我们感谢可乐实验室的成员对稿件进行有益的讨论和评论。这项研究得到了 NIH/NIEHS R44ES023521-02 和 CIRM DISC2-10110 对可乐博士的资助。

1. Id1 病毒的制备和感染
<ol><li>由转染 pCMVDR8.74、pMD2、pCDH-EF1-Id1-PGK-PuroR (Addgene: 质粒 #107735) 生成 Id1 overexpressing 慢病毒北疆 HEK293T 细胞。收集病毒粒子, 滤液, 从上清纯化, 并存储在-80 摄氏度, 如在次郎等。(2003)19. 或者, 通过向慢病毒北疆生产的供应商发送 pCDH-EF1-Id1-PGK-PuroR, 在商业上生产 Id1 慢病毒北疆。 注意: 请遵守慢病毒载体安全法规和标准操作协议。 注意: 重要的是: 最佳病毒效价应至少为 108至 109传感单位/毫升 (TU/毫升)。</li>
	<li>在感染之前, 用50µl 的涂层试剂涂上一层96的井板, 通常是由 Engelbreth-圣群小鼠肉瘤细胞分泌的凝胶蛋白混合物 (见材料表)。</li>
	<li>在6井板中, 加入1毫升的 PBS, 不含钙2 +镁2 +和0.5 毫米 EDTA, 将一井中的多能干细胞分离出来。 注: 离解时间从3–10分钟不等。当超过80% 的细胞从盘子的底部分离出来时, 继续下一步。</li>
	<li>将无菌吸管的细胞悬浮液收集到15毫升塑料离心管中。</li>
	<li>中和分离试剂与5毫升的 PBS 含钙2 +和镁2 +。</li>
	<li>颗粒细胞离心 (200 x g 3 分钟)。</li>
	<li>去掉上清, 轻轻地轻弹管子, 以去除颗粒。</li>
	<li>在1毫升的干细胞培养基中重新悬浮细胞。</li>
	<li>根据供应商说明使用自动单元格计数器计数单元格。</li>
	<li>在50µL 的干细胞介质中, 每层2万个可行细胞 (96 井板) 辅以2µM/岩石通路抑制剂 (见表1和 2)。</li>
	<li>24小时后, 监测细胞附件和替换介质与100µl 干细胞培养基补充2µM/岩石通路抑制剂和 6 ng/毫升铵溴 (见表 1和2), 一小时之前, 慢病毒载体感染。 注意: 细胞应该牢固地附着在盘子的底部。</li>
	<li>在冰上解冻 Id1-lentivirus。</li>
	<li>添加3µL 的纯化慢病毒北疆每井 (病毒滴度应介于 108到 109涂/毫升), 然后转移板块回细胞培养孵化器 (37 °c, 5% CO2, 20% O2)。</li>
	<li>24 h 病毒感染后, 将新鲜干细胞培养基的100µL 添加到受感染的细胞中。</li>
	<li>48 h 慢病毒载体感染后, 用200µL 新鲜干细胞培养基替换含有培养基的病毒, 辅以1µg/毫升嘌呤霉素。</li>
	<li>每天刷新媒体与200µL 干细胞介质补充1µg/毫升嘌呤霉素。</li>
	<li>当文化达到80% 融合, 离解细胞使用200µL 的 PBS 没有钙2 +和镁2 + 0.5 毫米 EDTA (为一个井的96井板)。</li>
	<li>将细胞悬浮液收集到离心管中。</li>
	<li>中和分离试剂与5毫升的 PBS 含钙2 +和镁2 +。</li>
	<li>颗粒细胞离心 (200 x g 3 分钟)。</li>
	<li>去掉上清, 轻轻地轻弹管子, 以去除颗粒。</li>
	<li>在1毫升的干细胞培养基中重新悬浮细胞, 辅以2µM/岩石通路抑制剂。</li>
	<li>将所有单元 (1 毫升) 转移到12井板的涂敷井中。</li>
	<li>24小时后, 在转导前, 用1毫升的干细胞介质, 用2µM/岩石通路抑制剂和 6 ng/毫升铵溴化酶, 对井内细胞附着进行监测, 并更换培养基。</li>
	<li>在冰上解冻 Id1-lentivirus。</li>
	<li>添加5µL 的纯化病毒每井 (12 井板)。</li>
	<li>24 h 后慢病毒载体感染, 取代含有1毫升新鲜干细胞培养基的病毒, 补充3µg/毫升嘌呤霉素。</li>
	<li>48 h 后慢病毒载体感染, 替换含有1毫升新鲜干细胞培养基的病毒, 辅以6µg/毫升嘌呤霉素。</li>
