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> 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。或更换自定义组件的"正血清替代品"的DMEM/F12或KO - DMEM（100％），L -丙氨酰- L - GLN或L -谷氨酰胺（2毫米），MNAA（1X），纪念必不可少的氨基酸（MEAA 1X），和β-巯基乙醇（100μM），过滤和储存在4 ° C，辅以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/毫升），人力激活素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> HESC心脏分化媒体:DMEM/F12（90％），定义FBS（10％），L -丙氨酰- L -谷氨酰胺或L -谷氨酰胺（2毫米）。 </li><li>媒体含有烟酰胺（NAM）:增加N上午HESC媒体或心脏HESC分化媒体终浓度为10毫米，过滤，储存于4 ° C和使用在2周的准备。 </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&gt; 75％的未分化的人类胚胎干细胞（SM所有紧凑型细胞），通常轻微不透明（不白堆砌的细胞，不明确的分化细胞）定义解剖显微镜底下的边缘。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。 </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/ hESC媒体包含殖民地件到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。心脏的胚胎干细胞的诱导下的定义与烟文化体系<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毫米烟酰胺（NAM）的媒体。 </li><li>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10毫米南隔日媒体替换旧媒体，并允许心脏诱导的人类胚胎干细胞殖民地成长为每天8-10。暴露在不结盟运动，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。殖民地将扩大规模，并每天8-10，D细胞就会开始堆积在一些地区的殖民地。 </li></ol> 5。悬浮培养持续心脏分化<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>切成小块的心脏诱导人类胚胎干细胞集落，并用无菌枪头或拉玻璃毛细管分离。 </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/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米L /以及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 ° C培养箱允许浮动（cardioblasts）蜂窝集群，形成4-5天。 </li></ol> 6。跳动的心肌细胞表型胶粘剂文化成熟<ol><li>池媒体一起包含在一个50毫升的锥形管的浮动cardioblasts。在5分钟1400转的离心机。吸的，你可以多旧媒体，并加入等量的新鲜胚胎干细胞的心肌分化含10 mM的不结盟运动的媒体。 </li><li>移液器上下混合浮动cardioblasts和等分4毫升/ 6孔板。板块转移到了37 °彗星培养箱允许cardioblasts附加过夜。 </li><li>更换心脏胚胎干细胞分化的介质含有10毫米不结盟运动隔日。 </li><li>在第8天，没有不结盟运动的心脏与人类胚胎干细胞分化的媒体取代，并继续以取代心脏HESC分化媒体发送很其他的一天。跳动的心肌细胞就会开始出现在连续种植约1-2周后退出不结盟运动，并随着时间的数字增加，并可以保留3个月以上的强烈而有节奏的收缩。 </li></ol> 7。代表性的成果: 烟酰胺（NAM）呈现足以引起人类胚胎干细胞保持在定义的文化体系，从多能性完全过渡到cardiomesodermal表型（图2B）。不结盟运动的未分化的人类胚胎干细胞的暴露后，殖民地内的所有细胞发生形态的变化，大分化的细胞，下调Oct - 4的表达，并开始表达心脏特异性转录因子（CSX）Nkx2.5和α-辅，与cardiomesoderm表型（ 图2B）一致。这些分化的细胞，继续繁殖和殖民地的大小增加。 Nkx2.5强度增加通常观察到的细胞开始堆积（ 图2B）的殖民地地区。后不结盟运动处理的胚胎干细胞的分离，将形成一个悬浮培养浮动蜂窝集群（cardioblasts），继续心脏的分化过程。允许cardioblasts重视和继续与不结盟运动治疗1周后，将开始跳动的心肌细胞出现约1-2周连续种植了效率的大幅增加后，退出不结盟运动相比，自发的多系分化人类胚胎干细胞治疗不超过同一时期（ 图2C）。跳动的心肌细胞簇内的细胞会表达标记心肌细胞的特性，包括Nkx2.5和α-辅肌动蛋白（ 图 2C ）。跳动的心肌细胞的收缩确认电气型材强，有节奏，以及协调，夹带与回忆的P经常冲动，- QRS - T复合物临床心电图体表电极（ 图2C，也见视频）。心肌细胞可保留3个月以上的强烈收缩。 <img alt="图1" src="/files/ftp_upload/3274/3274fig1.jpg" /> 图1。常规方法与小分子诱导方法对人类多能干专门的功能细胞的干细胞分化的总体计划。 （A）常规方法使用通过自发的胚层分化的人类多能干细胞的多系倾向示意图。 （B）原理控制的高效诱导人类多能干细胞专门为特定的临床相关的宗族提供简单的小分子。 <img alt="图2" src="/files/ftp_upload/3274/3274fig2.jpg" /> 图2。 烟诱导多能性心脏谱系在规定条件下的规范和对跳动的心肌细胞进展直接 。 （一）原理图，描绘了心脏的胚胎干细胞定向分化的协议的时间线。 （b）在未分化的人类胚胎干细胞暴露于烟酰胺（NAM）下定义的文化系统，大的区别的10月4（红色）阴性细胞内的殖民地开始出现，相比模拟处理（DMSO）为控制人类胚胎干细胞。南诱导10月4负的细胞开始表达Nkx2.5（绿色）和α-辅肌动蛋白（红色），与早期心脏分化相一致。 Nkx2.5强度逐渐增加，通常在殖民地的细胞开始堆积的地方观察。所有的细胞都表示其原子核的DAPI染色（蓝色）。 （三）南诱导心脏致力于人类胚胎干细胞产生与效率的大幅增加跳动的心肌细胞，细胞簇评估器显示的节律性收缩（见电剖面和视频），Nkx2.5（绿色）和α-辅肌动蛋白（红色）免疫阳性。承包心肌代表的代表大跳动的心肌细胞集群和更高功率的图像相图像显示（还可以看到相应的视频）。箭头指示的大跳动的心肌细胞集群电生理记录。跳动的心肌细胞电生理剖面显示定期，夹带，让人联想到在临床心电图的P - QRS - T复合物有节奏的冲动。比例尺:0.1毫米。 视频:从南诱导人类胚胎干细胞分化的心肌细胞 。 视频1和2显示代表约1个月和3个月大的跳动心肌细胞簇后附件。视频3和4代表，承包心脏肌肉在更高的功率。视频5显示了一个typica升小跳动的心肌细胞群自发地分化从胚胎干细胞没有不结盟运动作为治疗控制。 <a href="/files/ftp_upload/3274/3274video1.avi">点击这里观看视频1。</a> <a href="http://www.jove.com/files/ftp_upload/3274/3274video2.avi">点击这里观看视频2。</a> <a href="http://www.jove.com/files/ftp_upload/3274/3274video3.avi">点击这里观看视频3。</a> <a href="http://www.jove.com/files/ftp_upload/3274/3274video4.avi">点击这里观看视频4。</a> <a href="http://www.jove.com/files/ftp_upload/3274/3274video5.avi">点击这里观看影片5。</a>

