Name: assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts
Uploaded: 2017-03-22T14:00:00.000+00:00
Duration: 9 min 29 s
Description: Watch this Scientific Journal Video about Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts at JoVE.com

A.F.-P. was supported by the National Science Foundation Graduate Research Fellowship under Grant No.2015165336. N.V.M was supported by grants from the NIH (HL094699), Burroughs Wellcome Fund (1009838), and the March of Dimes (#5-FY14-203). We acknowledge Young-Jae Nam, Christina Lubczyk, and Minoti Bhakta for their important contributions to protocol development and data analysis. We also thank John Shelton for valuable technical input and members of the Munshi lab for scientific discussion.

涉及动物的做法，所有的实验程序进行的机构动物护理和使用委员会在UT西南医疗中心的批准。 1.隔离HCN4-GFP E12.5小鼠胚胎成纤维细胞（MEF中） <ol><li>建立纯合子HCN4-GFP男性和CD-1雌性间定时交配。 </li><li>在二氧化碳安乐死和随后的颈椎脱位E12.5牺牲怀孕的女性。 <ol><li>与解剖钳除去子宫角，如前所述22，23，并将其放置在不含Ca 2+或Mg上用1×磷酸盐缓冲盐水（PBS）冰上的培养皿2+。 </li></ol></li><li>执行使用无菌技术在组织培养罩的所有后续步骤。 <ol><li>取下子宫羊水和胚胎囊用剪刀和镊子解剖。请附更好地处理胎盘。孕NT CD-1雌性通常生出的幼崽10和14之间。 </li><li>使用解剖钳，取分离的胚胎，并迅速在70％（体积/体积）乙醇冲洗它们的两倍。 注:清洗要快，尽量减少细胞死亡。 </li><li>取出头部，四肢，尾巴，内脏器官，包括从隔离胚胎的心脏。 </li><li>使用无菌刀片在尺寸约为1mm 3放置剩余组织在10-cm培养皿用1ml 1X PBS中并精细地剁碎的。 </li><li>转移组织糜与PBS中的50毫升锥形管中。 </li><li>旋在300×g离心3分钟。小心吸多余的PBS。 </li><li>加入1毫升每胚胎无菌0.25％胰蛋白酶EDTA的。孵育在37℃水浴中的细胞15分钟。轻轻地混合管，每4分钟。在消化组织将显著降低产量。 </li><li>以最大速度（3200 RPM）为4秒涡细胞混合物。 </li><li>加入2毫升的成纤维细胞培养基，每个胚胎混匀。过滤ŧhrough用吸管通过过滤器，以帮助细胞100微米的细胞滤网。请参阅表1为所有后续介质的制定。 </li><li>旋转300 XG 4分钟。小心吸上清。 </li><li>加入10 mL新鲜的成纤维细胞培养基每3个胚胎和磨碎6-10倍。 </li><li>板1 15厘米的组织培养皿中的细胞制备的每3个胚胎。