XHP一直支持由国家卫生研究院（NIH）国家老龄问题研究所（NIHK01AG024496）和尤尼斯肯尼施莱佛国立儿童健康和人类发展研究所（NIHR21HD056530）的赠款。

1。解决方案和媒体准备<ol><li>明胶涂层解决方案：0.1％（W / V），蒸压在DDH 2 O的明胶和储存于4 ° C。 </li><li>基质胶涂层解决方案。联合解决方案：缓慢解冻基质胶（10毫升）于4 ° C过夜，加10毫升冰冷的DMEM或DMEM/F12，拌匀等分1毫升/管消毒的组织文化引擎盖底下，储存在-20 ° C.工作液：缓慢解冻1毫升基质胶分装在4 ° C 1-2小时，转移到14毫升冷冻的DMEM或DMEM/F12，拌匀涂装前消毒的组织文化引擎盖下方。 </li><li>人层粘连蛋白涂层解决方案：稀释1毫升人类层粘连蛋白溶液（0.5毫克/毫升的Tris缓冲盐，储存在-80℃，在4 ° C缓慢解冻1-2小时），用冰冷的DMEM或DMEM/F12到12.5毫升工作液（40微克/毫升）消毒的组织文化引擎盖下方紧接涂层。 </li><li>生长因子库存解决方案（500 - 1000X）：溶解在10微克生长因子/ ml的消毒缓冲（0.5％BSA，1.0毫米的数码地面电视，10％甘油，1XPBS）和存储50-100μL/管分装在-80 ° C </li><li>全反式维甲酸溶液（1000X）：10 -80毫米，分装店在DMSO ° C。 </li><li> HESC媒体的DMEM/F12或Ko - DMEM（80％），KO血清替代（20％），L -丙氨酰- L -谷氨酰胺和L -谷氨酰胺（2毫米），纪念不必要的氨基酸（MNAA 1X），并β-巯基乙醇（100微米），过滤和储存于4 ° C，使用前20毫微克/毫升碱性成纤维细胞生长因子的补充。或更换“击倒（KO）的血清替代品”的DMEM/F12或KO - DMEM（100％），L -丙氨酰- L - GLN或L -谷氨酰胺（2毫米），MNAA（1X），纪念必不可少的定义组件：氨基酸（MEAA 1X），和β-巯基乙醇（100μM），过滤和储存在4 ° C，碱性成纤维细胞生长因子（20毫微克/毫升），人胰岛素（20微克/毫升），抗坏血酸（50微克/为辅毫升），人类激活素A（50毫微克/毫升），人血白蛋白/ Albumax（10毫克/毫升），和人转（8微克/毫升），使用前。 </li><li>国科会媒体：DMEM/F12（100％），N - 2个supplemeNT（1X），肝素（8微克/毫升）。 </li></ol> 2。板的涂层<ol><li>涂层板明胶：添加2.5毫升/明胶溶液6孔板，在37 ° C培养箱孵育过夜。 </li><li>涂层板与层粘连蛋白：寒意明胶涂层钢板和删除明胶，添加2.5毫升/良好的基质胶或人类的层粘连蛋白涂层工作方案预冷糊化6孔板孵育在4 ° C过夜。 </li></ol> 3。在规定条件下未分化的人类胚胎干细胞的传代和播种<ol><li>允许人类胚胎干细胞菌落生长到5-7天左右，并采取胚胎干细胞培养板，解剖显微镜（剖析阶段预温至37 ° C）下方解剖消毒罩。 </li><li>选择被分裂的胚胎干细胞菌落。形态上，这些殖民地应该有定义的边缘联合国&gt; 75％，未分化的人类胚胎干细胞（小型紧凑细胞），通常稍不透明的（不是白堆砌的细胞，没有明确的分化细胞）derneath解剖显微镜。