	<li>当文化达到80% 融合, 离解细胞使用500µL 的 PBS 没有钙2 +和镁2 +和与0.5 毫米 EDTA (为一个井的12井板)。</li>
	<li>将细胞悬浮液的一半 (250 µL) 转移到一井中, 涂层试剂涂覆 6-井板, 含有2毫升的干细胞介质, 辅以2µM/岩石通路抑制剂。</li>
	<li>使用另一半 (250 µL) 的样品 RNA 提取和 qRT PCR, 以确定慢病毒载体介导的Id1表达水平, 如在可乐等。(2012)4。 注意: 重要的是: 如果Id1 mRNA 表达水平低于0.005 倍的GAPDH表达水平 (见表 3为 qRT PCR 引物), 重复感染过程如上所述, 直到 Id1 表达水平超过阈 值。</li>
</ol>2. hPSCsId1维护
<ol><li>培养 hPSCsId1在涂层6个井板和增长在3毫升每井干细胞媒介补充6µg/毫升嘌呤霉素。</li>
	<li>当 hPSCsId1达到80% 融合, 通道细胞作为骨料 (1:6 分裂比率) 使用无酶离解试剂遵循供应商指南和增长3毫升每井的干细胞培养基补充6µg/毫升嘌呤霉素。</li>
</ol>3. hPSCsId1的制备
<ol><li>涂层12井板与500µL 每井涂料试剂。</li>
	<li>当 hPSCsId1达到90% 融合离解细胞通过增加1毫升 PBS 没有钙2 +和镁2 +和以0.5 毫米 EDTA 每井为 5–10 min. 检查离解过程每分钟, 一旦80% 的细胞与板块分离, 请继续下一步。</li>
	<li>将细胞悬浮液收集到离心管中。</li>
	<li>中和分离试剂与5毫升的 PBS 含钙2 +和镁2 +。</li>
	<li>颗粒细胞离心 (200 x g 3 分钟)。</li>
	<li>去掉上清, 轻轻地轻弹管子, 以去除颗粒。</li>
	<li>在2毫升的干细胞培养基中重新悬浮细胞, 辅以2µM/岩石通路抑制剂。</li>
	<li>使用自动单元格计数器计数单元格。</li>
	<li>在1毫升的干细胞培养基中, 30万细胞每井均辅以2µM/岩石通路抑制剂和6µg/毫升嘌呤霉素, 并将其转移到组织培养孵化器中。</li>
	<li>每日刷新媒体与2毫升干细胞培养基补充6µg/毫升嘌呤霉素, 直到文化达到90% 融合。在这一点上, 启动心脏分化 (下一步)。</li>
</ol>4. hPSCsId1分化为第一心脏场样心脏祖细胞 (FHF CPs)
<ol><li>对于12井板格式, 启动分化 (0 天) 取代干细胞介质与1.5 毫升的感应介质 (表 1) 补充与激活 A (100 ng/毫升) (见表 2的建议体积和激活浓度不同的板格式)。</li>
	<li>1天 (在分化开始后24小时), 用2毫升的感应介质替换介质, 而不激活 a (每井12井板)。</li>
	<li>3天, 用2毫升的感应介质替换介质, 而不激活 a (每井12井板)。</li>
	<li>在5天, 收集 FHF CPs 进行冷冻保存 (下一步)。 注意: 重要的是: 在这一点上, 冷冻保存是首选, 因为它使拆的心脏祖生成从随后的心肌细胞生产和生物库大批电池。请注意, 另一种情况是, 5 天 FHF cps 可以传递到一个新的涂层板继续分化, 请参阅步骤5.1–5.5 到 passFHF CPs, 然后进行步骤6.5 的区别。</li>
</ol>5. FHF 的超低温保存
<ol><li>从含有5天 FHF CPs 的水井中吸取所有介质, 并快速添加1毫升的温 (37 °c) 1x 酶的离解试剂 (见材料表)。把盘子放在组织培养孵化器里。</li>
	<li>1分钟后, 在孵化器中摇板侧侧。</li>
	<li>2分钟 (总时间) 后, 从孵化器取出板, 并添加1毫升10% 的血清介质。</li>
	<li>用5毫升的吸管轻轻吸管细胞, 以促进细胞脱离板块。</li>
	<li>用 200 x g 离心法在离心管和颗粒细胞中收集细胞悬浮液3分钟</li>
	<li>在2毫升的冷冻保存试剂中重新悬浮细胞颗粒。</li>
	<li>使用自动单元格计数器计数单元格。</li>