培养过夜在37℃，5％CO 2培养箱。 </li><li>隔夜培养后，用新鲜的30毫升每平板培养基更换介质。将细胞放回培养箱中过夜。 注意:检查在荧光显微镜下HCN4-GFP +细胞污染。文化应该是GFP -也只有在重新编程成为GFP +。 </li><li>第二天，收获细胞用3mL新鲜预热0.25％胰蛋白酶-EDTA的。计数和冻结的细胞。通常情况下，冻结的细胞以每毫升3×10 6个细胞。预期一愕LD应每个胚胎3×10 6个细胞。 </li></ol></li></ol> 2.逆转录病毒生产和再编程注意:以下协议要求传染性逆转录病毒的生产和处理。在执行BSL-2准则和无菌技术在生物安全等级2机柜下面的步骤。用10％漂白粉处置暴露于逆转录病毒的所有材料。 <ol><li> 逆转录病毒的生产和MEF中准备 注意:以下协议是用于在6孔板逆转录病毒生产和MEF感染在24孔板中。对于其它的格式， 见表2。的MEF是在-1天镀，所以定时将需要为每个实验进行适当协调（参见第2.3节和图2）。 <ol><li>维持开发平台-E（PE）细胞根据制造商的建议。简言之，将培养的PE细胞在DMEM补充有10％FBS，1微克/毫升puromy霉素，10微克/毫升杀稻瘟菌素，青霉素和链霉素。传代细胞1:4时，培养物达到70-90％汇合每两天。 </li><li> -2天 :转染前一天，种子开发平台-E细胞以1×10 6个细胞/孔在转染培养基一个6孔板。细胞应在转染时70-80％汇合。 </li></ol></li><li> 转染使用市售的转染试剂。 注:商用试剂应在转染前室温（RT）。为DNA转染，添加每个逆转录病毒质粒DNA单独（G，H，M和T），以形成一个GHMT鸡尾酒9。 <ol><li> -1天:在15 mL锥形聚苯乙烯管中，用每个反应的转染试剂的6微升混合降低血清培养基60微升为一6孔板格式。孵育混合物，在室温下5分钟。 注意:由于在这里所使用的转染试剂结合到塑料，一的dd直接向低血清培养基，以避免在转染效率的任何降低。 </li><li>添加共2微克每反应GHMT鸡尾酒，并轻轻拍打至混合。不要旋涡。在室温下孵育15分钟进行反应。 </li><li>从步骤2.2.3中逐滴的方式PE细胞添加混合物。 </li><li>孵育转染的开发平台- E细胞过夜，37℃，5％ 二氧化碳培养箱。记录转染时。 </li></ol></li><li> HCN4-GFP小鼠胚胎成纤维细胞的播种。 <ol><li>电镀的MEF前1小时，准备免疫细胞化学24孔板。 <ol><li>添加每孔12mm的纤连蛋白盖玻片。 </li><li>大衣井用300微升牛胶原溶液（ 如 SureCoat）孵育在37℃培养箱1小时。 </li><li>立即MEF电镀之前抽吸涂覆溶液。 </li></ol></li><li>解冻HCN4-GFP的MEF的冷冻小瓶中并用预热fibrobl洗X1在500×g离心5分钟AST介质。 </li><li>采用台盼蓝染色或类似的染料确定细胞活力。计算使用下面的公式每毫升的活细胞培养的对数: ％存活细胞= [1.00 - （总细胞的蓝色细胞总数/总数）]×100 <ol><li>计算使用下面的公式存活细胞总数: 细胞悬浮液的活细胞=％存活细胞×稀释系数x 10,000×总体积</li></ol></li><li>种子3×10 4个细胞每孔到与先前制备牛胶原溶液，纤连蛋白盖玻片24孔板中。 </li></ol></li><li> 转导和的MEF重编程 注:根据制造商的注意事项，保养得当开发平台-E细胞产生的1×10 7感染单位/毫升的平均滴度。虽然滴度不直接对每个实验测定GFP的控制总是包括作为感染效率的替代品。