这项研究是由美国国立卫生研究院4R00HL094708-03支持，MCW的研究事务委员会新教师奖和克恩基金会（启动资金）在威斯康星医学院（RLG）;香港的主题为基础的研究计划T13-706 / 11（KRB）的研究资助局; U01 HL099776，CIRM TR3-05556，CIRM DR2A-05394，和美国心脏协会研究者贡献奖（JCW）;美国心脏协会博士后奖学金12POST12050254（印刷电路板）。我们感谢希望坎贝尔在威斯康星血液研究所的流式细胞仪的核心与数据收集和仔细审查稿件的援助。

1，解决方案和媒体准备<ol><li>人类胚胎干细胞合格基质涂层原液<ol><li>慢慢解冻胚胎干细胞在冰上合格基质（5毫升）中的4ºC过夜。分配等份放入预冷的1.5毫升的无菌微量离心管中并立即存储于-20℃。 注:在等分试样的体积将根据许多变化，并且通常为270〜350微升。制造商提供的步骤2.1中所述关于实现稀释后的1X浓度为25毫升需要等分的音量的详细信息。 </li></ol></li><li> HPSC媒体原液<ol><li>使用超纯水作为稀释剂，除非另有说明。消毒用0.22微米的过滤器的所有组件。存储以下的本体溶​​液在4ºC:碳酸氢钠（75毫克/毫升）;柠檬酸（10毫米，pH值= 3）。 </li><li>消毒用0.2微米的注射器过滤所有的组件，每个等分储存在-20℃:卢激酶（ROCK）抑制剂Y-27632（10毫在DPBS中，将100μl等分试样）; L-抗坏血酸2 - 磷酸盐（64毫克/毫升，500微升等分试样）;亚硒酸钠（70微克/毫升，100微升等分试样）;转铁蛋白（50毫克/毫升，107微升等分试样）;成纤维细胞生长因子2（FGF2 200纳克/微升的DPBS，250μL分装）;转化生长因子β1（TGFβ1，100纳克/微升冷的10mM柠檬酸，10微升等分试样）。 </li></ol></li><li> HPSC媒体E8溶液组成<ol><li>准备使用的等分试样在步骤1.2中制备的介质:DMEM / F12（含L-谷氨酰胺和HEPES 500毫升），碳酸氢钠（3.62毫升，最终:543微克/毫升），L-抗坏血酸2 - 磷酸盐（500微升;终:64微克/毫升），亚硒酸钠（100微升;最终:140毫微克/毫升），转铁蛋白（107微升;最后:10.7微克/毫升），胰岛素（1毫升;最后:20微克/毫升），FGF2（250微升;决赛:100毫微克/毫升），转化生长因子β1（10微升;决赛:2毫微克/毫升）。 </li><li>结合所有组件，过滤消毒，并储存在4°C下长达2周。 </li></ol></li><li> HPSC媒体E8与ROCK抑制剂<ol><li>加入15微升ROCK抑制剂来12毫升E8媒体按上述方法制备拌匀。确保最终浓度是除细胞/媒体在步骤3.7之后为10μM。 </li></ol></li><li> Wnt信号调制器储液<ol><li>存储以下分装置于-20℃。 CHIR 99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5​-methyl-1 H -imidazol-2-yl)-2-pyrimidinyl]amino]ethy​l]amino]-3-pyridinecarbonitrile; 10mM的DMSO中，将15μl等分试样）。 IWR-1（4-（和1,3,3a，4,7,7a-六氢-1,3 - 二氧代-4,7亚甲基-2H-异吲哚-2 - 基） - N-8-喹啉基 - 苯甲酰胺; 10mM的DMSO中，6微升等份）。 </li></ol></li><li>分化培养基<ol><li>制备分化培养基＃1的RPMI 1640（含L-谷氨酰胺）中有2％B27减去胰岛素补充。 </li><li>制备分化培养基＃2的RPMI 1640（含L-谷氨酰胺）中有2％B27加上胰岛素补充和2％FBS中。 </li></ol></li><li>细胞洗涤溶液F或细胞培养<ol><li>用Dulbecco氏磷酸盐缓冲盐水（DPBS），无钙或镁。 </li></ol></li><li>细胞清洗液的流式细胞仪<ol><li>用磷酸盐缓冲盐水（PBS，无钙或镁），有1％FBS。商店在4ºC。 </li></ol></li><li>在流式细胞仪电池维修解决方案<ol><li>用Hank氏平衡盐溶液（HBSS;没有钙或镁），用5％FBS中。