We are grateful for expert technical assistance from the UNC Flow Cytometry Core and UNC Microscopy Core. We thank members of the Qian lab and the Liu lab for helpful discussions and critical reviews of the manuscript. This study was supported by NIH/NHLBI R00 HL109079 grant to Dr. Liu and American Heart Association (AHA) Scientist Development Grant 13SDG17060010 and the Ellison Medical Foundation (EMF) New Scholar Grant AG-NS-1064-13 to Dr. Qian.

这里列出的协议采用新生小鼠。动物保健和实验是按照由科实验室的动物医学（DLAM）北卡罗来纳大学教堂山分校制定的准则进行。 1.准备缓冲区和媒体<ol><li>制备成纤维细胞（FB）介质：补编500毫升的IMDM培养基用100ml胎牛血清（FBS）和6ml青霉素/链霉素。 </li><li>制备ICM介质：补编400毫升DMEM培养基用100ml的M199培养基和50ml FBS中。 </li><li>准备B27介质：补充490毫升RPMI1640培养基用10ml B27媒体。 </li><li>制备开发平台-E培养基：补编500毫升DMEM培养基用50ml的FBS，5 ml青霉素/链霉素和5ml非必需氨基酸。 </li><li>制备开发平台-E转染培养基：补编500毫升DMEM培养基用50ml的FBS和5ml非必需氨基酸。 </li><li>镨epare明胶包被的24孔板：添加0.5 ml的0.1％明胶溶液至24孔板一个井，在37℃下使用前右至少10分钟，并吸出。 </li><li>制备FACS标记缓冲液：补编500毫升DPBS用10ml的FBS和2ml EDTA（0.5M），以达到2mM的最终浓度。通过一个0.22微米的过滤器，并储存在4℃。 </li><li>制备MACS分类缓冲器：补编500毫升的DPBS与2.5g的BSA和2ml EDTA（0.5M），以达到2mM的最终浓度。通过一个0.22微米的过滤器，并储存在4℃。 </li><li>制备2×HBS缓冲液为染：补充500毫升的HEPES缓冲液（50mM）配有280 mM氯化钠（8.1816克），10毫米氯化钾（0.37275克），1.5毫的 Na 2 HPO 4（0.10647克），12毫葡萄糖（1.08096克）。加约650微升10N NaOH将pH调节至7.05-7.12。通过在4℃的0.22微米孔径过滤和储存过滤。 </li><li>准备2.5M的氯化钙溶液转：添加18.376克钙Cl 2中至50ml双蒸2 O.通过在4℃的0.22微米孔径过滤和储存过滤。 </li><li>准备ICC（免疫细胞化学）固定缓冲液：添加5 ml的多聚甲醛（PFA，32％）到35 ml的DPBS达到4％的最终浓度。 </li><li>准备ICC透化缓冲液：添加0.1 ml的曲通至100毫升DPBS达到0.1％的最终浓度。 </li><li>准备ICC封闭缓冲液：添加5克牛血清白蛋白（BSA）到100毫升DPBS达到5％的最终浓度。 </li><li>准备ICC染色缓冲液：添加1克的BSA至100ml DPBS达到1％的最终浓度。 </li></ol> 2.代新生小鼠心肌成纤维细胞<ol style="margin-left: 40px;"><li>生成块培养方法心脏成纤维细胞<ol><li>清洁新生儿αMHC-GFP转基因小鼠（P1到P2）用75％的乙醇。用无菌剪刀剪了头。然后，让来自在一个腋下到另一水平切口。使用无菌钝弯曲钳解剖出心脏，并将其放置在含冰冷的PBS缓冲液的24孔板的一个孔。 <ol><li>另外，横向切开后挤出的心脏。 </li></ol></li><li>通过荧光显微镜检查心脏GFP表达。由于GFP的表达是由αMHC启动子是一种心脏特异性启动子驱动，从转基因小鼠的心脏应该在绿15，而负心脏是暗淡的，在任何荧光。 </li><li>需要3-4 GFP阳性的心成60毫米中心孔培养皿并剁碎成小块尺寸小于1毫米3通过无菌剪刀和镊子。 </li><li>将3-4碎的心，1个10 cm培养皿用2毫升FB媒体。让组织安顿下来3小时。 </li><li>慢慢加入8毫升预热FB媒体包含心脏组织的菜肴。请勿打扰组织了三天。 </li><li>更换介质每三天。 </li><li>第7天，aspir吃了培养液，洗涤细胞用DPBS。加入3 ml的0.05％胰蛋白酶，以每块板和消化在37℃下5分钟。 </li><li>添加5毫升FB媒体解渴胰蛋白酶。轻轻分离细胞与细胞刮刀。吸取媒体上下进一步机械分离组织。 </li><li>收集细胞，并通过40微米的细胞滤网过滤，以避免心脏组织碎片的污染，然后通过在200×g离心纺丝5分钟沉淀细胞。 </li><li>洗涤细胞一旦与MACS缓冲液和细胞准备进行排序。 </li></ol></li><li>生成的酶消化法心脏成纤维细胞<ol><li>收获GFP阳性的心为2.1.1。转移了所有的心情到10cm皿用10毫升冰冷的DPBS。 </li><li>挤心室用无菌镊子以除去血液并用冰冷的DPBS中冲洗一次。 </li><li>修剪的心是自由的其他组织和脂肪。 </li><li>横切心脏成仍松散连接的约4个。 </li><li>如果小于20心中，调心入15ml锥形管中以8毫升温的0.05％胰蛋白酶-EDTA，并在37℃下进行15分钟。 <ol><li>如果有20-30心中，转移心中到50ml锥形管中，用10毫升温的0.05％胰蛋白酶-EDTA，并在37℃下进行15分钟。 </li></ol></li><li>丢弃胰蛋白酶，并添加5毫升（对于小于20的心）或10毫升（20-30心）温暖的II型胶原酶（0.5毫克/毫升）在HBSS。 </li><li>涡旋上涡旋管为在约4至6的速度级1分钟（如果液体不起床到盖，则速度为细）。 </li><li>孵育管中37℃水浴3-5分钟。 </li><li>涡管1分钟。 </li><li>沉积物用于通过重力1分钟，直到组织碎片沉淀下来，收集液态到含有5毫升冷的FB培养基的新管中。 </li><li>重复步骤2.2.6-2.2.10 3-5倍。 </li><li>通过40微米的细胞过滤过滤把所有的收藏品使单细胞悬浮液。 </li><li>离心机在200×g离心5分钟，在4℃。 </li><li>悬浮细胞在10ml MACS缓冲液中，离心，在200×g离心5分钟，在4℃。 </li><li>可选：如果有很多血细胞，重悬细胞与1ml RBC裂解缓冲液（150mM的氯化铵 ，10毫KHCO 3和0.1mM EDTA）中，保持在冰上1分钟，然后稀释用10ml MACS缓冲液和离心机在200×g离心5分钟。洗一次用MACS缓冲。 </li></ol></li><li> Thy1.2 +成纤维细胞通过MACS分离（磁性激活细胞分选） <ol><li>通过台盼蓝染色测定存活细胞数。 <ol><li>取10微升的细胞从10 mL细胞悬液步骤2.2.14。用10微升0.4％台盼蓝溶液混合。 </li><li>该混合物添加到血球。允许它静置3-5分钟，然后在显微镜下立即检查。死细胞被染成蓝色，活细胞是未染色。 </li><l我&gt;计数的所有活细胞中的血细胞计数仪的四个1毫米角落正方形。确定活细胞数：每平方×2（稀释因子）平均计数×10 4×10（细胞悬浮液的总体积）。 </li></ol></li><li>为小于1×10 7个细胞，重悬细胞，在90微升10微升Thy1.2微珠冷冻的MACS缓冲液中。添加更多的珠按比例，如果有大于1×10 7个细胞。拌匀，孵育在冰箱中（2-8°C）30分钟。 </li><li>加入10 mL的MACS缓冲液和自旋样品在200×g离心5分钟;用10ml MACS缓冲液，再离心洗一次。 </li><li>带来的体积至2ml在MACS缓冲液中。 </li><li>设置一个MACS机在油烟机。插入一个LS柱到分离器。应用3 ml的MACS缓冲液向柱平衡它。 </li><li>通过平衡LS列通过细胞悬液。 </li><li>洗3次，用2ml MACS每次缓冲。 </li><li>就拿LS列关闭t他分离器和洗脱3次用2ml MACS缓冲液到一个新的50ml管中。 </li><li>旋转，在200×g离心5分钟。 </li><li>悬浮细胞在10毫升FB媒体，计数细胞和种子成板在适当的密度。用于从外植体培养方法产生的成纤维细胞，所述种子细胞密度可以是约2-3×10 4个细胞/孔的24孔板中。从酶消化的方法产生的成纤维细胞，将细胞密度可能约为4-5×10 4个细胞/孔的24孔板中。 </li></ol></li></ol> 3.逆转录病毒生成的ICM重新编程注意：执行在无菌条件下在一个BSL2生物安全柜以下步骤。妥善处置转染的细胞，吸头和管道的建议，以避免对环境和健康危害的风险。 <ol style="margin-left: 40px;"><li>保持开发平台-E细胞在开发平台-E的介质补充有1μg/ ml的嘌呤霉素和10μg/ ml的b在37℃下lasticidin用5％ 的 CO 2。 </li><li>第2天，更换，由缺乏嘌呤和稻瘟开发平台-E培养基中。 </li><li>在第0天，拆分开发平台-E细胞成10厘米的转染前一天以大约每盘4-5×10 6个细胞的菜肴。细胞应为〜上转染当天80-90％汇合。 </li><li>在第1天，改变培养基，以转染培养基上在1小时内转染前细胞。转染程序如下： <ol><li>使用商业试剂盒转染<ol><li>混合20微升转染试剂（例如脂质体）和500微升减少血清的培养基（例如，的Opti-MEM）。加10微克的逆转录病毒质粒的另一个500微升减少血清的培养基。在室温下孵育（RT）下各混合物5分钟。 </li><li>慢慢加入该质粒混合物转染混合物。这一步可能需要15-30秒。孵育20分钟后，混合物在RT（溶液可以出现混浊）。 </LI&gt; <li>添加混合物滴加至所述细胞。 </li><li>在37℃培养箱中孵育过夜。 </li></ol></li><li>转用磷酸钙。 <ol><li>混合40微升2.5M的氯化钙和440微升双蒸2 O，然后加入20微克的逆转录病毒质粒（调整DDH 2 O的量，使总量为500微升）。 </li><li>加入500微升2×HBS到一个15毫升的锥形管中。 </li><li> VORTEX哈佛商学院，同时添加钙DNA混合物到哈佛商学院滴加。此步骤需要约30秒，每个样品。因此，最大限度地同时做3-4个样品。 </li><li>在室温下孵育3分钟孵育较长时间导致更大的沉淀和更少的细胞会占用沉淀物。 </li><li>添加该混合物滴加至细胞和20X显微镜下观察。精细沉淀（很多小的是好的，但一些大的是不好的）应该可以看到。 </li><li>孵育过夜，在37℃培养箱中。 </l我&gt; </ol></li></ol></li><li>第2天，对细胞用8ml预热培养基更换培养基。孵育24小时。 </li><li>在第3天及4天，通过使用10毫升无菌一次性注射器，通过0.45μm孔径的醋酸纤维素过滤器过滤它，并转移到50毫升管收集从碗碟培养上清。加入2毫升逆转录病毒沉淀溶液到每8毫升含有病毒的上清液。轻轻地混合，并孵育过夜，4℃。 </li><li>在第5天，在1500×g下旋转该病毒混合物在4℃下30分钟。病毒颗粒应在管的底部出现白色沉淀。 </li><li>弃去上清液。降速离心残余溶液在1500×g下5分钟。吸卸下流体的所有痕迹时，注意不要打扰沉淀的逆转录病毒颗粒的沉淀。 </li><li>重悬逆转录病毒颗粒与每8毫升病毒上清100微升DPBS缓冲。分装冰上病毒。立即使用或储存在-80℃。 </li></ol> 4.重编程心脏成纤维细胞<ol><li>加入4微升10毫克/毫升聚凝胺溶液放入10毫升ICM介质，并通过上下抽吸轻轻混匀。聚凝胺的最终浓度为4微克/毫升。 </li><li>添加含有凝聚胺500微升ICM媒体的24孔板每孔接种成纤维细胞。 </li><li>添加10微升反转录病毒到每孔含有任2-3×10 4个，从外植体培养物或4-5×10 4个细胞的酶消化法的细胞。病毒的量应成比例地增加在不同的培养板或菜肴增加的细胞数量。把板在37℃的培养箱中24-48小时，以使病毒感染。心脏重新编程时间线示于图1。 </li><li>病毒转导后，用500微升常规ICM媒体替换包含媒体的病毒。 </li><li>更换介质每2-3天。阳性筛选病毒转导的细胞，在2微克/毫升添加补充有嘌呤ICM媒体到靶细胞。