高GFP表达和强度（GFP +&gt; 95％）通常与相关成功GHMT介导代iCLMs的。 <ol><li> 0天:24小时转染后，通过0.45微米孔径无表面活性剂的醋酸纤维素过滤器过滤在PE逆转录介质和转移到15毫升锥形管中。添加凝聚胺至8微克/毫升的最终浓度。仔细补充细胞2毫升新鲜染培养基。 注意:如果媒体被改变得太快细胞很容易地从板脱落。 </li><li>吸出培养的MEF的介质，并添加新鲜收集逆转录病毒介质;它应产生1.7毫升介质每孔一个6孔板的。每添加一个24孔板的〜800微升。返回MEF板的孵化器和孵化过夜。 </li><li> 第1天:重复步骤2.4.1和2.4.2。丢弃第二次病毒采集后的细胞。返回感染的MEF的孵化器，并让他们休息过夜。 </li><li> 第2天:48小时诱导后，吸用1X PBS的细胞条件媒体和洗X1。加每一个24孔板的孔500μL的预温热iCLM介质。 </li><li>更换iCLM媒体，每2 - 3天。病毒诱导的心脏重新编程的免疫细胞化学（ICC）分析后14天处理板。 </li></ol></li></ol> 3.重新编程的MEF免疫染色<ol><li> 14天的诱导后，小心吸媒体。 </li><li>用冰冷的1×PBS中的300μL的冲洗每个孔中。吸干多余的解决方案。 </li><li>固定的细胞用每孔4％低聚甲醛（PFA）溶液24孔板的250微升。在室温下孵育15分钟。 注:将固定的细胞可以存储在PBS在4℃下进行1 - 染色前2周。 </li><li>通透细胞用300微升0.1％PBS-100的TritonX（PBST）洗井X3。孵育在室温下洗涤之间5分钟。吸出最后一次洗涤后，多余的溶液。 </li><li> 10座分在室温用1X通用块缓冲区以300微升/孔。 </li><li>准备（ICC）染色缓冲液:加1:1 1X PBS和1x通用的封闭液。稀释在IC卡染色缓冲第一抗体，并在4℃下孵育抗体过夜。请参考推荐的稀释材料部分。 <ol><li>染色一对幻灯片鼠标α辅肌动蛋白，鸡αGFP和兔NPPA为IPM和IAM鉴定。 </li><li>染色一对幻灯片鼠标α辅肌动蛋白，鸡αGFP，并为IPM和IVM鉴定兔MYL2。 </li></ol></li><li>次日，洗涤孔×3用300微升0.1％的PBST。孵育在室温下洗涤之间5分钟。吸出最后一次洗涤后，多余的溶液。 </li><li>在准备ICC染色缓冲二级抗体稀释液。请参考推荐的稀释材料部分。孵育的二抗于室温1小时后，避光。 <ol><li>染色所有幻灯片具有以下二次antibodie小号:鼠标的Alexa-555，鸡的Alexa-488和Alexa的兔-647。 </li></ol></li><li>洗井X3 300微升0.1％PBST。孵育在室温下洗涤之间5分钟。避光。 </li><li>加含有1.5微克/毫升4&#39;，6-二脒基-2-苯基吲哚（DAPI）的到玻璃显微镜载片安装媒体的2.4微升。小心地从24孔板的孔取出盖玻片，除去过量溶液，并转移到载玻片带安装的介质。轻压盖玻片去除多余的量和空气。 </li><li>密封幻灯片与首选指甲油或塑料密封胶。商店安装在4℃避光幻灯片。 </li></ol> 4.识别心脏亚型的使用共聚焦显微镜注:对于成像，共焦显微镜配备有405能够光谱检测的至少2个荧光检测器，488，555，和639的波长是必要的，以便识别IPMS，IAMS和IVMS。 IMAG使用计划复消色差透镜20X / 0.75客观的或更好的E细胞。使用制造商的图像分析软件，扫描的图像缩放可以实现40X-油浸品质的图像。 <ol><li>图片库:取8位图像用DAPI，Alexa的-488，Alexa的-555和Alexa-647通道（CH）。