商店在4ºC。 </li></ol></li><li>固定解流式细胞仪<ol><li>使用Cytofix缓冲器作为TNNI3和IRX4抗体固定溶液。 </li><li>使用1X PBS中的4％多聚甲醛（使鲜）为TNNT2和MLC2a抗体固定的解决方案。 </li><li>用70％甲醇/ 30％丙酮作为MLC2v抗体固定溶液。 </li><li>存储所有的解决方案在4ºC。 </li></ol></li><li>透解流式细胞仪<ol><li>使用Phosflow彼尔姆缓冲三世通透溶液F或TNNI3和IRX4抗体。储存于室温（25℃）。 </li><li>在1X PBS用0.2％的Triton X-100作为TNNT2，MLC2v和MLC2a抗体通透的解决方案。商店在4ºC。 </li></ol></li><li>封闭液<ol><li>用10％山羊血清中的1X PBS。使新鲜和储存在4ºC备用。 </li></ol></li></ol> 2，涂层板<ol><li>慢慢解冻1等份的hESC合格基质，在4°C下30分钟，加至25ml冷的DMEM / F 12，然后立即涂敷的6孔板前在无菌组织培养罩拌匀。 </li><li>添加每一个6孔板的1毫升下灭菌组织培养罩（1等分的hESC合格基质足以外套4个6孔板）。 </li><li>允许的hESC合格基质在室温下罩30分钟设定。吸出过量的hESC合格基质和添加2毫升E8媒体与ROCK抑制剂到每个孔中。使用平板立即，或者如果德IRED，在4°C店镀长达1周后立即并平衡至室温，使用前30分钟。 </li></ol> 3，传代和未分化hPSCs的单层培养维护<ol><li>保持hPSCs在单层培养在6孔板中，并在无菌组织培养罩执行所有步骤。 </li><li>使用已经非常成熟HPSC线（&gt; P20），并没有表现出细胞的神经，上皮 - 或成纤维细胞样形态的存在同质形态。确保细胞增殖鲁棒性，并且平均通道，每三天，在75％汇合时，在0.75×10 6每接种好。 </li><li>通路hPSCs与解离为单细胞水平的至少5倍之前开始分化，以确保最高的分化效率。到通道hPSCs，吸E8媒体和两次用4ml 1X DPBS（预加热至室温）的洗细胞。加入1 ml的细胞分离的精神疾病的溶液（已预先加热至室温）至各孔中，离开井静置3-7分钟，直至小区边界开始到舍入。 </li><li>用棉栓的玻璃移液管与灯泡撞出细胞转移到15毫升锥形管中含有1ml的DMEM / F12培养基（以灭活细胞剥离溶液）。 </li><li>除去的细胞溶液10微升等分试样，并用10微升台盼蓝的微量离心管中混合。计数细胞（ 例如自动或手动的血球），而细胞的其余部分通过离心在130 XG收集5分钟，在室温下进行。通过此协议，预计使用自动血细胞计数器，还是今后，基地总细胞数的所有细胞数量的70-80％存活率。 </li><li>吸出介质并重新悬浮在DMEM / F 12细胞沉淀以每500μl的0.75×10 6个细胞的终浓度。 </li><li>加入500微升细胞悬浮液到6孔板的各孔中，其中每个孔周转箱NS2毫升E8媒体ROCK抑制剂，在步骤2.3准备。在拼接件离开板，轻柔地在前端至后端和侧到另一侧的运动，以均匀地分散细胞穿过井移动。回到细胞培养箱中在5％CO 2，37℃。 </li><li>传代后开始的24小时，每天使用2个毫升/ E8没有ROCK抑制剂更换介质。最佳接种密度达到之前100％汇合，开始分化。播种0.75×10 6个细胞/孔的四天内开始分化，但根据需要优化每个细胞系之前。 </li></ol> hPSCs 4。心肌细胞诱导的Wnt信号通路的选择性调节<ol><li>在天0-7每日更新媒体（2毫升/孔），和第8天之后每隔一天。 </li><li>在第0天，用分化培养基＃1替换E8媒体开始分化过程。加入1.2微升CHIR（6μM终浓度）至各孔中。重复第1天。 </li><li>在2-3天，更换新鲜不同的应tiation媒体＃1。 </li><li>在第4天，更换新鲜分化培养基＃1。加入1μlIWR-1（5μM终浓度）至各孔中。重复第5天。 </li><li>在6-7天，更换新鲜分化培养基＃1。 </li><li>在第8天，更换新鲜分化培养基＃2。 </li><li>继续更换分化培养基＃2隔日文化所需的时间。 </li><li>如果需要的话，使用该细胞剥离溶液以下在3.3-3.6中概述的步骤，但用分化培养基＃2代媒体通路心肌细胞。