Name: differentiation of atrial cardiomyocytes from pluripotent stem cells using the bmp antagonist grem2
Uploaded: 2016-03-10T15:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2 at JoVE.com

":在调查培训计划在心血管机制"（JB）这项工作是由美国国立卫生研究院资助HL083958和HL100398（AKH）和2T32HL007411-33支持。

1.准备细胞培养基，解决方案和试剂。 <ol><li>制成500毫升之小鼠胚胎干细胞的（MESC）通过混合和无菌过滤（0.2微米孔径）445毫升格拉斯哥最低必需培养基（GMEM），将50ml热灭活的小牛血清（FBS），媒体5毫升100X浓缩的L-谷氨酰胺置换，5微升的重组小鼠白血病抑制因子（LIF）以每毫升1×10 7个单位，和1.43微升β-巯基乙醇（终浓度为50μM）。在4℃保存待用。 </li><li>通过混合和无菌过滤制成500毫升之胚体（EB）分化培养基的391毫升Iscove氏改良的Dulbecco培养基（IMDM），将100ml热灭活的FBS，加入5ml 100X浓缩L-谷氨酰胺置换，加入5ml 100X浓缩非必需氨基酸（NEAA），和2.86微升β-巯基乙醇（终浓度为100μM）。储存于4˚C，直到准备使用。 </li><li>准备0.2％（重量）明胶/ v溶液通过混合500毫升过滤1克猪皮明胶粉末，蒸馏H 2 O高压灭菌。需要注意的是在RT明胶可能不完全溶于水。高压灭菌后的明胶应完全溶解。在室温下存放待用。 </li><li>通过溶解冻干Grem2 50微克粒料在100补充有0.1％BSA的做出0.5微克/微升原液微升磷酸盐缓冲盐水（PBS）制备Grem2重组蛋白质等分试样。等分试样20微升该储液的五个无菌1.5毫升离心管中并储存在-80˚C，直到准备使用。 </li><li>根据公布的协议或购买为10X库存准备PBS稀释1:10过滤，蒸馏水2 O做出1X工作稀释度19。通过在使用前高压灭菌消毒的PBS。在室温下存放待用。在一些步骤，DPBS不含Ca 2+和Mg 2+被使用。这是PUrchased作为1X解决方案。订购信息被包括在试剂的表。 </li></ol> 2.明胶的制备涂层10厘米组织培养皿<ol><li>在无菌组织培养罩溶于5ml 0.2％的明胶溶液中添加用于涂覆每个10厘米组织培养皿。地方菜在在37℃培养箱中在至少30分钟或长达4天。 </li></ol> 3.明胶的制备包被的6孔组织培养板<ol><li>在无菌组织培养罩1毫升0.2％明胶的添加至6孔组织培养板的每个孔中。代替板中在37℃培养箱中在至少30分钟或长达4天。 </li></ol> 4.胚胎干细胞培养<ol><li>解冻mESCs <ol><li>通过在无菌组织培养罩温热至RT制备卓制介质。 10毫升RT媒体添加到明胶涂层10cm皿。 </li><li>除去含有约1×10 6从细胞mESCs的1瓶cryostorage。拿着离心管用钳子，可在37℃水浴。轻轻旋转，直到细胞悬浮液解冻，只有一小冰晶保持在小瓶的中心。擦干小瓶用一次性无绒擦拭，并用70％乙醇消毒。 </li><li>在无菌组织培养罩取下解冻细胞利用离心管P1000的提示，并传输到4.1.1制备的10 cm培养皿）。 </li><li>放置10cm皿到37℃，5％CO 2的湿润的细胞培养箱中，并均匀地轻轻移动板前后散布的细胞，然后一边到另一边。允许细胞附着到板O / N或至少6小时。 </li><li>首先用明视场显微镜下一晨检细胞附着。一些细胞碎片和死细胞将在媒体中观察到。这个是正常的。从菜肴吸媒体和替换用10毫升新鲜卓制介质。 </li><li>每天都在改变细胞的媒体，以防止分化。 </li></ol></li><li&gt;传代mESCs <ol><li>传代细胞在60-70％汇合。通过吸媒体，并用5毫升无菌1X DPBS冲洗而不的MgCl 2和氯化钙准备传代细胞。加2ml的0.05％胰蛋白酶-EDTA溶液。在孵化器放置在37℃5分钟。 </li><li>当细胞培养，通过吸出明胶溶液，并加入10ml RT卓制媒体准备新10cm皿。 5分钟后检查细胞脱离。如果细胞保持连接，继续在37℃在一个时间孵育2分钟，直到完全脱离。 </li><li>通过加入3ml卓制媒体的猝灭胰蛋白酶-EDTA。转移细胞悬液至15ml离心管中并旋转，在200×g离心5分钟以沉淀。从颗粒吸媒体和10毫升MESC媒体再次暂停。 </li><li>根据所需的分割比例，添加0.5-1.0毫升细胞悬液，在步骤4.2.2准备每道菜）。将盘中孵化器均匀地轻轻回移动到前面，然后一边到另一边分发细胞。所有流细胞附着的O / N或至少12小时。 