	<li>添加冷冻剂 (见材料表) 在足够的体积, 以 cryopreserve 细胞在所需浓度 (通常 5–10 x 106细胞/毫升)。</li>
	<li>将细胞转移到冷冻瓶中, 将瓶子放在冷却装置中 (1 °c/分钟), 并将冷却装置放在-80 摄氏度冷藏柜中过夜。</li>
	<li>24小时后, 将冷冻小瓶转移到液氮中长期贮存。 注意: 协议可以在这里暂停。</li>
</ol>6. FHF 向心室状心肌细胞分化
<ol><li>转让日 5 FHF 从液氮储存到干冰容器的冷冻瓶。</li>
	<li>放置小瓶在37°c 水浴为2到3分钟, 直到细胞悬浮完全地解冻。 注意: 重要的是: 解冻过程是时间敏感的。将细胞暴露在冷冻介质中, 时间过长 (即10 分钟) 会降低细胞的存活能力。</li>
	<li>转移细胞到15或50毫升离心管, 取决于解冻瓶的数量。</li>
	<li>分配滴状 (每30秒5滴) 预预热 (37 °c) 心源介质 (表 1) 到细胞溶液。继续这个过程, 直到1:3 冷冻介质: 心源介质比率达到。此时, 增加心源性培养基的添加率直到1:10 冷冻介质: 心源介质比达到。 注意: 重要: 快速渗透改变会增加细胞在解冻过程中的死亡;因此, 强烈建议, 如上文所述, 滴状添加心源性介质。</li>
	<li>离心细胞悬浮和颗粒细胞离心 (200 x g 3 分钟)。</li>
	<li>移除上清液, 轻轻轻拍离心管, 取出细胞颗粒。</li>
	<li>再悬浮细胞与心源介质补充2µM/岩石通路抑制剂。 注: 细胞活力可以从解冻到解冻的不同, 一般来说, > 65% 的可行性预测良好的电镀效率。</li>
	<li>用心源介质稀释浓缩细胞悬浮液, 辅以2µM/岩石通路抑制剂, 以获得所需的电镀细胞浓度 (见表 2为不同的板材格式)。 注意: 重要的是: 注意, 如果细胞种子太稀少, 心脏分化效率可能会下降。</li>
	<li>种子细胞在板上。</li>
	<li>将盘子放在组织培养罩中20分钟, 然后将其转移到组织培养孵化器。</li>
	<li>在6天的分化, 监测细胞附着。 注: 心脏祖细胞保存在分化的5天。24 h 在解冻以后细胞将是在6天分化。 注意: 漂浮 (死) 细胞的存在是常见的。</li>
	<li>在7天, 吸入50% 的媒体, 并取代相同数量的新鲜心源性媒体。 注: 在这一点之后, 不需要在心源性介质中添加一条。</li>
	<li>每隔一天用心源介质替换50% 的介质。</li>
	<li>注意在11天和13天之间出现的自发跳动。</li>
	<li>在15天, 用 DAPI (核染色) 和 ACTC1 (泛心标记) 对免疫荧光定量测定心脏分化效率。</li>
</ol>7. 心室状心肌细胞的传递和维持
注意: 14–16天, 应获得一层自发收缩心室状心肌细胞。在这一点上, 建议分离和重新板细胞, 以融汇的文化和防止细胞脱离板块。
<ol><li>从水井中吸取所有介质, 并快速添加1毫升预热 (37 °c) 1x 酶的离解试剂。 注: 1x 含酶的离解试剂体积因板材格式而异, 但应完全覆盖井表面的底部。</li>
	<li>将板块转移到组织培养孵化器中。</li>
	<li>在孵化箱内每隔2分钟轻轻摇动板侧, 加速脱离过程。</li>
	<li>在5–15分钟后, 当80% 到100% 的细胞从盘子的底部分离出来时, 从孵化器中取出盘子, 并通过增加等量的10% 种血清介质来抑制1x 酶的离解试剂活性。 注: 这一过程中含有1x 酶离解的孵化时间将因批次而异。</li>
	<li>将细胞悬浮液收集到离心管上。</li>
	<li>使用自动单元格计数器将单元格悬浮与吸管和计数单元格混合。</li>
	<li>稀释细胞悬浮物到期望的细胞集中 (参见表 2为不同的板材格式) 与维护媒介补充与2µM/岩石通路抑制剂。</li>
	<li>将心肌细胞播种后, 均匀分布, 使细胞在转印前10–20最小。</li>
	<li>24小时后再电镀, 监控细胞的附着和恢复。 注意: 漂浮 (死) 细胞的存在是常见的。48–72在重新电镀后, 心肌细胞应恢复自发收缩。</li>
	<li>为了保持心肌细胞的培养, 每隔一天将50% 的培养基替换为维持介质, 直到使用心室状心肌, 进行后续实验。</li>
</ol>