传代过程中，游离心肌成小群〜3-10细胞。种子在〜6×10 5％使用人类胚胎干细胞以及合格的矩阵涂井和分化培养基＃2单元。 </li></ol> 5，收集细胞进行流式细胞仪<ol><li>执行5-9的实验台（ 即 ，未育）的步骤，所有剩余的问题。 </li><li>吸生长介质，并用1×PBS洗涤细胞两次。加入1 ml的细胞剥离溶液（预先温热至室温）至每孔，并留下静置3-7分钟，直至小区边界开始到舍入。用棉花塞住硼硅玻璃一次性9"吸管球撞出细胞转移到15 mL锥形管上的冰。 </li><li>在整个协议的其余部分，维持细胞在冰上并在200 XG在4ºC进行离心5分钟。 </li><li>收集细胞，离心，吸出的解决方案。用10ml的血清吸液管反复研磨，以确保细胞团块重悬细胞于10ml细胞洗涤溶液中分散成单个细胞。计数细胞，在步骤3.5，而细胞的其余部分通过离心收集。 </li><li>重悬的细胞在细胞洗涤溶液照顾到完全解聚的细胞沉淀并等分每圆底管1×10 6个细胞。收集细胞，离心，吸出的解决方案。 </li></ol> 6，固定和细胞内抗原染色的透<ol><li>加入100μl固定的解决方案，以细胞团。加入溶液中逐滴连续温和的震荡，然后在冰上置15分钟。 </li><li>加入3 ml的细胞洗涤液。收集细胞，离心，吸出的解决方案。 </li><li>加入100μl通透的解决方案，以细胞团。加入溶液中逐滴连续温和涡旋然后在冰上置30分钟。使用固定和透化条件如表1中概述了每种抗体。 </li><li>加入3 ml的细胞洗涤液。收集细胞，离心，吸出的解决方案。重复一共有两个洗透了。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td rowspan="2"> 初级抗体（克隆） </td><td rowspan="2"> 免疫/抗原表位识别的 </td><td rowspan="2"> Istotype控制 </td><td colspan="2"&gt; 在100μl每1×10 6个细胞的一抗的量 </td><td rowspan="2"> 固定液 </td><td rowspan="2"> 通透的解决方案 </td><td rowspan="2"> 二抗 </td><td rowspan="2"> 二次抗体的量/ 1×10 6个细胞于100μl </td></tr><tr><td> 对于％的正测量 </td><td> 抗原定量 </td></tr><tr><td> TNNI3（284（19C7） </td><td> ISASRKLQL（人） </td><td>小鼠IgG2b </td><td> 1.0微克</td><td> 3.0微克</td><td> BD Cytofix </td><td> BD Phosflow烫发三</td><td>山羊抗小鼠IgG2b-Alexa488 </td><td> 600纳克</td></tr><tr><td> TNNT2（1C11） </td><td>全长纯天然人肌钙蛋白T蛋白。 </td><td>小鼠IgG1 </td><td> 1.0微克</td><td> 2.0微克</td><td> 4％PFA在1％的PBS中</td><td> 0.2％的Triton X-100在1％的PBS中</td><td>山羊抗小鼠IgG1 - 的Alexa 488 </td><td> 600纳克</td></tr><tr><td> MLC2v（330G5） </td><td> FDPEGKG </td><td>小鼠IgG2a </td><td> 2.0微克</td><td> 3.0微克</td><td> 70％甲醇/ 30％丙酮</td><td> 0.2％的Triton X-100在1％的PBS中</td><td>山羊抗小鼠IgG2a - 的Alexa 647 </td><td> 600纳克</td></tr><tr><td> MLC2a（4E7） </td><td>在大肠杆菌中产生的人MYL7的全长人重组蛋白大肠杆菌 </td><td>小鼠IgG1 </td><td> 0.5微克</td><td> 3.0微克</td><td>在1％的PBS中的4％PFA中</td><td> 0.2％的Triton X-100在1％的PBS中</td><td>山羊抗小鼠IgG1 - 的Alexa 488 </td><td> 600纳克</td></tr><tr><td> IRX4 </td><td> LQEHRKNP YPTKGEKI MLAIITKM TLTQVST </td><td>兔IgG </td><td> 0.5微克</td><td> 0.5微克</td><td> BD Cytofix </td><td> BD Phosflow烫发三</td><td>山羊抗-RABBIT的IgG-PE </td><td> 600纳克</td></tr></tbody></table> 表1中的抗体浓度。