保持三天。在此之后，保持嘌呤霉素在ICM介质以1微克/毫升的浓度。 </li><li>病毒感染后三天，取板从培养箱并把它的倒置荧光显微镜（20X）下观察GFP表达。 注意：病毒感染后10天，细胞可以收获FACS分析和ICC分析，以确定重编程效率（在步骤5和6）。 </li><li>病毒感染后14天，B27媒体取代ICM媒体。更换介质每三天。自发跳动的细胞位点可能会出现三个（细胞酶消化法）或病毒转导后四（细胞外植体培养方法）周。 </li></ol>重编程效率5.免疫细胞化学分析<ol><li>冲洗细胞用冰冷的PBS三次;去除多余的解决方案。 </li><li>添加0.5 ml的IC卡的固定缓冲液至24孔板各孔中。固定细胞在RT 15-20分钟。 </li><li>冲洗细胞，用PBS三次。 </li><li>添加0.5 ml的IC卡透化缓冲液到24孔板的每个孔中。透化的细胞在室温15分钟。 </li><li>冲洗细胞，用PBS三次。 </li><li>添加0.5 ml的IC卡封闭缓冲到24孔板各孔中，在室温下孵育1小时。 </li><li>稀释的主要抗体与ICC染色缓冲液。加入200μl抗体溶液，每孔孵育过夜，在4℃。抗体的稀释因素中列出的材料。 </li><li>在第二天，用PBS洗涤细胞三次。 </li><li>加入200μl次级抗体溶液至细胞，并孵育1小时，在室温在暗处。 </li><li>用PBS三次洗涤细胞。 </li><li>添加150微升安装含有DAPI到各孔溶液中。允许染色细胞核1分钟。然后圆形的玻璃盖片以及覆盖各个轻轻按压在底面防滑用钳去除气泡。 </li><li>吸出多余的溶液，样品准备用于成像。的代表图像表示重编程的细胞表达心脏标志物cTnT和αActinin在D14示于图2B。 </li></ol>重编程效率的6流式细胞仪分析<ol><li>彻底DPBS洗一次细胞。加0.3 ml胰蛋白酶（0.05％），以每孔并在37℃下5分钟。 </li><li>轻轻敲击板以方便吊装细胞。检查显微镜下细胞脱离。加入1毫升FACS缓冲液到每个孔中，当大多数细胞被离解。移液器上下进一步机械解离的细胞。 </li><li>在24孔板孔转移细胞到96孔深孔板中，以良好。旋，在200×g离心5分钟，在4℃下以收集细胞。 </li><li>洗涤细胞一次，流式细胞仪缓冲。 </li><li>加入100微升固定/透化溶液重悬细胞进行20分钟，在4℃。 </li><li>用1X洗涤液，颗粒洗涤细胞两次，并去除上清。 </li><li>制备在1×洗涤缓冲液中对GFP和肌钙蛋白初级抗体溶液。彻底重悬用50μl抗体溶液各样品，孵育在4℃下30分钟。 </li><li>在1X洗涤液，颗粒一次洗涤细胞，并去除上清液。 </li><li>添加包含的Alexa Fluor 488缀合的驴抗兔IgG和Alexa Fluor 647缀合的驴抗小鼠IgG向每个样品加入50μl次级抗体溶液。孵育在4℃下，在黑暗中30分钟。 </li><li>在1X洗涤液，颗粒一次洗涤细胞，并去除上清液。 </li><li>悬浮细胞于400μl固定缓冲液（DPBS用1％PFA）。细胞转移到FACS管与细胞滤网盖。样品是准备流式细胞仪检测。重新编程efficienc代表FACS分析只有使用Y pMxs-嘌呤霉素MGT构造具有或不具有嘌呤选择示于图2A。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblast:+the+renaissance+cell.">Cardiac fibroblast: the renaissance cell.</a> Circ Res. 105, 1164-1176 (2009).</li><li>Ieda, 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=Cardiac+fibroblasts+regulate+myocardial+proliferation+through+beta1+integrin+signaling.">Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling.</a> Dev Cell. 16, 233-244 (2009).</li><li>Moore-Morris, 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=Resident+fibroblast+lineages+mediate+pressure+overload-induced+cardiac+fibrosis.">Resident fibroblast lineages mediate pressure overload-induced cardiac fibrosis.</a> J Clin Invest. 124, 2921-2934 (2014).</li><li>Addis, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+direct+fibroblast+reprogramming+to+cardiomyocytes+using+calcium+activity+as+a+functional+measure+of+success.">Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success.</a> J Mol Cell Cardiol. 60, 97-106 (2013).</li><li>Chen, J. 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=Inefficient+reprogramming+of+fibroblasts+into+cardiomyocytes+using+Gata4,+Mef2c,+and+Tbx5.">Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, and Tbx5.</a> Circ Res. 111, 50-55 (2012).