小心地勾勒出选定的殖民地和删除分化成纤维细胞层周围的殖民地和所有有区别的部分（如果任何殖民地）与P2的无菌枪头边缘拉玻璃毛细管。 （请注意，多能胚胎干细胞是在瞬态阶段，经历即使在最佳条件下自发分化，虽然首选，它的难以细胞确定论文100％无差别解剖显微镜下，&gt; 75％的未分化是通常的传递点考虑。“分化的细胞通常不会种子和持续增长，在传代过程中被淘汰） </li><li>取下吸浮动分离分化的细胞含有的旧媒体。人类胚胎干细胞介质（无碱性成纤维细胞生长因子），一次洗干净。加入3毫升/以及含有20 ng / ml的碱性成纤维细胞生长因子的新鲜胚胎干细胞媒体。 </li><li>切成小块的未分化的胚胎干细胞菌落，用无菌枪头或拉玻璃毛细管和分离。</li><li>池媒体中含有50毫升的锥形管分离的殖民地拼凑。洗板一次1毫升/人类胚胎干细胞以及媒体含有20毫微克/毫升碱性成纤维细胞生长因子和池一起。 </li><li>吸新鲜的涂层板的基质胶或人类的层粘连蛋白溶液。分装4毫升/包含殖民地件以及人类胚胎干细胞媒体6孔板。轻轻板转移到孵化器，无晃动，让殖民地件的种子没有令人不安的气氛中5％的CO 2，饱和湿度37℃培养箱过夜。 </li></ol> 4。文化系统定义维甲酸诱导下的人类胚胎干细胞神经<ol><li>在播种后第3天，从每个板块以及删除最旧的媒体和留下足够的媒体，让人类胚胎干细胞的殖民地被淹没（决不允许人类胚胎干细胞向干出）。更换4毫升/新鲜胚胎干细胞含有20 ng / ml的碱性成纤维细胞生长因子和10微米维甲酸的媒体。 </li><li>替换旧媒体与新鲜胚胎干细胞含有20 ng / ml的碱性成纤维细胞生长因子和媒体10μM维甲酸隔日，并让神经诱导人类胚胎干细胞的菌落长到7或8天。殖民地内的所有细胞将发生大分化的细胞，继续繁殖的形态变化。殖民地的大小会增加，以涵盖7天或8板，细胞就会开始堆积在一些地区的殖民地。 </li></ol> 5。悬浮培养持续神经元分化<ol><li>以人类胚胎干细胞培养板解剖显微镜消毒罩下面剖析（剖析阶段预温至37 ° C）。小心地勾勒出殖民地和成纤维细胞层周围的殖民地（分化的细胞迁移的殖民地）与P2的无菌枪头边缘删除或拉玻璃毛细管。 </li><li>取下吸浮动分离的成纤维细胞中含有的旧媒体。人类胚胎干细胞介质（无碱性成纤维细胞生长因子），一次洗干净。加入3毫升/以及新鲜的人类胚胎干细胞介质（无碱性成纤维细胞生长因子）。 </li><li>剪下的神经- Induced人类胚胎干细胞成小块殖民地，并用无菌枪头分离或拉玻璃毛细管。 </li><li>池媒体中含有50毫升的锥形管分离的殖民地拼凑。洗板一次1毫升/以及人类胚胎干细胞介质（无碱性成纤维细胞生长因子）和汇集。 </li><li>分装4毫升/无血清胚胎干细胞媒体殖民地件含有6以及超低附件板和孵化在37 °彗星培养箱允许浮动（神经母细胞）细胞集群，形成4-5天。 </li></ol> 6。胶粘剂文化中的神经细胞表型成熟<ol><li>池媒体浮动神经母细胞含有50毫升的锥形管。在5分钟1400转的离心机。吸，你可以旧媒体和含血管内皮生长因子（20毫微克/毫升），NT - 3（10毫微克/毫升），并BDNF（10纳克/毫升），加等量新鲜国科会媒体。 </li><li>移液器上下混合浮动的神经母细胞和分装4毫升/ 6孔板。也可以本身的神经母细胞在一个层粘连蛋白/胶原（基质胶）或人类的层粘连蛋白聚合的三维矩阵​​含有血管内皮生长因子（20毫微克/毫升），NT - 3（10毫微克/毫升）和BDNF（10毫微克/毫升）在无血清的国科会媒体eded 。