<ol>
	<li>Chong, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic-stem-cell-derived+cardiomyocytes+regenerate+non-human+primate+hearts.">Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.</a> Nature. 510, 273-277 (2014).</li><li>Kodo, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPSC-derived+cardiomyocytes+reveal+abnormal+TGF-beta+signalling+in+left+ventricular+non-compaction+cardiomyopathy.">iPSC-derived cardiomyocytes reveal abnormal TGF-beta signalling in left ventricular non-compaction cardiomyopathy.</a> Nature cell biology. 18, 1031-1042 (2016).</li><li>Kim, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+arrhythmogenic+right+ventricular+dysplasia+with+patient-specific+iPSCs.">Studying arrhythmogenic right ventricular dysplasia with patient-specific iPSCs.</a> Nature. 494, 105-110 (2013).</li><li>Colas, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-genome+microRNA+screening+identifies+let-7+and+mir-18+as+regulators+of+germ+layer+formation+during+early+embryogenesis.">Whole-genome microRNA screening identifies let-7 and mir-18 as regulators of germ layer formation during early embryogenesis.</a> Genes &amp; development. 26, 2567-2579 (2012).</li><li>Cunningham, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Id+genes+are+essential+for+early+heart+formation.">Id genes are essential for early heart formation.</a> Genes &amp; development. , (2017).</li><li>McKeithan, W. L., Colas, A. R., Bushway, P. J., Ray, S., Mercola, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+generation+of+multipotent+mesoderm+(Kdr+)+progenitor+cells+in+mouse+embryonic+stem+cells+for+functional+genomics+screening.">Serum-free generation of multipotent mesoderm (Kdr+) progenitor cells in mouse embryonic stem cells for functional genomics screening.</a> Current protocols in stem cell biology. , 13 (2012).</li><li>McKeithan, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+platform+for+assessment+of+congenital+and+drug-induced+arrhythmia+with+hipsc-derived+cardiomyocytes.">An automated platform for assessment of congenital and drug-induced arrhythmia with hipsc-derived cardiomyocytes.</a> Frontiers in physiology. 8, 766 (2017).</li><li>Sharma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+of+tyrosine+kinase+inhibitor+cardiotoxicity+with+human+induced+pluripotent+stem+cells.">High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells.</a> Science translational medicine. 9, (2017).</li><li>Burridge, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+generation+of+human+cardiomyocytes.">Chemically defined generation of human cardiomyocytes.</a> Nature methods. 11, 855-860 (2014).</li><li>Kattman, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+optimization+of+activin/nodal+and+BMP+signaling+promotes+cardiac+differentiation+of+mouse+and+human+pluripotent+stem+cell+lines.">Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.</a> Cell stem cell. 8, 228-240 (2011).</li><li>Laflamme, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocytes+derived+from+human+embryonic+stem+cells+in+pro-survival+factors+enhance+function+of+infarcted+rat+hearts.">Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts.</a> Nature biotechnology. 25, 1015-1024 (2007).</li><li>Lian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+via+temporal+modulation+of+canonical+Wnt+signaling.">Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 1848-1857 (2012).</li><li>Willems, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule-mediated+TGF-beta+type+II+receptor+degradation+promotes+cardiomyogenesis+in+embryonic+stem+cells.">Small molecule-mediated TGF-beta type II receptor degradation promotes cardiomyogenesis in embryonic stem cells.</a> Cell stem cell. 11, 242-252 (2012).</li><li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cardiovascular+progenitor+cells+develop+from+a+KDR++embryonic-stem-cell-derived+population.">Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.</a> Nature. 453, 524-528 (2008).</li><li>Palpant, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+high-purity+cardiac+and+endothelial+derivatives+from+patterned+mesoderm+using+human+pluripotent+stem+cells.">Generating high-purity cardiac and endothelial derivatives from patterned mesoderm using human pluripotent stem cells.</a> Nature protocols. 12, 15-31 (2017).</li><li>DeLaughter, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+resolution+of+temporal+gene+expression+during+heart+development.">Single-cell resolution of temporal gene expression during heart development.</a> Developmental cell. 39, 480-490 (2016).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+genetics+of+hypoplastic+left+heart+syndrome.">The complex genetics of hypoplastic left heart syndrome.</a> Nature genetics. 49, 1152-1159 (2017).</li><li>Corrado, D., Link, M. S., Calkins, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrhythmogenic+right+ventricular+cardiomyopathy.">Arrhythmogenic right ventricular cardiomyopathy.</a> New England journal of medicine. 376, 1489-1490 (2017).</li><li>Kitamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrovirus-mediated+gene+transfer+and+expression+cloning:+powerful+tools+in+functional+genomics.">Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics.</a> Experimental hematology. 31, 1007-1014 (2003).</li></ol>