像素停留的第6号第1024帧大小，行步骤时间在2和2的平均是足够的高清晰度的图像。 </li><li>对于每个滑动，从一个边缘开始，并开始扫描上下为α辅肌动蛋白+肌节+细胞红色荧光通道（Ch。）（参见图3和图5A为实施例）。肌条纹更容易在555纳米波长的视觉识别。 <ol><li>一旦α辅肌动蛋白肌节+ + T细胞已被确定，切换到绿色通道。并保持注意如果它是正数（IPM）。切换到计算机，以评估远红647章。 （IAM或IVM）。 注:细胞是α辅肌动蛋白+ / +肌节/ Hnc4-GFP + / NPPA - / MYL2 -被指定为IPMS。 GFP表达将在整个细胞（ 图4A）中可以看出。 </li><li>染色幻灯片与α辅肌动蛋白（鼠Alexa555的），HCN4-GFP（鸡Alexa488），NPPA（兔Alexa647）和DAPI。是α辅肌动蛋白+ / +肌节/ Hnc4-GFP的细胞- / + NPPA是IAM。 NPPA染色会出现核周和点状（ 图4B）。 </li><li>染色幻灯片与α辅肌动蛋白（鼠Alexa555的），HCN4-GFP（鸡Alexa488），MYL2（兔Alexa647）。阳性细胞α辅肌动蛋白+ / +肌节/ Hnc4-GFP - / + MYL2 IVMS是。 MYL2染色将展出沿肌纤维横纹一个形式。由于在染色及Z平面的质量的变化，条纹可能不总是容易看到（ 图4C）。 </li></ol></li></ol>注意:为了评估潜在重新编程的MEF的实际数量，2个孔24孔平板的接种在平行于实验孔和电镀后收获一天。铺板的细胞的总数量，然后通过平均两个孔来确定。这成为镀实际的总的细胞（aTotal）。 <ol><li> 肌+ <ol><li>目视检查进行正确α辅肌动蛋白+ /肌节+（右面板图3）和记录（ 图5B-i）的盖玻片每个小区。 </li><li>由镀实际的总的细胞（aTotal）制表α辅肌动蛋白+ /肌节+对每个盖玻片和除法的总数（ 图5B-ⅲ）。例如，如果aTotal = 12500细胞，100细胞，α-辅肌动+ /肌节+然后，镀敷的MEF的0.8％被重新编程。平均reprograming实验将产生1％α -肌动蛋白+ /肌节+细胞（ 图5C）。 </li></ol></li><li> 亚型+ 注:对于下面的步骤，请参考图5B / C为代表性iCLM定量的工作流程。简单地说，对于每个小节+细胞，制表，如果它是为任一亚型（ 图5B-ⅰ）是唯一的。除以亚型+细胞的数量比的平均肌节+细胞×100（ 图5B-i）的计算％亚型（ 图5B-ⅲ）。为了计算绝对％亚型效率（ 图5B-iv）中 ，通过接种×100（ 图5B-ⅱ）细胞的总数量从图划分亚型+细胞数5B-i的 。 <ol><li>对于每个α辅肌动蛋白的+ /肌节+细胞，评估它们是否GFP +，NPPA +或MYL2 <sUP&gt; +。 </li><li>制表α辅肌动蛋白+ /肌节+ / Hnc4-GFP + / NPPA总数- / MYL2 - ，辅肌动蛋白+ /肌节+ / Hnc4-GFP - / NPPA +和α辅肌动蛋白+ /肌节+ / Hnc4-GFP - / MYL2 +细胞（ 图5B-1）。 </li><li>计算每个亚型的百分比，通过以+ /肌节+细胞孔的α辅肌动蛋白的总数除以亚型+细胞的数目和由100 GHMT相乘产生IPMS，IAMS和IVMS以大致相同的比例（ 图图5B-ⅲ）。 </li><li>计算亚型+细胞的绝对数量，除以亚型+细胞的总数通过aTotal实验条件和100（ 图5B-ⅱ）相乘。平均IPMS占总感染细胞群体的0.3％，IAMS 0.3％，和网络视频监控软件0.25％（ 图5B-IV）。 </li></ol></li></ol>