</li><li>Christoforou, N., 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=Transcription+factors+MYOCD,+SRF,+Mesp1+and+SMARCD3+enhance+the+cardio-inducing+effect+of+GATA4,+TBX5,+and+MEF2C+during+direct+cellular+reprogramming.">Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming.</a> PLoS One. 8, e63577 (2013).</li><li>Fu, J. D., 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=Direct+reprogramming+of+human+fibroblasts+toward+a+cardiomyocyte-like+state.">Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state.</a> Stem Cell Reports. 1, 235-247 (2013).</li><li>Hirai, H., Katoku-Kikyo, N., Keirstead, S. A., Kikyo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+direct+reprogramming+of+fibroblasts+into+cardiomyocyte-like+cells+with+the+MyoD+transactivation+domain.">Accelerated direct reprogramming of fibroblasts into cardiomyocyte-like cells with the MyoD transactivation domain.</a> Cardiovasc Res. 100, 105-113 (2013).</li><li>Ieda, 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=Direct+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors.">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142, 375-386 (2010).</li><li>Inagawa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cardiomyocyte-like+cells+in+infarct+hearts+by+gene+transfer+of+Gata4,+Mef2c,+and+Tbx5.">Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5.</a> Circ Res. 111, 1147-1156 (2012).</li><li>Islas, J. F., 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=Transcription+factors+ETS2+and+MESP1+transdifferentiate+human+dermal+fibroblasts+into+cardiac+progenitors.">Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors.</a> Proc Natl Acad Sci U.S.A. 109, 13016-13021 (2012).</li><li>Jayawardena, T. 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=MicroRNA-mediated+in+vitro+and+in+vivo+direct+reprogramming+of+cardiac+fibroblasts+to+cardiomyocytes.">MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes.</a> Circ Res. 110, 1465-1473 (2012).</li><li>Muraoka, N., 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=MiR-133+promotes+cardiac+reprogramming+by+directly+repressing+Snai1+and+silencing+fibroblast+signatures.">MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.</a> Embo J. 33, 1565-1581 (2014).</li><li>Nam, Y. 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=Induction+of+diverse+cardiac+cell+types+by+reprogramming+fibroblasts+with+cardiac+transcription+factors.">Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors.</a> Development. 141, 4267-4278 (2014).</li><li>Nam, Y. 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=Reprogramming+of+human+fibroblasts+toward+a+cardiac+fate.">Reprogramming of human fibroblasts toward a cardiac fate.</a> Proc Natl Acad Sci USA. 110, 5588-5593 (2013).