板块转移到了37 °彗星培养箱，让神经母细胞附加过夜。 </li><li>更换NSC媒体含有血管内皮生长因子（20毫微克/毫升），NT - 3（10毫微克/毫升），和BDNF（10毫微克/毫升），隔日。含突起的神经元细胞和色素细胞的广泛的网络将随着时间的数字不断培育和增加2个星期内开始出现，并可以持续3个月以上。 </li></ol> 7。代表性的成果： 维甲酸（RA）呈现足以吸引从多能性维持在定义文化系统过渡的人类胚胎干细胞向神经外胚层表型（ 图 2A）独家。 RA未分化的人类胚胎干细胞的暴露后，殖民地内的所有细胞进行形态学变化- 4月，大分化的细胞停止表达多能性相关的标记，并开始表达神经外胚层相关的各种标志物，如HNK1，AP2，和TrkC（ 图2B） 。这些大分化的细胞会继续繁殖和殖民地将扩大规模，进行自发地表达早期神经元的标记β- III -微管蛋白（ 图 2B ）。更加成熟的神经元标记MAP - 2细胞越积越多（ 图 2B）菌落地区将开始出现。与外观的神经外胚层细胞和神经细胞的分化重合，神经元特异性转录因子NURR1，多巴胺能神经元分化和激活的酪氨酸羟化酶（TH） 基因 16牵连，将转运到细胞核内（ 图2B） 。 RA的治疗的胚胎干细胞分离后，将形成漂浮在悬浮邪教的细胞簇（神经母细胞）URE继续进行神经分化过程中。碱性成纤维细胞生长因子去除后允许附加到一个组织培养板或在层粘蛋白/胶原聚合3维矩阵接种于无血清定义介质，β三，微管蛋白和地图- 2表达，突起的神经母细胞后，含细胞和色素细胞就会开始出现大幅增加，自发的多系分化的人类胚胎干细胞治疗不超过同一时期相比，在效率，并可以持续超过3个月（图2C）。 <img alt="图1" src="/files/ftp_upload/3273/3273fig1.jpg" /> 图1控制专门为特定的临床相关的宗族提供简单的小分子的高效诱导人类多能干细胞的示意图。 <img alt="图2" src="/files/ftp_upload/3273/3273fig2.jpg" /> 图2 <stro吴&gt;维甲酸诱导神经谱系规范和神经细胞的全能性在规定条件下直接的进展（ 一 ）原理图描绘的人类胚胎干细胞定向神经分化的协议的时间线。 （b）在未分化的人类胚胎干细胞暴露在维甲酸（RA）的定义的文化系统，大的区别的Oct - 4的殖民地内（红色）阴性细胞开始出现，如模拟处理（DMSO）为控制人类胚胎干细胞相比。 RA诱导差异10月4负的细胞开始表达HNK - 1（红色），AP2（红色），TrkC（绿色），然后β- III -微管蛋白（红色），与早期神经外胚层分化一致。这些细胞不断走向成熟，最终表达神经元标记MAP - 2（绿色），通常在细胞开始堆积的地方，。与外观的神经外胚层细胞和神经细胞的分化重合，神经元特异性转录因子NURR1（绿色），牵连多巴胺能神经元分化和激活的酪氨酸羟化酶（TH）基因，易位到细胞核。所有细胞都表现出其原​​子核的DAPI染色（蓝色）。 （三）治疗RA诱导突起轴承细胞表达β-Ⅲ-微管蛋白（红色）和MAP - 2（绿色显示，在3二维矩阵）的广泛网络评估朝着高效率的神经细胞谱系分化。箭头指示的色素细胞在中枢神经系统的典型。所有细胞都表现出其原​​子核中的insets DAPI染色（蓝色）。比例尺：0.1毫米。