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Biotechnol. 25, 1015-1024 (2007).</li><li>Caspi, O., Huber, I., Kehat, I., Habib, M., Arbel, G., Gepstein, A., Yankelson, L., Aronson, D., Beyar, R., Gepstein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+embryonic+stem+cell-derived+cardiomyocytes+improves+myocardial+performance+in+infarcted+rat+hearts.">Transplantation of human embryonic stem cell-derived cardiomyocytes improves myocardial performance in infarcted rat hearts.</a> J. Am. Coll. Cardiol. 50, 1884-1893 (2007).</li><li>Akazawa, H., Komuro, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+transcription+factor+Csx/Nkx2-5:+Its+role+in+cardiac+development+and+diseases.">Cardiac transcription factor Csx/Nkx2-5: Its role in cardiac development and diseases.</a> Pharmacol. Ther. 107, 252-268 (2005).</li><li>Leda, M., Fu, J. D., Delgado-Olguin, P., Vedantham, V., Hayashi, Y., Bruneau, B. G., Srivastava, D. <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+fibroblasts+into+functional+cardiomyocytes+by+defined+factors.">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142, 375-386 (2010).</li><li>Efe, J. A., Hilcove, S., Kim, J., Zhou, H., Ouyang, K., Wang, G., Chen, J., 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=Conversion+of+mouse+fibroblasts+into+cardiomyocytes+using+a+direct+reprogramming+strategy.">Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy.</a> Nat. Cell. Biol. 13, 215-222 (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地在体外分化成胚层细胞通过多血统的聚合阶段，人类胚胎干细胞衍生的多系聚合（胚体），只有一个非常小的一部分细胞（&lt;2 ％）自发分化成心肌13-15（ 图1A）。继免疫选择，心肌细胞丰富的人口能够作为生物起搏器的功能来抢救损坏的心肌功能，机械和电子注入动物模型的心脏13。在啮齿动物的心肌梗死模型，嫁接人类胚胎干细胞衍生的心肌后代能够生存和成熟的长达12周，部分remuscularIZE受伤的心脏，并改善收缩功能 14,15 。然而，通过多系分化多能干细胞，移植后的致瘤性的高风险的人类心脏承诺细胞中产生的低效率，阻碍了进一步的临床翻译。渠道广泛的多能胚胎干细胞分化潜能，完全可预见的心脏表型的发展战略是利用人类胚胎干细胞生物学在心脏领域的力量至关重要。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1B）。我们发现，烟从P直接诱发心系承诺根据定义文化luripotent的人类胚胎干细胞，进一步发展到与高效率（ 图2）跳动心肌细胞。 Nkx2.5是正常的心脏发育的基因突变是必不可少的一个进化保守的同源盒转录因子与人类先天性心脏疾病（CHD）的，人类最常见的出生缺陷16。 Nkx2.5表达到目前为止，检查所有脊椎动物最早是心脏前体细胞的标志和必要的适当的心脏septation和形成/成熟的电气传导系统16。在脊椎动物中，发病早期Nkx2.5表达模式大致配合的时间和面积在早期胚胎的心脏规范，Nkx2.5基因继续通过发展中的心脏 16表示。在我们的人类胚胎干细胞模型，不结盟运动的出现引发心脏诱导多能性胚胎干细胞直接促进的心肌特异性transcri表达ption因子Nkx2.5，一个过程，可能会效仿从人类胚胎cardiogenesis的多能干外胚层cardiomesoderm规范。未来的研究将揭示人类心脏发育的遗传和表观遗传控制分子作为替代品，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。没有不结盟运动治疗，&lt;2％的人类胚胎干细胞进行自发分化成跳动的心肌细胞 12-15 。不结盟运动治疗，我们已经能够产生大于95％的胚胎的心脏前体跳动的心肌细胞&gt; 50％，并从下一个定义文化的人类胚胎干细胞保持在一个过程，可能会仿效人类胚胎心脏发育12。近日，已知的心脏命运决定基因已被用于transdifferentiate产后击败了&lt;0.5％17，18的低效率的心肌细胞的小鼠成纤维细胞&lt;/ SUP&gt;。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有限的多能胚胎干细胞内细胞团（ICM）或人类囊胚4,5的外胚层衍生，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（8细胞）胚胎阶段的22日和23人为地重新编程的细胞。

