化还原1（SRXN1）。 （E） Nrf2蛋白定位（绿色）的代表性图像。所有值均显示为3个生物重复的平均值±SEM。 * p &lt;0,05，** p &lt;0.01;从参考文献7中修改。 <a href="http://ecsource.jove.com/files/ftp_upload/55702/55702fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 在这些浓度和处理时间下，鱼藤酮还引起星形胶质细胞（GFAP + ）细胞百分比（ 图7A和图7B ）的浓度依赖性增加，而不影响NSCs（巢蛋白+ ）和神经元（MAP2 + ）的比例（ 图7B ）。通过观察特定神经元亚型的比例，鱼藤酮治疗（10nM和100nM）显着减少多巴胺能神经元（TH + ）的数量（ 图7C和D ），而GABA能（GABA + ）和谷氨酸能（VGlut1 + ）神经元的百分比没有变化（ 图7D ）。类似地，以前的体内和体外研究已经描述了鱼藤酮依赖性和选择性多巴胺能神经元细胞死亡21,22,23 。 <img alt="图7" class="xfigimg" src="/files/ftp_upload/55702/55702fig7.jpg" /> 图7.鱼藤酮对胶质细胞和多巴胺能神经元的影响。 （A） GFAP +细胞（红色）的代表性图片，在插图中放大40倍，未处理或用100nM鱼藤酮处理24小时。 （B）量化<html>的巢蛋白+ ，MAP2 +和GFAP +细胞，对未处理的细胞进行归一化（对照，Ctr）。 （C）多巴胺能TH +神经元（绿色）的代表性图片，在插图中放大40倍，未处理或用100nM鱼藤酮处理24小时。 （D） GABA + ，VGlut1 +和TH +神经元细胞的量化，对未处理的细胞进行标准化（Ctr）。所有值均显示为3个生物重复的平均值±SEM。 * p &lt;0,05，** p &lt;0.01，*** p &lt;0.001;从参考文献7中修改。 <a href="http://ecsource.jove.com/files/ftp_upload/55702/55702fig7large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 统计显着性通过单因素方差分析（Dunnett&#39;s Multiple Comparison Test）作为后检验（比较所有柱与对照柱）xref"&gt; 24或根据分析类型通过双尾不配对或配对t检验，所有数据表示至少三个生物复制品的平均值±平均值（SEM）的标准误差，条形码上的星号表示与对照组有显着差异。* p &lt;0.05，** p &lt;0.01，*** p &lt;0.001。 表格1。 关于解离的花环电镀密度的注意事项:如果玫瑰花序片段看起来不完全解离，为了达到约15.000个细胞/ cm 2的细胞镀覆密度，可以将衍生自约50个EB / 1×60mm皿的解离的花环片段重新悬浮于将50毫升完整的NRI培养基按如下方法（取决于平板格式）: <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 多孔板/ MEA </td><td> 生长面积（cm 2 </s向上&gt; /孔） </td><td> 细胞悬液至每孔板（或MEA芯片） </td><td> 可镀的最大板数（用50ml细胞悬浮液） </td></tr><tr><td> 96口井</td><td> 0.3 </td><td> 100ul </td><td>五</td></tr><tr><td> 48口井</td><td> 0.7 </td><td> 220ul </td><td> 4 </td></tr><tr><td> 24口井</td><td> 2 </td><td> 625 ul </td><td> 3 </td></tr><tr><td> 12口井</td><td> 4 </td><td> 1.25毫升</td><td> 3 </td></tr><tr><td> 6口井</td><td> 10 </td><td> 3.125毫升</td><td> 2 </td></tr><tr><td>单井MEA芯片</td><td> 3.5 </td><td> 1.1毫升</td><td> 45 </td></tr></tbody></table> 表2:验收标准 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 标记/抗体</td><td> 28 DIV后的百分比（在DAPI +（活体）细胞上） </td></tr><tr><td> B-III-微管蛋白（Tuj1） </td><td> 35-45％ </td></tr><tr><td> MAP2 </td><td> 50-60％ </td></tr><tr><td> NF200 </td><td> 45-55％ </td></tr><tr><td> GFAP </td><td> 10-25％ </td></tr><tr><td>巢</td><td> 15-25％ </td></tr></tbody></table>

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

人类诱导的多能干细胞（hiPSC）被认为是用于药物和化学筛选以及开发用于毒性测试的新体外模型（包括神经毒性）的有力工具。这里描述了将hiPSC分化为神经元和神经胶质的详细方案。

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

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

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Cell Biology

Developmental Biology

Unit 1

Embryonic and Induced Pluripotent Stem Cells

SCIENTISTS IN ACTION

23.0K 次观看.  Joint Research Centre. 人类诱导的多能干细胞（hiPSC）被认为是用于药物和化学筛选以及开发用于毒性测试的新体外模型（包括神经毒性）的有力工具。这里描述了将hiPSC分化为神经元和神经胶质的详细方案。 

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

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

基于发育毒性测定化学品安全筛选和系统生物学数据生成人类多能干细胞

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

科研

发育生物学

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

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

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

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

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

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

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

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

神经石生长分析与神经毒性评估与人类神经原生细胞衍生神经元

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

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, ...

神经科学

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

分化后再蛋白的人类多能干细胞衍生神经元，用于高含量的内尿成生和突合性成熟筛查

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

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

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

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

在三维悬浮生物反应器中产生人神经原蛋白2诱导神经元

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

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

通过集成活细胞监测，自动生产人类诱导多能干细胞衍生皮质神经元和多巴胺神经元

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

生物学

Differentiating Human Induced Pluripotent Stem Cells into Neurons on Micro-Electrode Arrays

Source: Frega, M., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54900">Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.</a> J. Vis. Exp. (2017)
The video demonstrates a method for generating excitatory cortical neurons from human-induced pluripotent stem cells (hiPSCs). Upon doxycycline induction, the hiPSCs overexpress neurogenin-2, a neural-specific transcription factor. By using specific media and co-culturing with astrocytes, this process facilitates the hiPSCs&#39; differentiation into neurons with synaptic connections.

在微电极阵列上将人诱导多能干细胞分化为神经元

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Differentiation of ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

Differentiating Non-Transgenic and Transgenic Human Pluripotent Stem Cells into Neural Progenitor Cells

Source: Cvetkovic, C., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates the generation of neural progenitor cells from non-transgenic and transgenic human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural progenitor cells using a neuronal differentiation medium. While differentiation is induced in non-transgenic cells using small molecules, the transgenic hPSCs are differentiated by adding an antibiotic that induces the promoter for a neuronal differentiation-specific transcription factor.

将非转基因和转基因人多能干细胞分化为神经祖细胞

Ethic approvals were obtained when performing the experiments.
1. Cell Culture and Reagent Preparation

	Prepare coated plates for cell culture.
	
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