列出的是初级抗体，克隆（如单克隆）用于流量，优化的浓度和固定和透化条件的每个初级和次级抗体流式细胞仪分析。作为抗体的库存浓度可以在同一克隆的供应商之间变化，最终浓度每1×10 6个细胞的各抗体的固定测定体积，而不是稀释液中，提供了。免疫原用于产生抗体，或识别由抗体的表位，由制造商所提供的，被列出，但未经实验此处验证。 7抗体染色<ol><li>对于每一个实验中，包括一种未染色的对照每固定/透化条件和相应的同种型对照为每个使用的初级抗体。包括非心肌细胞的细胞类型作为阴性对照（例如 ，多能干细胞或成纤维细胞）。使用同种型对照在相同的浓度对应的第一抗体（ 见表1）。 </li><li>悬浮细胞在100μl封闭液使用的是P200的吸管分解细胞。在冰上25分钟，并轻轻摇动样本集。 </li><li>无需拆卸封闭液，加入一抗，或按照表1的准则同型对照。在冰上轻轻摇动45分钟。 </li><li>加入3 ml的细胞洗涤液。收集细胞，离心，吸出的解决方案。重复总共初级抗体标记后洗涤两次。 </li><li>如果一个荧光标记的一抗的情况下，进入8.1。当未结合的第一抗体被使用（如在这里所描述的所有标记物），与缀合至荧光基团如下的二次抗体进行标记。 </li><li>使用P200的吸管DISA悬浮细胞在100μl封闭液ggregate细胞。 </li><li>每添加表1中的指引二级抗体。在冰上用温和的摇摆30分钟。 </li><li>加入3 ml的细胞洗涤液。收集细胞，离心，吸出的解决方案。重复一共有两个洗二次抗体标记后。 </li></ol> 8，制备细胞流式细胞仪<ol><li>使用P1000的吸管分解细胞重悬细胞于400μl细胞的维护解决方案。 </li><li>通过预湿50微升细胞保养液准备在圆底管中的细胞过滤器盖。冰上集管。用细胞过滤网帽，以防止细胞聚集体堵塞的流式细胞仪。 </li><li>转移细胞溶液至细胞过滤帽，并允许细胞悬浮液通过重力排水。挖掘管的底部轻轻地放在台面作为必要的，这样的细胞被收集到管中，并置回冰上尽可能快。冲洗滤网用250微升细胞维持这样lution，以确保电池的最大恢复。 </li><li>维持细胞在冰上避光，直到流式细胞仪分析。 </li></ol> 9，流式细胞仪分析详细的收购设置将仪器各不相同。基本参数考虑为最佳的数据收集进行说明。 <ol><li>为了获得最佳的流稳定，确保喷嘴大小超过5-6x被分析的细胞的直径。 注意:这可能文书之间变化，但是一个重要的考虑因素都对分析仪和细胞分选选择适当的喷嘴尺寸。第10天心肌细胞的平均直径为11微米（范围8-14微米）;因此，在分析仪标准的150微米的样品进样管效果很好。 </li><li>紧接之前的数据的分析，涡流每个管短暂地分散细胞聚集体。 </li><li>优化前向和侧向散射光的电压设置为不同的细胞类型的基础上未染色的对照的实验中，使得所感兴趣的种群上规模和居中（ 图2A）中使用的每个固定/透化条件。 <ol><li>保持整个单次实验中这些设置，并从实验一致的实验在同一台仪器，因为显著转变可能表明流式细胞仪和样品制备潜在的问题。 </li></ol></li><li>对于每个荧光团，分析同种型对照和调整相应的激光的电压来确定，以获得可显示的峰的左，右边缘的荧光直方图所需的最小强度。相应抗体染色标本采集数据时，要保持每个同型对照激光设置。 注:信号漂移往往发生在对的时间在同一台仪器。关键是在基于适当的同种型对照各实验开始时，以调整激光的设置。 </li><li>收取最低10,000个事件。 50,000个事件是优选的。 </li><li>对于统计分析，门的活细胞群，以排除碎片（ 图2A）。基于标记放置，允许≤2％贡献来自同种型对照， 如图2B所示，C中的门控群体中确定阳性细胞百分数。 </li></ol>