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The authors have nothing to disclose.

本研究通过心脏转录的逆转录病毒介导的表达提供的MEF转换的直接重新编程战略转化为一组不同的亚型心脏因素GATA4，MEF2C，Tbx5中，和HAND2（GHMT）。在使用具有特定的PM记者鼠标组合多重染色的方法，我们能够确定（IAM），网络视频监控软件，并在IPMS单细胞分辨率。这种试验可以在隔离能力个别转录因子对亚型多样性和肌节发展的贡献的体外系统的实验研究。与此同时，这可能会带?...

心脏是在胚胎1，2，开发的第一个功能器官。与循环系统相结合，其提供氧，营养物，和开发过程中的废物处理机制。受精后三个星期，人的心脏跳动，第一次和适当的调整则是由心肌细胞（CMS）维护。因此，这些特殊细胞的不可逆损失是潜在的进行性心脏衰竭的根本问题。而一些生物如斑马鱼和爪蟾具有用于心脏再生的潜力，成年哺乳动物心脏较为有限3，5，6。因此，由于心脏的重要功能，是不令人吃惊的是心脏疾病是全世界死亡的首要原因，占仅7美国60万人死亡。该refore，基于细胞的疗法有效地修理或更换损伤心肌是伟大的具有临床意义。 山中和同事8的开创性的研究表明，四种转录因子的强制表达足以完全分化的成纤维细胞转化为多能干细胞。不过，所有的多能干细胞的策略的致瘤能力已在其用于治疗目的使用的一个关键问题。这促使科学领域寻找替代方法转分化的细胞，同时避免多能的阶段。最近，一些团体已经通过与转录异位表达显示小鼠成纤维细胞直接转化为诱导的心肌样细胞（iCLMs）显示了这一战略的可行性因素GATA4，MEF2C，Tbx5中，后来，HAND2（GMT和GHMT分别）9，10。 Furthermore，同样的策略可以在体内和人衍生的组织9，11，12来进行。最近的研究已强调了额外的因素或可被调制以进一步改善心脏重编程效率13，14，15的信号通路。总之，这些研究表明定向转分化为再生疗法的潜力。然而，CM重新编程，未知的分子机制，重复性不一致由于方法上的差异16低效率，iCLMs的异质性仍未得到解决。 为了直接评价iCLM异质性，我们设计了一个离散的和强大的单细胞测定为肌节发展和心脏谱系specificatio的识别正两个功能的心肌细胞的必要特征。存在由它们的位置和独特的电特性定义在心脏的至少三个主要类型的CM:心房（AM），心室（VM）和起搏器（PM）17，18，19，20。在一个精心策划的组合，他们让血液正常抽水。在心脏损伤，一个或所有亚型可能会受到影响，细胞治疗的类型，需要根据具体情况逐案予以解决。目前，大多数战略，注重整体代心肌细胞，而少量的工作正在做研究，调节亚型规范的分子机制。 下面详细研究如何正确量化井井有条肌节，并确定一组不同的亚型心肌的。使用心脏起搏器（PM）专用鼠标的记者，我们可以申请一个我mmunocytochemical方法来区分诱发房状细胞（IAM），诱发室状细胞（IVM），并诱导般的PM肌细胞（IPMS）21。根据我们的观察，只表现出肌组织细胞能够自发跳动。这种独特的重新编程平台可以评估的作用 在肌组织，子规范，CM重编程效率的某些参数在单细胞分辨率。