注:1:10的分路比 - 1:20是最卓制线常见。这个比率可根据所使用的线路，通道号，以及在新的盘所需合流而变化。对于1:10的分路比将细胞悬液1毫升到9毫升卓制媒体和板新制备的明胶涂层10cm皿。 </li></ol></li></ol> 5.准备胚体注:除离心所有步骤在无菌组织培养罩中进行。 <ol><li>在EB媒体细胞悬浮液的制备<ol><li>在70％汇合（通常24-48小时后通过），用于EB形成细胞。 4.2.3） - 通过分离，并根据章节4.2.1）制粒准备EB形成细胞。 注:（ 例如 ，很少细胞死亡，没有污染，适当的形态），这是最好的传代细胞至少一次解冻后，检查健康和强劲的增长（如克，每天发生的24-48小时前群体倍增）用于拨打日圆。 </li><li>重悬在5ml EB介质颗粒。计数使用血球或自动细胞计数细胞。 </li><li>稀释细胞悬浮液中的EB媒体的需要的体积，使25个细胞/微升（或2.5×10 3细胞/ ml）的终浓度。注意，培养皿的每个盖子保持约60的EB，并且需要大约1.3 ml的细胞悬浮液。在此步骤中，制备细胞悬浮液的足够的体积，以产生胚的期望数量。例如，如果需要进行实验240的EB，以2.5×10 3个细胞/ ml制备5.2毫升。 注:如果不用于EB形成mESCs可以通过旋转下来，重新悬浮于卓制媒体和第4.2节电镀用于将来的实验。 </li></ol></li><li>胚体创作<ol><li>通过加入2ml 1X PBS对电子商务的底部准备无菌10厘米细菌培养皿为悬滴形成ACH。 </li><li>传送来自步骤5.1 EB悬浮液），以无菌溶液盆。 </li><li>从制备的培养皿中移除盖并小心沉积六十20 EB悬浮微升滴上的内表面。做到这一点有效地使用装有6提示多通道移液管。每20微升滴现在有500个细胞。 </li><li>轻轻盖上盖子上的细菌培养皿的下半部分，小心不要打跑悬滴。放置成菜细胞培养培养箱中于37˚C. 2天后，细胞准备如5.3所描述的冲洗）。 </li></ol></li><li>胚体冲落 注意:所有冲落的步骤在无菌组织培养罩中进行。 <ol><li>术后第2天EB的准备清洗下来，转移〜6厘米的细菌培养皿中。预暖3毫升每个培养皿EB媒体RT的。每个6厘米细菌培养皿可以有效地保持2盖子值得挂滴。 注意:一定要使用非涂布培养皿˚F或该步骤（见材料表）。不要使用组织培养皿或鼓励细胞粘附任何物体的表面。如第6条所述应EB中保持悬浮在媒体直到准备进行电镀。 </li><li>从培养箱除去10cm陪替皿并放入无菌的组织培养罩。小心地取下锅盖胚，并在45度角倾斜。 </li><li>使用装有3毫升RT的EB培养基5ml的血清吸管，轻轻在盖的底部洗胚成池。仔细检查被粘表面，并在必要时重新冲洗胚盖子。 </li><li>洗所有的EB转移到一个6cm培养皿中后。 </li><li>所有的EB重复步骤5.3.2）-5.3.4）。每个6cm培养皿中应该从两个10厘米盖包含的EB。将所有的菜放回孵化器在37℃两天。 注:胚过于接近在一起，悬浮物粘在一起，并形成大的聚集。这可以不利地影响心脏的分化效率以及显著减少可用胚的数量。为了防止聚集，胚应每天检查并轻轻来回移动分开，然后一边到另一边。在早期分化步骤中使用的细菌的菜肴有必要防止EB附着于培养皿和允许的EB，以在悬浮液中生长。 </li></ol></li></ol> 6.电镀和治疗胚。随着Grem2 <ol><li>通过加入足量的库存（0.5微克/微升）Grem2至RT EB培养基为1-5微克/ ml的最终稀释度制备Grem2处理介质。 注:有效浓度用于治疗的EB的变化取决于细胞系和当前很多Grem2的比活性。它建议使用剂量之前滴定找到最大效用。对于CGR8卓制线，我们经常使用3-5微克/毫升Grem2的。 Grem2的有效浓度的心房肌细胞的产生会产生之间的快速萎缩细胞第7-8天是贯穿每个普遍良好。如果订约表型没有在处理的培养物观察到的，建议的Grem2剂量指示的范围内进行滴定。 </li><li>从每个孔抽吸明胶包衣溶液井和加入2ml RT Grem2处理介质的制备6孔组织培养板中。从孵化器中取出胚，并放置到无菌组织培养罩。 </li><li>使用P1000从6厘米培养皿设定为100微升转移30的EB到每个孔中。地方胚进入孵化器均匀地分散轻轻移动板后到前，然后一边到另一边。 <ol><li>胚已连接到明胶涂井后，更换Grem2媒体每两天，直到10 10天的一天后，中止Grem2治疗和补充新鲜EB媒体每两天要保持健康的细胞。 注:连接后，胚将沿涂板的表面变平。 </li></ol></li></ol> 7.细胞解离用于RT-qPCR的ANALYSIS <ol><li>细胞解离与胰蛋白酶-EDTA <ol><li>从每孔吸媒体和每无菌DPBS负的Ca +和Mg2 +的孔1 mL冲洗两次。 </li><li>将0.5ml的0.25％的无菌胰蛋白酶-EDTA溶液，以从该单元将被收集各孔中。 </li><li>放置在培养箱板在37℃5分钟。 </li><li> 5分钟后检查细胞分离。如果细胞仍保持附着在井抽头的底部轻轻地松开。如果攻不放松的细胞，放回孵化器和监控每2分钟，直到细胞完全不沾边。 </li><li>通过加入0.5ml的EB培养基到每个孔中和胰蛋白酶-EDTA溶液。 </li><li>传送全部的1 ml的细胞悬浮液到1.5ml微量离心管中。 </li><li>沉淀细胞通过在台式离心机纺丝在300×g下5分钟。 </li><li>从吸颗粒媒体和在-70℃下继续前进立即溶解或存储颗粒，直到准备裂解。长期供应不推荐粒料m个存储，因为这可能会导致RNA降解。 </li></ol></li><li>细胞裂解和分子分析<ol><li>在这一点上的细胞用市售柱提取试剂盒或根据由制造商提供的方案使用RNA提取试剂（见材料表）已准备好用于RNA分离。一旦分离，反向转录的RNA，并使用标准技术20,21使用的cDNA用于RT-qPCR的分析。 注:此实验室RT-qPCR的引物使用如表1房上市身份，使用RT-qPCR的组合来看待的心房和心室基因的表达和电生理记录到动作电位时程（见第8）证实。基因的选择被包括在该协议作为可靠心房和心室的标记的例子。关于在多能干细胞分化的上下文心房和心室的基因表达进一步信息在参照图14中找到。&lt;/ LI&gt; </ol></li></ol> 8.电生理分析<ol><li>细胞分离与胶原酶<ol><li>从每个孔，并与每无菌PBS孔的1 mL冲洗两次抽吸介质。 </li><li>加入0.5毫升1毫克/毫升胶原酶溶液每个孔中。 </li><li>放置在培养箱板在37℃10分钟。 </li><li> 5分钟后检查细胞分离。如果细胞仍保持彼此附着或阱抽头的底部轻轻地松开。轻轻研磨带P1000进一步解离的细胞。 注意:为方便片钳记录细胞应研磨直到的单细胞悬浮液来实现的。 </li><li>将0.5ml的EB培养基到每个孔中，并通过移液混合。 </li><li>传送全部的1 ml的细胞悬浮液到1.5ml微量离心管中。 </li><li>沉淀细胞通过在台式离心机纺丝在300×g下5分钟。 </li><li>从细胞沉淀吸媒体。 </li><li>再暂停500个细胞μLPBS中有10％FBS，作为确保磨碎成单细胞悬浮液。 </li></ol></li><li>电表征。 注意:许多优秀的协议已发表在本杂志上使用单细胞膜片钳测定细胞的膜电位。有关如何执行单细胞膜片钳新手读者详细信息被称为这些协议21-24。下面包含此协议，这将有助于有经验的电生理准备和处理衍生卓制为CM的表征亚型的具体信息。 <ol><li>制备下列组成的细胞内记录溶液:10mM的HEPES缓冲液，5mM的MgATP，2毫EGTA，110毫谷氨酸钾，10mM的氯化钾和10mM NaCl等。调整溶液至用NaOH的pH为7.2。 </li><li>制备细胞外记录（浴）溶液以下组成:2毫米的HEPES，10mM葡萄糖，1mM的MgCl 2的，2毫氯化钙 ，5.4毫米氯化钾，和137毫米氯化钠。调整溶液至用NaOH的pH值为7.4。 </li><li>当准备用标准的硼硅酸盐玻璃长丝录音解决方案回填测量2-5兆欧电阻玻璃移液管。 </li><li>将100微升悬浮细胞进入录音室包含使用P200细胞外记录解决方案。 注:细胞可以在悬浮液中进行修补或，便于搬运和使用药物治疗和/或灌注测定，可以接种到如在8.2.5概述了明胶包被盖玻片）。 </li><li>通过将10毫米直径的玻璃盖玻片到6孔板的每个孔中，并与明胶溶液覆盖作为该协议的3.1节中所述）制备明胶涂覆的玻璃盖玻片上。 <ol><li>至少30分钟抽吸明胶溶液后，加入100微升细胞悬浮液直接向盖玻片和整个6孔板放入培养箱，至少2小时或直到细胞已经附着。细胞已连接后，Remove盖玻片和地点与细胞外的录音解决方案录音室。 注意:如果使用αMHC-红色荧光蛋白荧光心脏记者卓制线，CM也通过其红色荧光的细胞核进行识别。如果不使用心脏记者线，自发收缩的细胞能够观察并分离为测量。多能干细胞衍生的CM通常更小更圆润，缺乏完全形成成人的心肌细胞的特性矩形形态。 </li></ol></li><li>让所有的记录，在37℃。修补用全细胞膜片钳在电流钳模式2-5兆欧电阻的硼硅玻璃吸管单细胞。使用温和的负压达到之前获得整个小区接入千兆欧姆密封。采用快速负压破裂膜，并获得前记录膜电位的全细胞的访问。 </li><li>唤起使用4毫秒的去极化电流注射的动作电位。 注意:对于PSC衍生的CM所需的电流的量可重复触发的动作电位可以变化。为了找到最佳的阈值电流，开始在以逐步的方式相对低的电流和增加触发电流幅度，直到获得可再现的动作电位。 注:操作为Grem2诱导心房肌细胞电位的持续时间通常是在20〜40毫秒的范围内，并且缺乏一个平台期（ 图6B）。 </li></ol></li></ol>