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关键的分化方案的成功是利用hPSCs的高品质文化已在微分之前的开始传代在单细胞水平为至少五个通道。类似的分化效率都在各种HPSC行常规观察到，如果他们是100％汇合，在分化开始时，独立细胞系。如果细胞在分化开始时的汇合是≤95％或&gt; 100％的次最佳效率是观察。因此，接种密度和传代的定时应仔细优化为每个线路达到100％汇合的单层细胞在分化的开始。在这方面，镀槽的明确数目/孔，?...

心肌细胞从hPSCs，包括胚胎干细胞和hiPSC，生成可作为非常早期的人类心脏发育过程中的体外模型，提供了洞察阶段不进行机理研究，否则访问。该模型系统提供了独特的机会来研究控制心脏谱系的承诺和细胞命运规范的分子途径。近年来，为了有效地生成心肌细胞从hPSCs的能力大大改善了1-15。然而，其中的协议存在的细胞系的变化相对于在产生心肌细胞和定时，在该细胞表达腔特异性标记物（ 例如 ，心室和心房）的效率。理想情况下，该模型系统的未来应用，功能上定义的细胞更均匀种群是期望的。与以前的方法中，心肌细胞的分化这里描述的协议不要求CELL聚集或添加激活素A和骨形态发生蛋白4（BMP4）和强劲的生成TNNI3，TNNT2，IRX4，MLC2v和MLC2a文化非常积极的一天10细胞穿过测试日的所有人类胚胎干细胞和hiPSC线。该策略在技术上容易实现，尤其是相对于三维培养，大量培养，或胚状体基础的策略4-9，并且在一项研究描述了一种小分子具有选择性毒性，对hPSCs（Boheler 等人最近被限定。 ）65。该协议的功能包括使用人类胚胎干细胞资格基质（基质胶）的单层，利用小分子完全定义的媒体来调节Wnt信号（类似，但有别于1,2,7,13）单层培养hPSCs的分化，流式细胞仪染色方法的分化效率和小区标识评价的优化流程。综上所述，本协议的优点相比之前的报道，包括其高性价比eness，重现性，以及其高效率，用于产生在多个HPSC线，包括胚胎干细胞和hiPSC线心肌细胞。 流式细胞仪是评估细胞的培养质量，确定亚群同质化的强大的分析工具，以及适当的实验设计，可以提供定量测量。与所有的抗体为基础的策略，实验结果准确解释要求的试验设计元素，包括抗体浓度和固定和通透条件（针对细胞内的抗原时），经过认真的检查每一个抗体作为次优条件显著影响抗体的效结合，因此，结果的解释。重要的是，如果需要进行定量的，单克隆抗体是至关重要的，因为多克隆抗体可以识别多个表位，并且容易发生批次到批次的变化。目前，各种抗体（POlyclonal和单克隆）和染色方案已经描述了用于在体外分化的评估，从而难以比较中协议1,2,9,11 cardiomyogenesis的效率。出于这个原因，单克隆抗体用于仅当对于所有流式细胞仪分析。展望未来，预计这些染色方法的标准化，特别是关于定量，最好允许差异化战略之间的比较。 标记物的选择，以及它们相应的抗体，用于评估在体外 cardiomyogenesis纯度之间的报告而有所不同。 TNNT2一直被认为是细胞致力于心肌命运的指标和常规用于评价心肌分化方法的效率。然而，TNNT2也早期小鸡和大鼠发展16,17表达在骨骼肌中，它是存在于人平滑肌18 </sup&gt;。因此，TNNT2不一定是人类cardiomyogenesis 在体外的特异性标志物。 MLC2v和MLC2a通常用于为心室和心房亚型的替代指标，分别为。然而，与依托MLC2v和MLC2a确定在体外分化的上下文心肌亚型挑战源自以下事实:这些基因产物可以不通过成人限于整个心脏发育的特定腔室，从心脏管。在啮齿动物心脏循环，MLC2a基因是主要的心房/流道面积和MLC2v基因主要是在室/流出道地区。在环状心脏，共表达MLC2a和MLC2v的mRNA中的流入道，房室通道，并流出道19,20进行观察。由出生后3天，MLC2v mRNA表达被限制在心室和由出生后10天，MLC2a被限制到心房中的新生大鼠心脏19。因此，解释关于cardiomyogenesis效率和亚型的身份不但要考虑的参考标志物水平的存在和数量，但必须考虑到发展阶段（县）的分化所分析的时间点对应的数据。这是特别重要的考虑是由hPSCs的体外分化产生心肌细胞的成熟阶段最相似的那些胚胎/胎儿发育21-25。因此，依靠标记的空间表现在出生后心脏可能不适合HPSC源性细胞的评估，至少在某些情况下。 在努力促进更具体标准的发展，用于定义在体外心肌细胞的身份，TNNI3被认为是一种有价值的标志物，用于评估cardiomyogenesis 体外 ，它被限制为心肌整个胚胎发育中的小鸡和斑马鱼15,20并且不存在于人胎儿骨骼肌26。而TNNI1是存在于人类胎儿心脏，TNNI3是唯一TNNI存在于正常成人心脏27,28亚型。对于心肌细胞亚型的身份，IRX4 29-31是细胞与心室命运的信息标记。在蛋白质水平上，IRX4最近已显示被限制在从线性心脏管通过新生儿阶段的心室中的鼠标32。因此，优化的染色方法为TNNI3的通过流式细胞术分析和IRX4进行说明。就我们所知，这是高效率的基于抗体染色并在人心肌IRX4水平的流式细胞仪分析的方法的第一次描述。

