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。

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

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

科研

发育生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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, ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...