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C., Wernig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+conversion+of+fibroblasts+to+functional+neurons+by+defined+factors.">Direct conversion of fibroblasts to functional neurons by defined factors.</a> Nature. 463, 1035-1041 (2010).</li><li>Kim, J., Efe, J. A., Zhu, S., Talantova, M., Yuan, X., Wang, S., Lipton, S. A., Zhang, K., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+reprogramming+of+mouse+fibroblasts+to+neural+progenitors.">Direct reprogramming of mouse fibroblasts to neural progenitors.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 7838-7843 (2011).</li><li>Kim, K., Doi, A., Wen, B., Ng, K., Zhao, R., Cahan, P., Kim, J., Aryee, M. J., Ji, H., Ehrlich, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+memory+in+induced+pluripotent+stem+cells.">Epigenetic memory in induced pluripotent stem cells.</a> Nature. 467, 285-290 (2010).</li><li>Gore, A., Li, Z., Fung, H. L., Young, J. E., Agarwal, S., Antosiewicz-Bourget, J., Canto, I., Giorgetti, A., Israel, M. A., Kiskinis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+coding+mutations+in+human+induced+pluripotent+stem+cells.">Somatic coding mutations in human induced pluripotent stem cells.</a> Nature. 471, 63-67 (2011).</li><li>Feng, Q., Lu, S. J., Klimanskaya, I., Gomes, I., Kim, D., Chung, Y., Honig, G. R., Kim, K. S., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemangioblastic+derivatives+from+human+induced+pluripotent+stem+cells+exhibit+limited+expansion+and+early+senescence.">Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence.</a> Stem. Cells. 28, 704-712 (2010).</li><li>Klimanskaya, I., Chung, Y., Becker, S., Lu, S. -. J., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+human+embryonic+stem+cells+from+single+blastomeres.">Derivation of human embryonic stem cells from single blastomeres.</a> Nat. Protocols. 2, 1963-1972 (2007).</li><li>Park, I. H., Zhao, R., West, J. A., Yabuuchi, A., Huo, H., Ince, T. A., Lerou, P. H., Lensch, M. W., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+somatic+cells+to+pluripotency+with+defined+factors.">Reprogramming of human somatic cells to pluripotency with defined factors.</a> Nature. 451, 141-146 (2008).</li></ol>