<table border="0" cellpadding="0" cellspacing="0" width="922">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:431px;">Name of Reagent / Equipment</td>
			<td style="width:152px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:231px;">Comments/Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;"></td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Cell Culture</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">BD Matrigel, hESC-qualified matrix</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:111px;">354277</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Accutase cell detachment solution</td>
			<td style="width:152px;">Stem Cell Technologies</td>
			<td style="width:111px;">7920</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:431px;">Dubelcco&#39;s phosphate buffered saline (DPBS), no calcium, no magnesium</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Dimethyl sulfoxide (DMSO, Hybri-Max, sterile-filtered)</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">D2650</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Sodium bicarbonate</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">53817</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Citric acid</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">C2404-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Y-27632 dihydrochloride selective p160ROCK inhibitor</td>
			<td style="width:152px;">R&#38;D Systems</td>
			<td style="width:111px;">1254</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">L-ascorbic acid-2-phosphate sesquimagnesium salt hydrate</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">A8960-5G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Sodium selenite</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">S5261-10G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Transferrin (Optiferrin &#8211; Defined, Animal-free, Recombinant Human)</td>
			<td style="width:152px;">Invitria</td>
			<td style="width:111px;">777TRF029</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Fibroblast growth factor 2 (FGF2)</td>
			<td style="width:152px;">R&#38;D Systems</td>
			<td style="width:111px;">4144-TC-01M</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Transforming growth factor beta 1 (TGF&#946;1)</td>
			<td style="width:152px;">Peprotech</td>
			<td style="width:111px;">100-21</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">DMEM/F12 with L-Glutamine and 15 mM HEPES</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Insulin</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">I9278-5ML</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">CHIR-99021 HCl</td>
			<td style="width:152px;">Sellekchem</td>
			<td style="width:111px;">S2924</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">IWR-1</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">I0161</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">RPMI 1640 with L-Glutamine</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">B-27 Supplement Minus Insulin</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">A1895601</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">B-27 Supplement (with Insulin)</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Fetal bovine serum</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">10437</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;"></td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Flow cytometry reagents</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Phosphate buffered saline (10X)</td>
			<td style="width:152px;">Quality Biological Inc.</td>
			<td style="width:111px;">119-069-151</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:431px;">Hank&#39;s balanced salt solution without Ca2+ and Mg2+</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">14175-095</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">BD Cytofix</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:111px;">554655</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">BD Phosflow perm buffer III</td>
			<td style="width:152px;">BD Biosciences</td>
			<td style="width:111px;">558050</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">16% Paraformaldehyde</td>
			<td style="width:152px;">Thermo Scientific</td>
			<td style="width:111px;">28906</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Methanol</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:111px;">179957-4L</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Acetone</td>
			<td style="width:152px;">Mallenckrodt Chemicals&#160;</td>
			<td style="width:111px;">2440-16</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Triton X-100</td>
			<td style="width:152px;">Amresco</td>
			<td style="width:111px;">M236-10ML</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Goat serum</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">16210-064</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Trypan blue solution, 0.4%</td>
			<td style="width:152px;">Life Technologies</td>
			<td style="width:111px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Materials</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">5 ml round bottom polystyrene tube (without cap)</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">352008</td>
			<td>for preparation of cells for flow cytometry</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">5 ml round bottom polystyrene tube (with 35 &#181;M cell strainer snap cap)</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">352235</td>
			<td>for preparation of cells for flow cytometry</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">2 ml rubber bulbs</td>
			<td style="width:152px;">Fischer Scientific</td>
			<td style="width:111px;">03-448-24</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">9&#34; borosilicate unplugged glass Pasteur pipets</td>
			<td style="width:152px;">BioExpress</td>