作者没有什么可透露的。

为成功鉴别, 请务必密切遵循上面列出的指示。此外, 我们在这里强调的关键参数, 强烈影响分化的结果。在开始分化之前, 应观察以下三种形态学参数: hPSCsId1的茎形态、高细胞压实和0天的高汇流 (> 90%)。在这方面, 最佳分化条件最好是以电镀分离 hPSCsId1为单细胞, 并允许它们形成紧密的聚类膜, 在2到3天的过程中增长到90% 汇合。反之, 如果 hPSCId1文化表现出较差的细胞和菌落...

大规模生产人多潜能干细胞 (hPSCs) 源性心脏祖细胞和心肌细胞是干细胞治疗的先决条件1, 疾病建模2,3和快速表征新的路径调节心脏分化4,5,6和生理学7,8。虽然一些研究9,10,11,12,13,14,15以前描述高效心脏分化协议从 hPSCs, 没有解决导致心肌细胞的心脏领域起源, 尽管辨认重要分子区别左 (第一心脏领域) 和权利 (第二心脏领域)心室心肌细胞16与心场特异性先天性心脏病的存在;即发育不良左心综合征17或致心律失常右心室发育不良18。因此, hPSCs 的心源和心肌细胞的产生是提高其作为治疗和疾病建模工具相关性的必要条件。
这个协议依赖于 Id1 的本构表达, 最近确定的5第一心脏领域-指定提示, 结合激活 a, 是必要的, 足以启动 cardiogenesis 在 hPSCs。值得注意的是, 坎宁安等。(2017)5显示 Id1-induced 祖细胞特别表达第一心脏领域 (HCN4, TBX5), 但不是第二心脏领域标记 (SIX2, ISL1), 当他们接受心脏分化。此外, 作者还表明, 转基因小鼠胚胎缺乏完整的基因家族 (Id1-4), 发展不形成第一心脏领域的心脏祖细胞, 而更多的内侧和后心脏祖细胞 (第二心脏领域) 仍然可以形成, 从而表明, Id 蛋白是必不可少的启动第一心脏领域 cardiogenesis在体内。方便地, Id1-induced 祖细胞可冷冻保存, 自发分化为心肌样特征, 包括心室特异性标记 (IRX4、 MYL2) 表达和心室状动作电位。
在这里, 我们描述了一个简单的和可伸缩的方法, 以产生第一个心脏场样 (FHF) 心脏祖细胞和心室样心肌 Id1 overexpressing hPSCs。该协议的一个重要特点是, 通过一个方便的超低温保存步骤, 拆的心肌祖细胞产生的可能性。总之, 本协议详细说明了必要步骤 (1) 生成 Id1-overexpressing hPSCs, (2) 从 hPSCs 生成 FHF 心祖细胞, (3) cryopreserve 产生祖细胞, (4) 恢复 FHF-l 心祖分化, 生成高度丰富的 (> 70–90%) 跳动的心室状心肌细胞。