<table><tbody><tr><td>DMEM</td><td>Sigma</td><td>D5796</td><td>Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td></tr><tr><td>Medium 199</td><td>Thermo Fisher Scientific</td><td>11150059</td><td>Component of iCLM media</td></tr><tr><td>Fetal bovine serrum (FBS)</td><td>Sigma</td><td>F2442</td><td>Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td></tr><tr><td>Insulin-Transferrin-Selenium G</td><td>Thermo Fisher Scientific</td><td>41400-045</td><td>Component of iCLM media</td></tr><tr><td>MEM vitamin solution</td><td>Thermo Fisher Scientific</td><td>11120-052</td><td>Component of iCLM media</td></tr><tr><td>MEM amino acids</td><td>Thermo Fisher Scientific</td><td>1601149</td><td>Component of iCLM media</td></tr><tr><td>Non-Essential amino acids</td><td>Thermo Fisher Scientific</td><td>11140-050</td><td>Component of iCLM media</td></tr><tr><td>Antibiotic-Antimycotics</td><td>Thermo Fisher Scientific</td><td>15240062</td><td>Component of iCLM media</td></tr><tr><td>B-27 supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td>Component of iCLM media</td></tr><tr><td>Heat-Inactivated Horse Serum</td><td>Thermo Fisher Scientific</td><td>26050-088</td><td>Component of iCLM media</td></tr><tr><td>NaPyruvate</td><td>Thermo Fisher Scientific</td><td>11360-70</td><td>Component of iCLM media</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>1514022</td><td>Component of Plat-E media and fibroblast media</td></tr><tr><td>Puromycin&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A11139-03</td><td>Component of Plat-E media</td></tr><tr><td>Blasticidin&amp;nbsp;&amp;nbsp;</td><td>Gemini Bio-Products</td><td>400-128P</td><td>Component of Plat-E media</td></tr><tr><td>Glutamax</td><td>Thermo Fisher Scientific</td><td>35050-061</td><td>Component of Fibroblast media</td></tr><tr><td>Confocal laser scanning LSM700</td><td>Zeiss</td><td></td><td>For confocal analysis</td></tr><tr><td>FuGENE 6 transfection Reagent</td><td>Promega</td><td>E2692</td><td>Transfection reagent&amp;nbsp;</td></tr><tr><td>Opti-MEM Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985-070</td><td>Transfection reagent&amp;nbsp;</td></tr><tr><td>Polybrene</td><td>Millipore</td><td>TR-1003-G</td><td>Induction reagent. Use at a final concentration of 8 &amp;mu;M/mL</td></tr><tr><td>Platinium-E (PE) Retroviral Packagin Cell Line, Ecotropic</td><td>CellBiolabs</td><td>RV-101</td><td>Retroviral pacaking cell line</td></tr><tr><td>Trypsin 0.25% EDTA</td><td>Thermo Fisher Scientific</td><td></td><td>For MEFs and Plat-E dissociation</td></tr><tr><td>Mouse anti &amp;alpha;-Actinin (Clone EA-53)</td><td>Sigma</td><td>A7811</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Chicken anti-GFP IgY</td><td>Thermo Fisher Scientific</td><td>A10262</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Rabbit Pab anti-NPPA&amp;nbsp;</td><td>Abgent</td><td>AP8534A</td><td>Antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Rabbit Pab anti Myl2 IgG&amp;nbsp;</td><td>ProteinTech</td><td>10906-1-AP</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Vectashield solution with DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Vector Labs</td><td>H-1500</td><td>Dye for confocal analysis</td></tr><tr><td>Superfrost Plus Microscope slides</td><td>Thermo Fisher Scientific</td><td>12-550-15</td><td>25 x 75 x 1.0 mm</td></tr><tr><td>BioCoat Fibronectin 12 mm coverslips</td><td>NeuVitro Corp</td><td>GG-12-1.5</td><td>Coverslips for confocal analysis</td></tr><tr><td>100 &amp;mu;m cell strainer</td><td>Thermo Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>0.45 &amp;mu;m Syringes filters SFCA 25MM</td><td>Thermo Fisher Scientific</td><td>09-740-106</td><td>For virus filtration</td></tr><tr><td>6 mL Syringes</td><td>Covidien</td><td>8881516937</td><td>For virus filtration</td></tr><tr><td>Goat anti-Chicken IgY (H&amp;amp;L) A488</td><td>Abcam</td><td>AB150169</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Donkey anti-rabbit A647 IgG(H+L)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A31573</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Goat anti-mouse IgG(H+L) A555</td><td>Thermo Fisher Scientific</td><td>A21422</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>93443-100ml</td><td>For cell permeabilization&amp;nbsp;</td></tr><tr><td>Dulbecco&#039;s PBS without CaCl&lt;sub&gt;2&lt;/sub&gt; and MgCl&lt;sub&gt;2&lt;/sub&gt; (D-PBS)</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Power Block 10x Universal Blocking reagent</td><td>Thermo Fisher Scientific</td><td>NC9495720</td><td>Dilute to 1x in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>16% Paraformaldehyde aqueous solution (PFA)</td><td>Electro Microscopy Sciences&amp;nbsp;</td><td>15710</td><td>Use at 4% diluted in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>6 cm&amp;nbsp; plates</td><td>Olympus</td><td>25-260</td><td></td></tr><tr><td>6-well plates</td><td>Genesee Scientific</td><td>25-105</td><td></td></tr><tr><td>24-well plates</td><td>Genesee Scientific</td><td>25-107</td><td></td></tr><tr><td>10 cm Tissue culture dishes</td><td>Corning</td><td>4239</td><td></td></tr><tr><td>15 cm&amp;nbsp; Tissue culture dishes</td><td>Thermo Fisher Scientific</td><td>5442</td><td></td></tr><tr><td>15 mL Conical tubes</td><td>Corning</td><td>4308</td><td></td></tr><tr><td>50 mL Conical tubes</td><td>Corning</td><td>4249</td><td></td></tr><tr><td>0.4% Trypan blue solution</td><td>Sigma</td><td>T8154</td><td>For viability</td></tr><tr><td>Ethyl Alcohol 200 proof</td><td>Thermo Fisher Scientific</td><td>7005</td><td></td></tr><tr><td>Bleach</td><td>Thermo Fisher Scientific</td><td>6009</td><td></td></tr></tbody></table>