作者声明竞争的利益。 XHP是Xcelthera创始人。 XHP和余仁生有关人类胚胎干细胞的知识产权。

的发育研究和临床翻译的重大挑战之一是如何进行渠道广泛的分化潜能的多能干的人类干细胞所需的表型，高效和可预见的的。虽然这种细胞可以自发分化成胚层细胞在体外通过多血统的聚合阶段，只有一小部分细胞追求一个给定的血统 1,4 。在这些胚胎干细胞衍生的聚合，同时出现了大相径庭的不受欢迎的可能驻留在三个胚层的细胞类型的大量经常出现不仅效率低，所需的表型，但不可控和不可靠的。虽然心脏和神经系已在以前的报告中得出的，但是，在通过胚层诱导多能干细胞的致瘤性的高风险的专门细胞效率低下以下transplantatioN有阻碍进一步的临床翻译。 人类胚胎干细胞线最初是根据共培养保持增长被捕的小鼠胚胎成纤维细胞（MEFs）4 。几个人类馈线，馈线，化学制定文化系统已发展为11-13人胚胎干细胞，虽然必要的，足以维持人类多能干细胞的自我更新的元素仍然没有得到解决。这些外源性饲养层细胞和生物试剂，有助于保持未分化的人类胚胎干细胞的长期稳定增长，而面具，多能干细胞的能力，以应对发育信号。保持在未分化的人类胚胎干细胞定义的生物制剂无文化系统，让忠实的扩张和可控性的直接分化是其治疗的效用和潜在的关键因素之一。类风湿性关节炎是不够的诱导未分化的人类胚胎干细胞神经分化的维持根据以前报道的康迪特含有外源性饲养层细胞的离子。虽然神经谱系出现在人类胚胎干细胞分化的相对早期阶段，与RA治疗的胚胎干细胞分化的多系的聚合（胚体）仅略有增加产量低的神经元1， 14，15。为了实现统一的一个特定的血统的人类多能干细胞的转换，我们雇用了一个定义文化系统有能力投保未分化的人类胚胎干细胞的增殖完全确定条件，以及控制多能胚胎干细胞的有效诱导到一个特定的临床相关的谱系提供简单的小分子（ 图1，图2）。在人类中枢神经系统的发展，未来的研究将揭示遗传和表观遗传控制分子，作为替代品，这可能为人类胚胎干细胞多能性的命运的小分子介导的直接控制和调制方式时所产生再生疗法的临床相关的谱系。如果没有治疗类风湿性关节炎，1-5％的人类胚胎干细胞将发生自发分化成神经元1，14，15。 RA的治疗，我们已经能够产生大于95％的胚胎神经祖细胞和神经细胞从人类胚胎干细胞保持在一个过程，可能会模仿人类的胚胎发育14下一个定义文化。近日，已知的神经命运决定基因已被用于transdifferentiate成人神经祖细胞和神经元的低效率0.5-8％，17，18的小鼠成纤维细胞。不过，体细胞重新编程历史上一直与基因表达异常加速衰老和受损的治疗公用事业 19-21 。最后，我们在这里建立的协议是从内细胞团（ICM）或人类囊胚4外胚层衍生的多能胚胎干细胞，可能并不适用于其他的多能干细胞，包括动物起源的胚胎干细胞，胚胎干细胞从早期的桑椹胚的（八细胞）胚胎22，artificially重新编程的细胞23 。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>明胶</td><td>西格玛</td><td> G1890 </td><td></td></tr><tr><td>基质胶</td><td> BD生物科学</td><td> 356231 </td><td>生长因子减少</td></tr><tr><td>人权层粘连蛋白</td><td>西格玛</td><td> L6274 </td><td></td></tr><tr><td>全反式维甲酸</td><td>西格玛</td><td> R2625 </td><td></td></tr><tr><td> DMEM/F12 </td><td> Invitrogen公司</td><td> 10565018 </td><td></td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 31053036 </td><td></td></tr><tr><td> DMEM - KO </td><td> Invitrogen公司</td><td> 10829018 </td><td></td></tr><tr><td>淘汰血清替代</td><td> Invitrogen公司</td><td> 10828028 </td><td></td></tr><tr><td>纪念不必要的氨基酸溶液（MNAA，100X） </td><td> Invitrogen公司</td><td> 11140050 </td><td></td></tr><tr><td>纪念Å美浓酸溶液（MEAA，100X） </td><td> Invitrogen公司</td><td> 11130050 </td><td></td></tr><tr><td> β-巯基乙醇</td><td> Invitrogen公司</td><td> 21985023 </td><td></td></tr><tr><td> Albumax </td><td> Invitrogen公司</td><td> 11020021 </td><td></td></tr><tr><td>抗坏血酸</td><td>西格玛</td><td> A4403 </td><td></td></tr><tr><td>人转</td><td>西格玛</td><td> T8158 </td><td></td></tr><tr><td>人类碱性成纤维细胞生长因子</td><td> PeproTech </td><td> AF - 100 - 18B </td><td></td></tr><tr><td>人胰岛素</td><td> Invitrogen公司</td><td> 12585014 </td><td></td></tr><tr><td>人类激活素A </td><td> PeproTech </td><td> 120 - 14E </td><td></td></tr><tr><td>人BDNF </td><td> PeproTech </td><td> AF - 450 - 02 </td><td></td></tr><tr><td>人VEGF </td><td> PeproTech </td><td> AF - 100 - 20 </td><td></td></tr><tr><td>人力NT - 3 </td><td> PeproTech </td><td> 450-03 </td><td></td></tr><tr><td>肝素</tD&gt; <td>西格玛</td><td> H5284 </td><td></td></tr><tr><td> N - 2个补充（100X） </td><td> Invitrogen公司</td><td> 17502048 </td><td></td></tr><tr><td> 6超低附件板</td><td>康宁</td><td> 3471 </td><td></td></tr><tr><td> 6孔板</td><td>康宁</td><td> 3516 </td><td></td></tr></tbody></table>

efficient derivation of human neuronal progenitors and neurons from pluripotent human embryonic stem cells with small molecule induction