</li><li>Protze, S., 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=A+new+approach+to+transcription+factor+screening+for+reprogramming+of+fibroblasts+to+cardiomyocyte-like+cells.">A new approach to transcription factor screening for reprogramming of fibroblasts to cardiomyocyte-like cells.</a> J Mol Cell Cardiol. 53, 323-332 (2012).</li><li>Qian, L., Berry, E. C., Fu, J. D., Ieda, M., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+mouse+fibroblasts+into+cardiomyocyte-like+cells+in+vitro.">Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.</a> Nat Protoc. 8, 1204-1215 (2013).</li><li>Wang, H., 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+Molecules+Enable+Cardiac+Reprogramming+of+Mouse+Fibroblasts+with+a+Single+Factor,+Oct4.">Small Molecules Enable Cardiac Reprogramming of Mouse Fibroblasts with a Single Factor, Oct4.</a> Cell Rep. , (2014).</li><li>Wang, 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=Stoichiometry+of+Gata4,+Mef2c,+and+Tbx5+Influences+the+Efficiency+and+Quality+of+Induced+Cardiac+Myocyte+Reprogramming.">Stoichiometry of Gata4, Mef2c, and Tbx5 Influences the Efficiency and Quality of Induced Cardiac Myocyte Reprogramming.</a> Circ Res. , (2014).</li><li>Song, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+repair+by+reprogramming+non-myocytes+with+cardiac+transcription+factors.">Heart repair by reprogramming non-myocytes with cardiac transcription factors.</a> Nature. 485, 599-604 (2012).</li><li>Qian, 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=In+vivo+reprogramming+of+murine+cardiac+fibroblasts+into+induced+cardiomyocytes.">In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.</a> Nature. 485, 593-598 (2012).</li><li>Mathison, 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=In+vivo+cardiac+cellular+reprogramming+efficacy+is+enhanced+by+angiogenic+preconditioning+of+the+infarcted+myocardium+with+vascular+endothelial+growth+factor.">In vivo cardiac cellular reprogramming efficacy is enhanced by angiogenic preconditioning of the infarcted myocardium with vascular endothelial growth factor.</a> J Am Heart Assoc. 1, e005652 (2012).</li><li>Srivastava, D., Ieda, M., Fu, J., Qian, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+repair+with+thymosin+beta4+and+cardiac+reprogramming+factors.">Cardiac repair with thymosin beta4 and cardiac reprogramming factors.</a> Ann NY Acad Sci. 1270, 66-72 (2012).</li><li>Muraoka, N., Ieda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+transcription+factors+is+critical+for+cardiac+reprogramming.">Stoichiometry of transcription factors is critical for cardiac reprogramming.</a> Circ Res. , 116-216 (2015).</li><li>Srivastava, D., Ieda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+factors+for+cardiac+reprogramming.">Critical factors for cardiac reprogramming.</a> Circ Res. 111, 5-8 (2012).</li></ol>