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.

服用特定的PM记者鼠标的优势，我们开发了多重免疫策略，确定为如图1所示多样化的内源性肌细胞。以下在图2所示的重新编程的步骤，亚型特异性的CM的诱导可以早在4第21天虽然是在低速率检测。在第14天，在实验可以停止并评估肌组织（ 图3）和亚型规范（ 图4）。 图5总结玻片制备的用于I...

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

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 ...

assessing-cardiomyocyte-subtypes-following-transcription-factor

Universidad San Sebastian

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JoVE Journal

Developmental Biology

7.4K 次观看.  UT Southwestern Medical Center. 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. 

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

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. &#8220;Biological pacemakers&#8221; generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines &#8220;forward programming&#8221; of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of &#62;80% physiologically functional pacemaker cells. These &#8220;induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These &#8220;Induced-sinoatrial-bodies&#8221; (&#8220;iSABs&#8221;) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

基于ES-Cell的编程组合MYH6启动子，选择小鼠心脏起搏器细胞聚集一代

Treatment of the &#8220;sick sinus syndrome&#8221; is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, ...

科研

发育生物学

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

诱发心肌细胞的使用多顺反子构建表达GATA4，MEF2C和Tbx5中的最优比率改善发电

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

促纤维化信号增强因子介导的小鼠胚胎成纤维细胞再编程诱导心肌细胞的抑制

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Assessing Cardiac Reprogramming using High Content Imaging Analysis

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a reprogramming technique. Cardiac reprogramming provides a strategy to generate new cardiomyocytes. By introducing core cardiogenic transcription factors into fibroblasts; fibroblasts can be converted to iCMs without transition through the pluripotent stem cell state. However, the conversion rate of fibroblasts to iCMs still remains low. Accordingly, there have been numerous additional approaches to enhance cardiac reprogramming efficiency. Most of these studies assessed cardiac reprogramming efficiency using flow cytometry, while at the same time performed immunocytochemistry to visualize iCMs. Thus, at least two separate sets of reprogramming experiments are required to demonstrate the success of iCM reprogramming. In contrast, automated high content imaging analysis will provide both quantification and qualification of iCM reprogramming with a relatively small number of cells. With this method, it is possible to directly assess the quantity and quality of iCMs with a single reprogramming experiment. This approach will be able to facilitate future cardiac reprogramming studies that require large-scale reprogramming experiments such as screening genetic or pharmacological factors for enhancing reprogramming efficiency. In addition, the application of high content imaging analysis protocol is not limited to cardiac reprogramming. It can be applied to reprogramming of other cell lineages as well as any immunostaining experiments which need both quantification and visualization of immunostained cells.

We present a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a ...

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

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

人类多能干细胞的高效分化为心肌细胞，并鉴定流式细胞仪

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

使用GCaMP3报告器测量心脏特异性钙通量，对心脏重编程进行体外评估

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...