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			<td height="20" style="height:20px;width:204px;">ACTC1 antibody</td>
			<td style="width:144px;">Sigma</td>
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			<td height="20" style="height:20px;">Activin A</td>
			<td>Stem Cell Technologies</td>
			<td>Hu Recom Activin A</td>
			<td></td>
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			<td height="20" style="height:20px;">Antibiotic Antimycotic (Anti-Anti)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
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			<td height="20" style="height:20px;">B27 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement w/o - insulin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement w/o - vitamin A</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CDH5 antibody</td>
			<td style="width:144px;">R&#38;D Systems</td>
			<td>AF938</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CryoStor CS10</td>
			<td>Stem Cell Technologies</td>
			<td>7930</td>
			<td style="width:444px;">Cryopreservation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM high Glucose</td>
			<td>Mediatech</td>
			<td>10-013-CV&#160;</td>
			<td></td>
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			<td height="20" style="height:20px;">DPBS w/ Ca &#38; Mg</td>
			<td>Corning</td>
			<td>21-030-CV</td>
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			<td height="20" style="height:20px;">EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575-038</td>
			<td></td>
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			<td height="20" style="height:20px;">FBS</td>
			<td>VWR</td>
			<td>89510-186</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">FluoVolt membrane potential kit&#160;&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>F10488</td>
			<td>For optical action potential acquisition, please refer to McKeithan et al. 2017</td>
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		<tr height="20">
			<td height="20" style="height:20px;">KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel, Growth Factor Reduced</td>
			<td>Corning</td>
			<td>356231</td>
			<td style="width:444px;">Coating reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mTeSR1 media kit</td>
			<td>Stem Cell Technologies</td>
			<td>5850</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">PBS w/o Ca &#38; Mg</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
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			<td height="20" style="height:20px;">Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Puromycin&#160;</td>
			<td>Acros</td>
			<td>227422500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ReLeSR</td>
			<td>Stem Cell Technologies</td>
			<td>5872</td>
			<td style="width:444px;">Enzyme-free dissociation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TAGLN antibody</td>
			<td style="width:144px;">Abcam</td>
			<td>ab14106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thiazovivin</td>
			<td>Stem Cell Technologies</td>
			<td>72254</td>
			<td style="width:444px;">RHO/ROCK pathway inhibitor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE Express</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605 -010</td>
			<td style="width:444px;">1X enzyme-containing dissociation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tyrodes solution mix packets&#160;&#160;</td>
			<td>Sigma</td>
			<td>T2145-10X1L</td>
			<td>(For optical action potential acquisition, please refer to McKeithan et al. 2017)</td>
		</tr>
	</tbody>
</table>

generation of first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells

大量的功能性人类多潜能干细胞的产生是基于细胞的心脏治疗和疾病建模的先决条件。我们最近表明, Id 基因是必要的, 足以指定第一个心脏领域祖细胞在脊椎动物的发展。这种分化协议利用这些发现, 并使用 Id1 过度表达结合激活 A 作为有力的指定线索, 以产生第一个心脏场状 (FHF) 祖细胞。重要的是, 由此产生的祖细胞有效地区分 (~ 70–90%) 成心室状心肌。这里我们描述了一个详细的方法到 1) 生成 Id1-overexpressing hPSCs 和 2) 区分可伸缩数量的 cryopreservable FHF 祖细胞和心室样心肌。

hPSCs 的产生Id1线条 hPSCs 感染慢病毒北疆调解 Id1 过度表达 (图 1a)。一旦 hPSCId1生成, 转基因的表达是量化的 qRT PCR (图 1B)。只有 hPSCId1线表达Id1 mRNA 的水平大于0.005 倍的GAPDH应用于分化。
<p class="jove_content" fo:keep-together.within-...

Watch this Scientific Journal Video about Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells at JoVE.com

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

在这里, 我们描述了一种可伸缩的方法, 使用激活 a 和慢病毒北疆介导的 Id1-overexpression 的简单组合, 从人类多潜能干细胞中生成第一个心场样的心脏祖细胞和心室样心肌细胞。

人多潜能干细胞第一心场样心脏祖细胞和心室样细胞的生成

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

generation-first-heart-field-like-cardiac-progenitors-ventricular

Research

JoVE Journal

Developmental Biology

6.9K 次观看.  Sanford Burnham Prebys Medical Discovery Institute. 在这里, 我们描述了一种可伸缩的方法, 使用激活 a 和慢病毒北疆介导的 Id1-overexpression 的简单组合, 从人类多潜能干细胞中生成第一个心场样的心脏祖细胞和心室样心肌细胞。

视频: Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

从人类诱导多能干细胞使用小分子调制分化和随后的葡萄糖饥饿高度纯化心肌推导

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

科研

发育生物学

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 ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

从多能干细胞使用BMP拮抗剂Grem2心房心肌细胞分化

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

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 ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...