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The authors of this manuscript have no competing financial interests.

该协议通常产生培养物与那些心房谱系特征的CM的高百分比。正如任何分化方案之前，分化mESCs的质量应给予特别的注意。 mESCs应定期监测适当的形态（ 图1A）。该形成的EB的前发生的任何自发分化将严重限制心脏发生的效率，应传代（ 图1B）之前被移除。 EB大小也影响心脏发生。 200和1000元之间的EB启动细胞的数量已经过测试，每500 EB常规的细胞产生的CM的最高数字。被前...

多能干细胞用于产生和从困难宿主研究细胞到访问组织的基础研究和临床前研究，特别是在人类中1-5的有力工具。发育信号通路的合适的调制可以直接多能干细胞分化为所需表型的命运。许多协议已被开发，以产生从多能干细胞6-14心肌（CMS）。这些协议通常包括TFGβ超家族（活化素，BMP和TGF-β）和Wnt通过定时加入外源性生长因子和/或小分子10,12-15途径调制。这些协议是在增加了成为的CM细胞的百分比通常是有效的，但缺乏特异性，产生代表心房，心室和节点/传导系统谱系细胞的混合群。为了研究心肌特异性亚型更定向分化Appro公司ACH是必需的。 Gremlin2（Grem2，也被称为蛋白相关丹和地狱犬或PRDC的简称）是一种分泌BMP拮抗剂，在斑马鱼16心脏发育过程中是必要进行适当的心脏分化和心房腔的形成。治疗分化胚胎干细胞与Grem2在分化第4天，只是中胚层标记物的T的Brachyury和Cerberus的样1的峰表达后，增加的CM的产率，并产生主要心房谱系14的细胞池。 重组Grem2用于治疗的分化细胞，并且可以使用标准蛋白质的生产技术17制成，或者可以商购买到。它是在水溶液中高度可溶，并且可以在期望的时间点外源添加到培养物。 分化可以利用RT-qPCR的量化标记代表的表达来跟踪心血管祖细胞，心脏祖细胞，并致力于CMS。免疫也​​可用于识别和可视化心脏细胞类型的空间分布。 使用报告系统，以鉴定和分离的CM时，需要纯群体应用更容易进行。为此，我们推出了αMHC-DsRedNuc建设成为鼠标CGR8胚胎干细胞系14。 CGR8细胞生长和保持多能性无饲养细胞，促进扩增和分化测定18。在ES细胞系含有与心脏特异性的α-肌球蛋白重链（αMHC或MYH6）基因启动子下一个核定位信号的DsRed荧光蛋白编码序列。利用这些细胞，CM的可以容易地鉴定和分离为定量，细胞分选，电生理学，药物筛选，并研究心房分化的机制。