			<td style="width:111px;">P-2904-2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">0.65 mL microcentrifuge tubes, graduated, green</td>
			<td style="width:152px;">GeneMate</td>
			<td style="width:111px;">C-3259-G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">1.5 mL microcentrifuge tubes, graduated, red</td>
			<td style="width:152px;">GeneMate</td>
			<td style="width:111px;">C-3260-R</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">TC20 automated cell counter</td>
			<td style="width:152px;">BioRad</td>
			<td>145-0102</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Counting slides for TC20</td>
			<td style="width:152px;">BioRad</td>
			<td style="width:111px;">145-0011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">6 well flat bottom tissue-culture treated plate</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">353046</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">20 mL syringes</td>
			<td style="width:152px;">BD Plastipak</td>
			<td style="width:111px;">300613</td>
			<td>For filter sterilization of aliquots</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Sterile syringe filters, 0.2 &#181;m polyethersulfone</td>
			<td style="width:152px;">VWR</td>
			<td style="width:111px;">28145-501</td>
			<td>For filter sterilization of aliquots</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">250 mL filter system, 0.22 &#181;m polyethersulfone, sterilizing, low binding&#160;</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">431096</td>
			<td>For filter sterilization of bulk media&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">500 mL filter system, 0.22 &#181;m polyethersulfone, sterilizing, low binding&#160;</td>
			<td style="width:152px;">Corning</td>
			<td style="width:111px;">431097</td>
			<td>For filter sterilization of bulk media&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Antibodies and Isotype Controls</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Anti-TNNI3 (clone: 284 (19C7))</td>
			<td style="width:152px;">Abcam</td>
			<td>ab19615</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-TNNT2 (clone: 1C11)</td>
			<td>Thermo Scientific</td>
			<td>MA1-16687</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-MYL2 (MLC2v, clone: 330G5)</td>
			<td>Synaptic Systems</td>
			<td>310111</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-MYL7 (MLC2a, clone: 4E7)</td>
			<td>Abcam</td>
			<td>AB131661</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Anti-IRX4&#160;&#160;</td>
			<td>Bioss</td>
			<td>BS-9464R</td>
			<td>Primary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Alexa Fluor 488 Goat anti-mouse IgG1&#160;</td>
			<td style="width:152px;">Life Technologies</td>
			<td>A21121</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Alexa Fluor 647 Goat anti-mouse IgG2a</td>
			<td style="width:152px;">Life Technologies</td>
			<td>A21241</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Alexa Fluor 488 Goat anti-mouse IgG2b</td>
			<td style="width:152px;">Life Technologies</td>
			<td>A21141</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">Phycoerythrin(PE) Goat anti-rabbit IgG</td>
			<td style="width:152px;">R&#38;D Systems</td>
			<td style="width:111px;">F0110</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mouse IgG1</td>
			<td>BD Biosciences</td>
			<td>557273</td>
			<td>Isotype Control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mouse IgG2a</td>
			<td>eBiosciences</td>
			<td>16-4724-81</td>
			<td>Isotype Control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rabbit IgG-PE</td>
			<td>R&#38;D Systems</td>
			<td>IC105P</td>
			<td>Isotype Control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;"></td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">TaqMan Probesets for qRT-PCR</td>
			<td style="width:152px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ACTB</td>
			<td>Life Technologies</td>
			<td>Hs01060665</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Brachyury T</td>
			<td>Life Technologies</td>
			<td>Hs00610080</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CASQ2</td>
			<td>Life Technologies</td>
			<td>Hs00154286</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IRX4</td>
			<td>Life Technologies</td>
			<td>Hs01100809</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ISL1</td>
			<td>Life Technologies</td>
			<td>Hs00158126</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MESP1</td>
			<td>Life Technologies</td>
			<td>Hs01001283</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYH11 (SMMHC)</td>
			<td>Life Technologies</td>
			<td>Hs00224610</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:431px;">MYH6</td>
			<td>Life Technologies</td>
			<td>Hs01101425</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYL2 (MLC2v)</td>
			<td>Life Technologies</td>
			<td>Hs00166405</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYL7 (MLC2a)</td>
			<td>Life Technologies</td>
			<td>Hs01085598</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MYOG</td>
			<td>Life Technologies</td>
			<td>Hs01072232</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NKX2.5</td>
			<td>Life Technologies</td>
			<td>Hs00231763</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NPPA</td>
			<td>Life Technologies</td>
			<td>Hs01081097</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">POU5F1</td>
			<td>Life Technologies</td>
			<td>Hs04260367</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">SOX17</td>
			<td>Life Technologies</td>
			<td>HS00751752</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNI1</td>
			<td>Life Technologies</td>
			<td>Hs00913333</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNI2</td>
			<td>Life Technologies</td>
			<td>Hs00268536</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNI3</td>
			<td>Life Technologies</td>
			<td>HS00165957</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TNNT2</td>
			<td>Life Technologies</td>
			<td>Hs00165960</td>
			<td></td>
		</tr>
	</tbody>
</table>

high efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry

目前迫切需要开发用于修复受损的心脏，发现没有对心脏毒性作用的新的治疗药物，改善策略，以准确地模拟心脏疾病的方法。利用人类诱导多能干细胞（hiPSC）技术，"一盘"为这些应用程序生成心肌的潜力继续产生很高的热情。近年来，为了有效地生成人类多能干细胞（hPSCs）心肌细胞的能力有很大的提高，为我们提供了新的机会，以模拟人类心脏发育的早期阶段，否则无法访问。相反，许多以前的方法中，心肌细胞的分化这里描述的协议不需要的细胞聚集或添加激活素A或BMP4和鲁棒生成细胞的，这对于心肌肌钙蛋白高度阳性培养物I和T（TNNI3，TNNT2），iroquois-一流的同源蛋白IRX-4（IRX4），肌球蛋白调节轻链2，室/心肌亚型（MLC2v）和肌球蛋白调节轻链2，心房异构体（MLC2a）由10天对所有的人胚胎干细胞（胚胎干细胞）和hiPSC行测试，以日期。细胞可以传代并保持在培养90天以上。该战略在技术上实现简单，具有成本效益。从多能干细胞的心肌细胞的特性通常包括参考标记的分析，无论是在mRNA和蛋白质水平。对于蛋白质分析，流式细胞仪是评估细胞的培养质量，确定亚群同质化的强大的分析工具。然而，在样品制备技术变化可能显著影响流式细胞仪检测数据的质量。因此，染色方法的标准化应该促进各种差异化策略之间的比较。因此，对于IRX4，MLC2v，MLC2a，TNNI3和TNN的分析而优化的染色方法T2通过流式细胞术进行了描述。

在第0天，将细胞100％汇合以紧凑的形态和最小的细胞碎片。于第1-2天，通常观察显著细胞死亡（40-50％），但附着的细胞将保留紧凑的形态（ 图1A）。在这段时间内，媒体是橙色和浑浊的。粉红媒体表示过度的细胞死亡，并且在这种情况下，确认用锥虫蓝和停止，如果细胞死亡高于70％。细胞会在3-4天恢复和密度就会增加。在5-6天，最小的细胞死亡，并出现密集的修补程序可能开始出...

Watch this Scientific Journal Video about High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry at JoVE.com

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

该文章描述的详细方法通过选择性调节Wnt通路，随后通过流式细胞仪的参考标记分析来评估群体的均匀性和同一性的有效分化人多能干细胞分化为心肌细胞。

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 ...

high-efficiency-differentiation-human-pluripotent-stem-cells-to

Research

JoVE Journal

Biology

30.1K 次观看.  Medical College of Wisconsin. 该文章描述的详细方法通过选择性调节Wnt通路，随后通过流式细胞仪的参考标记分析来评估群体的均匀性和同一性的有效分化人多能干细胞分化为心肌细胞。 

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

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

科研

生物学

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

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 ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

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 ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

在肾小管上皮细胞胞浆钙的测量流式细胞仪

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.