The authors have nothing to disclose.

对于成功的ICM代使用此协议时，存在具有对整体效率产生影响的几个重要因素。尤其在开始成纤维细胞的条件和逆转录病毒编码MGT的质量可以极大地影响重编程效率。 产生的成纤维细胞作为新鲜健康尽可能是重要的。外植体培养方法，成纤维细胞可用于前外植七天后铺于菜肴。酶消化法，成纤维细胞，可以早在隔离的次日使用。冻结或传代的成纤维细胞进行重新编程，不推?...

Cardiovascular disease remains the leading cause of death worldwide, accounting for 17.3 million deaths per year1. Loss of cardiomyocytes resulting from myocardial infarction (MI) or progressive heart failure is a major cause of morbidity and mortality2. Due to limited regenerative capacity, adult mammalian hearts usually suffer from impaired pump function and heart failure following injury3-6. As such, efficient (re)generation of cardiomyocytes in vivo and in vitro for treatment of heart disease and for disease modeling is a critical issue needing to be addressed.
Recent development of direct reprogramming, which directly reprograms cells from one differentiated phenotype to another without transitioning through the pluripotent state, offers a promising alternative approach for regenerative medicine. The mammalian heart contains abundant cardiac fibroblasts (CFs), which account for approximately half of the cells in heart and massively proliferate upon injury7-9. Thus, the vast pool of CFs could serve as an endogenous source of new CMs for regenerative therapy if they could be directly reprogrammed into functional CMs. It has been shown that a combination of transcription factors, such as Gata4 (G), Mef2c (M) and Tbx5 (T), with or without microRNAs or small molecules can reprogram fibroblasts into iCMs10-26. Importantly, this conversion can also be induced in vivo, and results in an improvement in cardiac function and a reduction in scar size in an infarcted heart16,27-29. These studies indicate that direct cardiac reprogramming may be a potential avenue to heal an injured heart. However, the low efficiency of iCM reprogramming has become a major hurdle for further mechanistic studies. In addition, the reproducibility of cardiac reprogramming is another controversial issue of this technology11,30,31. 
Very recently, we generated a complete set of polycistronic constructs encoding G,M,T in all possible splicing orders with identical 2A sequences in a single mRNA. These polycistronic constructs yielded varied G, M and T protein expression levels, which led to significantly different reprogramming efficiency25. The most efficient construct, named MGT, which showed a relatively high Mef2c and low Gata4 and Tbx5 expression, significantly improved reprogramming efficiency and produced large amounts of iCMs with CM markers expression, robust calcium oscillation and spontaneous beating25. Moreover, by using MGT polycistronic construct, our study avoided the use of multiple vectors and generated cells with homogenous expression ratio of G,M,T, thus providing an improved platform for cardiac reprogramming research. To increase experimental reproducibility, here we describe in detail how to isolate fibroblasts, produce retrovirus carrying MGT cassette, generate iCMs and evaluate the reprogramming efficiency.