<table><tbody><tr><td>GMEM</td><td>Life Technologies</td><td>11710</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>10082</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050</td><td></td></tr><tr><td>LIF</td><td>EMD Millipore</td><td>ESG1107</td><td></td></tr><tr><td>&amp;szlig;-Mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>IMDM</td><td>Sigma</td><td>I3390</td><td></td></tr><tr><td>Non-Essential Amino Acids</td><td>Sigma</td><td>M7145</td><td></td></tr><tr><td>10 cm Tissue Culture Plates</td><td>Sarstedt</td><td>83.3902</td><td></td></tr><tr><td>10 cm Bacterial Petri Dishes</td><td>VWR</td><td>25384-342</td><td></td></tr><tr><td>6 cm Bacterial Petri Dishes</td><td>VWR</td><td>25384-092</td><td></td></tr><tr><td>6-well tissue culture plates</td><td>Sarstedt</td><td>83.3920</td><td></td></tr><tr><td>Gremlin 2 recombinant protein</td><td>R&amp;amp;D Systems</td><td>2069-PR-050</td><td></td></tr><tr><td>Sterile filter units</td><td>Thermo Fisher</td><td>09-741-02</td><td></td></tr><tr><td>Gelatin (from porcine skin)</td><td>Sigma</td><td>G1890</td><td></td></tr><tr><td>10X PBS, Sterile</td><td>Sigma</td><td>P5493</td><td></td></tr><tr><td>BSA&amp;nbsp;</td><td>Sigma</td><td>5470</td><td></td></tr><tr><td>0.05% Trypsin-EDTA solution</td><td>Life Technologies</td><td>25300054</td><td></td></tr><tr><td>DPBS, no Calcium, no Magnesium</td><td>Life Technologies</td><td>14200</td><td></td></tr></tbody></table>

differentiation of atrial cardiomyocytes from pluripotent stem cells using the bmp antagonist grem2

用于从多能干细胞的心肌细胞的种群的协议已被开发，但是这些通常会产生混合的表型的细胞。热衷于追求涉及特定肌细胞亚型研究的研究人员需要更定向分化的方法。通过处理小鼠胚胎干细胞（ES）与Grem2，分泌的BMP拮抗剂，它是必要的体内心房腔室的形成，可以产生具有一个心房的表型的大量心脏细胞的。改造的MYH6-DsRed的-NUC多能干细胞系的使用允许识别，选择和心肌细胞的纯化。在这个协议中胚状体是从使用悬滴法MYH6-DsRed的-NUC细胞产生的，并保持在悬浮液中，直到分化第4天（D4）。在D4细胞用Grem2处理并铺板于明胶包被的平板。 D8之间-D10大承包领域的文化观察，继续扩大和M通过D14 ATURE。分子，组织学和electrophysiogical分析表明细胞Grem2处理的细胞获得心房状特性提供一种在体外模型来研究心房心肌细胞的生物学和它们对各种药剂的响应。

在微分之前，多能干细胞应紧凑和自由自发分化。在图1A所示的细胞准备被singularized并用于在协议部分5.1中所述悬滴。在图1B所示的细胞自发分化，不应该被用于制备悬滴的。 包括在图面板2示成功地形成的EB在分化天2.质量的EB通常形成最佳内均匀地隔开，以及圆形挂下降， <st...

Watch this Scientific Journal Video about Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2 at JoVE.com

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

从多能干细胞使用BMP拮抗剂Grem2心房心肌细胞分化

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.

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

differentiation-atrial-cardiomyocytes-from-pluripotent-stem-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

7.7K 次观看.  Vanderbilt University School of Medicine. 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.

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

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

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 ...

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 ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

在微孔聚集尺寸优化人类多能干细胞的基于悬浮心脏分化

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

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 ...

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 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 ...