<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>烟</td><td>西格玛</td><td> N0636 </td><td></td></tr><tr><td> DMEM/F12 </td><td> Invitrogen公司</td><td> 10565042 </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>纪念氨基酸的解决方案（MEAA，100X） </td&gt; <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>定义胎牛血清</td><td> Hyclone公司</td><td> SH30073 - 03 </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 cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction

至目前为止，一直缺乏一个合适的人类心脏细胞来源的人类心肌再生的重大挫折，通过细胞移植或心脏组织工程1-3。心肌细胞分化成为末期出生后不久，失去增殖能力。没有证据显示，从其他来源如骨髓或脐带血来源的干/祖细胞，都能够引起收缩的心脏肌肉细胞移植到心脏 1-3 。需要再生或修复受损的心脏肌肉没有得到满足成人干细胞疗法，无论是内源性或通过细胞传递1-3。基因稳定的人类胚胎干细胞（胚胎干细胞）的无限扩张能力和无限制的可塑性，无心，听者有意 ，在体外培养的人体细胞的大量供应受到限制，以在需要的血统的推导多能干水库4,5修复和再生。由于全球心血管疾病和急性器官捐赠短缺的患病率，有浓厚的兴趣开发基于人类胚胎干细胞的疗法作为一种替代办法。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，造成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6-8 （见原理图图1a）。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能直接使用这种细胞的带鱼ZED移植患者问题9-11。为了克服这些障碍，我们已经解决了定义的文化系统必要和足够维持的人类胚胎干细胞的外胚层pluripotence的元素，服务从头推导临床适合人类胚胎干细胞作为一个平台，有效地指导对临床相关的谱系，这样的人类胚胎干细胞均匀12小分子（在图中看到的示意图 。1B ）。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的诱导多能胚胎干细胞，进一步发展到产生高效率的人力跳动的心肌细胞的cardioblasts 的直接cardiomesoderm规范（图2烟酰胺（NAM） ）。我们定义为cardioblasts多能胚胎干细胞直接诱导的条件下，不干预多系胚体阶段，从而使控制效率的推导整个基于细胞疗法的发展阶段的频谱的人体心脏细胞的大量供应。

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

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes 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，使推导一个人类心脏的祖细胞和心血管修复功能心肌的大量供应定义的条件下保持直接从cardioblasts协议。

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

efficient-derivation-human-cardiac-precursors-cardiomyocytes-from

Research

JoVE Journal

Biology

19.4K 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，使推导一个人类心脏的祖细胞和心血管修复功能心肌的大量供应定义的条件下保持直接从cardioblasts协议。

视频: Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

科研

生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

人类多能干细胞的神经元在一个Multititre的板格式的快速和高效地生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

人类心肌细胞的生成:从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

从人类Lipoaspirates脂肪来源干细胞的手动隔离

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...