<table border="0" cellpadding="0" cellspacing="0" width="1088">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">anti-cardiac troponin T</td>
			<td style="width:236px;">Thermo Scientific</td>
			<td style="width:200px;">MS-295-PO</td>
			<td style="width:191px;">1:200 for FACS and 1:400 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-GFP</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11122</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti- aActinin</td>
			<td>Sigma-Aldrich</td>
			<td>A7811</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Connexin43</td>
			<td>Sigma-Aldrich</td>
			<td>C6219</td>
			<td>1:500 for&#160; ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anit-Mef2c</td>
			<td>Abcam</td>
			<td style="width:200px;">ab64644</td>
			<td>1:1000 for ICC&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Gata4</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:200px;">sc-1237</td>
			<td>1:200 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Tbx5</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-17866</td>
			<td>1:200 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 488&#8211;conjugated donkey anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Inc</td>
			<td>711-545-152</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 647&#8211;conjugated donkey anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Inc</td>
			<td>715-605-150</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Cytofix/Cytoperm kit for intracellular staining</td>
			<td style="width:236px;">BD Biosciences</td>
			<td style="width:200px;">554722</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rhod-3 Calcium Imaging Kit</td>
			<td>Life Technologies</td>
			<td style="width:200px;">R10145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thy1.2 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-049-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vectashield solution with DAPI</td>
			<td>Vector labs</td>
			<td style="width:200px;">H-1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td style="width:200px;">F-2442</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.05%)</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">25-052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PRMI1640 medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement</td>
			<td>Life Technologies</td>
			<td style="width:200px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IMDM</td>
			<td>Life Technologies</td>
			<td style="width:200px;">12440-053</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">31985-070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">10-060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, high glucose</td>
			<td>Life Technologies</td>
			<td style="width:200px;">10-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-streptomycin</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">30-002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Non-essential amino acids</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11130-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11668500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">blasticidin</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11139-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">puromycin</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11138-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase II</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polybrene</td>
			<td>Millipore</td>
			<td style="width:200px;">TR-1003-G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td style="width:236px;">Fisher</td>
			<td style="width:200px;">BP151-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>PX1405</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Na2HPO4</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Fisher</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>EMS</td>
			<td style="width:200px;">15714</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Retrovirus Precipitation Solution</td>
			<td>ALSTEM</td>
			<td style="width:200px;">VC-200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.4%Trypan blue solution</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">T8154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">gelatin</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">G1393</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s PBS without CaCl2 and MgCl2 (D-PBS, 1x)</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">D8537</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS (Hanks Balanced Salt Solution)</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">21022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LS column</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#956;m cellulose acetate filter&#160;</td>
			<td>Thermo Scientific</td>
			<td style="width:200px;">190-2545</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24-well plates</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">3524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm Tissue culture dishes&#160;</td>
			<td>Thermo Scientific</td>
			<td style="width:200px;">172958</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60mm center well culture dish&#160;&#160;</td>
			<td>Corning</td>
			<td style="width:200px;">3260</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 Well Clear V-Bottom 2mL Polypropylene Deep Well Plate</td>
			<td>Denville Scientific</td>
			<td style="width:200px;">P9639</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Polystyrene round-bottom tubes with cell-strainer cap&#160;</td>
			<td style="width:236px;">BD Biosciences</td>
			<td style="width:200px;">352235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td style="width:236px;">Eppendorf</td>
			<td style="width:200px;">5810R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortexer MINI</td>
			<td style="width:236px;">VWR</td>
			<td style="width:200px;">58816-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EVOS&#174; FL Auto Cell Imaging System</td>
			<td>Life Technologies</td>
			<td style="width:200px;">AMAFD1000</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-303</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MidiMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-302</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Round glass cover slip</td>
			<td style="width:236px;">Electron Microscopy Sciences</td>
			<td style="width:200px;">72195-15</td>
			<td></td>
		</tr>
	</tbody>
</table>

