Name: generation of first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells
Uploaded: 2018-06-19T14:00:00.000+00:00
Duration: 8 min 37 s
Description: 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

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>

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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。

<table><tbody><tr><td>ACTC1 antibody</td><td>Sigma</td><td>A7811</td><td></td></tr><tr><td>Activin A</td><td>Stem Cell Technologies</td><td>Hu Recom Activin A</td><td></td></tr><tr><td>Antibiotic Antimycotic (Anti-Anti)</td><td>Thermo Fisher Scientific</td><td>15240062</td><td></td></tr><tr><td>B27 supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>B27 supplement w/o - insulin</td><td>Thermo Fisher Scientific</td><td>A1895601</td><td></td></tr><tr><td>B27 supplement w/o - vitamin A</td><td>Thermo Fisher Scientific</td><td>12587001</td><td></td></tr><tr><td>CDH5 antibody</td><td>R&amp;amp;D Systems</td><td>AF938</td><td></td></tr><tr><td>CryoStor CS10</td><td>Stem Cell Technologies</td><td>7930</td><td>Cryopreservation reagent</td></tr><tr><td>DMEM high Glucose</td><td>Mediatech</td><td>10-013-CV&amp;nbsp;</td><td></td></tr><tr><td>DPBS w/ Ca &amp;amp; Mg</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>EDTA</td><td>Thermo Fisher Scientific</td><td>15575-038</td><td></td></tr><tr><td>FBS</td><td>VWR</td><td>89510-186</td><td></td></tr><tr><td>FluoVolt membrane potential kit&amp;nbsp;&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>F10488</td><td>For optical action potential acquisition, please refer to McKeithan et al. 2017</td></tr><tr><td>KnockOut Serum Replacement</td><td>Gibco</td><td>10828010</td><td></td></tr><tr><td>Matrigel, Growth Factor Reduced</td><td>Corning</td><td>356231</td><td>Coating reagent</td></tr><tr><td>mTeSR1 media kit</td><td>Stem Cell Technologies</td><td>5850</td><td></td></tr><tr><td>PBS w/o Ca &amp;amp; Mg</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td></td><td></td></tr><tr><td>Puromycin&amp;nbsp;</td><td>Acros</td><td>227422500</td><td></td></tr><tr><td>ReLeSR</td><td>Stem Cell Technologies</td><td>5872</td><td>Enzyme-free dissociation reagent</td></tr><tr><td>RPMI 1640</td><td>Thermo Fisher Scientific</td><td>11875-093</td><td></td></tr><tr><td>TAGLN antibody</td><td>Abcam</td><td>ab14106</td><td></td></tr><tr><td>Thiazovivin</td><td>Stem Cell Technologies</td><td>72254</td><td>RHO/ROCK pathway inhibitor</td></tr><tr><td>TrypLE Express</td><td>Thermo Fisher Scientific</td><td>12605 -010</td><td>1X enzyme-containing dissociation reagent</td></tr><tr><td>Tyrodes solution mix packets&amp;nbsp;&amp;nbsp;</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。
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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 Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

使用多电极阵列（MEA）的人多能干细胞来源的心肌细胞（hPSC-CM）的电生理分析

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

小鼠心脏场特定心脏祖细胞的体外生成

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

产生来自多能干细胞的自组装人心脏类器官

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

生物化学

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

患者外周血单核细胞中人心肌细胞的生成和扩增

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

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

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 transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

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 ...

生物学

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

人诱导多能干细胞来源的心脏球体的生成、高通量筛选和生物样本库

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

生物工程

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 ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

生成心室样HiPSC衍生的心肌细胞和用于钙处理特征的高质量细胞制剂

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...