有一个大的需要尚未得到满足的临床适合人类神经细胞修复或再生受损的中枢神经系统（CNS）的结构和电路在今天的医疗保健行业的源。细胞疗法具有很大的承诺，以挽回失去的神经组织和中枢神经系统疾病的功能。但是，基于对中枢神经源性神经干细胞的细胞疗法遇到供应的限制和困难，在临床上使用，由于他们的文化和失败的可塑性有限扩张后的广泛传代1-3能力。尽管取得了一些有益的结果，出现中枢神经系统派生的人类神经干细胞（hNSCs）通过生产的营养和神经保护分子施加其治疗效果，主要是由非神经细胞的后代救援的内源性细胞1-3。另外，多能干的人类胚胎干细胞（胚胎干细胞）毫无顾忌治愈supplyin广泛的神经系统疾病克人类在开发中枢神经系统再生1,4-7的神经细胞类型的多样性。然而，如何进行渠道广泛的分化潜能的多能胚胎干细胞是有效的和可预见的所需的表型的发育研究和临床翻译的一个重大挑战。依靠传统的方法多通过自发的胚层分化的多能干细胞系的倾向，导致效率低下和不可控的谱系承诺，往往是由表型异质性和不稳定性，因此，一个高风险的致瘤性7-10。此外，未定义外国/动物生物补充和/或通常被用于隔离，扩建，和人类胚胎干细胞分化的馈线可直接利用等专门的细胞移植患者问题 11-13 。为了克服这些障碍，我们已经解决了一个定义的文化系统中的元素的必要和足够为sustaining外胚层的胚胎干细胞pluripotence，作为临床适合人类胚胎干细胞的新的De Novo推导的平台，有效地指导临床相关的谱系对小分子14（请在图中看到的示意图。1）这种人类胚胎干细胞均匀。维甲酸（RA）不会诱发1，14馈线保持未分化的人类胚胎干细胞神经分化。小鼠胚胎干细胞不同，治疗胚胎干细胞分化的胚状体（EBS）仅略有增加产量低的神经元1，14，15 。然而，各种小分子和生长因子的筛选后，我们发现，这些规定的条件提供足够维甲酸（RA）诱导，进一步发展到在人类神经祖细胞和神经元产生的神经母细胞的多能胚胎干细胞的神经外胚层规范直接开发高效率的中枢神经系统（ 图2） 。我们定义为神经诱导条件多能胚胎干细胞不干预多系胚体阶段，使控制大量供应了整个人类神经细胞的发育阶段的细胞为基础的疗法的频谱效率的推导，直接从爆炸。

Watch this Scientific Journal Video about Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction at JoVE.com

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

我们已经建立了诱导多能干人类胚胎干细胞与小分子物质，使推导的大量供应人类神经祖细胞和神经细胞类型，在发展中国家中枢神经系统神经修复定义的条件下保持直接的神经母细胞的协议。

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

efficient-derivation-human-neuronal-progenitors-neurons-from

Research

JoVE Journal

Neuroscience

15.2K 次观看.  San Diego Regenerative Medicine Institute. 我们已经建立了诱导多能干人类胚胎干细胞与小分子物质，使推导的大量供应人类神经祖细胞和神经细胞类型，在发展中国家中枢神经系统神经修复定义的条件下保持直接的神经母细胞的协议。

视频: Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

科研

神经科学

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

从人类多能干细胞的规范在前脑谷氨酸能神经元

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

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

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

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

从人多能神经嵴前体细胞的无饲养推导干细胞

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

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

从人多能干细胞定向多巴胺能神经元分化

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

利用 PiggyBac 载体将人诱导的多能干细胞 (Ipsc) 转化为功能性脊柱和颅内运动神经元

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

人类诱导多能干细胞脑微血管内皮细胞的衍生、扩张、冷冻保存和特征化

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

猫大型动物模型中人类胚胎干细胞来源的视网膜组织的视网膜下移植

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

人小胶质细胞样细胞：使用人突触体从诱导多能干细胞和 体外 活细胞吞噬测定中分化

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...