improved generation of induced cardiomyocytes using a polycistronic construct expressing optimal ratio of gata4, mef2c and tbx5

心脏成纤维细胞（CFS）成心肌细胞诱导直接转化（ICMS），通过治疗心脏疾病提供替代战略保持着再生医学的巨大潜力。这个转换已通过确定的因子如GATA4（G），MEF2C（M）和Tbx5中（T）的强制表达来实现。传统上，ICMS是由病毒表达这些个人因素鸡尾酒产生。然而，重新编程的效率是相对低的，大部分的体外 G，M，T转导的成纤维细胞不成为完全重新编程，使得难以研究的重编程机制它。我们最近已经表明，G，M的化学计量，T是用于有效ICM重新编程的关键。 G，M，T的具有M和低水平的G和T的通过使用我们的多顺反子MGT向量（以下称为MGT）显著增加重新编程的效率达到了较高水平的最佳化学计量学和体外改善ICM质量。在这里，我们提供用于产生ICMS与从心脏成纤维细胞MGT构建体的方法的详细描述。心脏成纤维细胞，代病毒重编程和重编程过程的评价的隔离也包括以提供有效和可再现代ICMS的一个平台。

重新编程步骤在图1中总结的示意图。MGT转导后，在重新编程的细胞的GFP表达可以早在第3天嘌呤选择转导的细胞的从第3天开始，并在头两个星期保持检测如果PMX-普罗-MGT构建体被使用。在第10天至14天，心肌标记物像cTnT和αActinin的表达可以由两个IC卡（图2B，步骤5）和FACS（图2A，步骤6）被检测到，这表明起始成纤维细胞正在经历重编程朝向心脏细胞命运。 <...

Watch this Scientific Journal Video about Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5 at JoVE.com

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

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 ...

improved-generation-induced-cardiomyocytes-using-polycistronic

Research

JoVE Journal

Developmental Biology

8.6K 次观看.  University of North Carolina, Chapel Hill. 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. 