Name: visualization of cell cycle variations and determination of nucleation in postnatal cardiomyocytes
Uploaded: 2017-02-24T14:00:00.000+00:00
Duration: 9 min 41 s
Description: Watch this Scientific Journal Video about Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes at JoVE.com

We thank S. Gr&#252;nberg (Bonn, Germany) and P. Freitag (Bonn, Germany) for their technical assistance.

该协议涉及动物的所有程序均符合波恩大学的道德标准，并从2010年指令欧洲议会/ 63 / EU的用于科学目的的动物保护准则的规定。 1. 在产后心肌细胞周期活动的体外可视化<ol><li>产后心肌细胞分离<ol><li>实验前准备工作<ol><li>制备培养基（IMDM，1％青霉素/链霉素，1％非必需氨基酸，0.1％β巯基乙醇）;介质1:加FCS至2％;介质2:添加FCS至20％。 </li><li>涂层的96孔培养皿（半生长面积:15 平方毫米）用在PBS中的溶液0.1％明胶。 </li></ol></li><li>心脏解剖<ol><li>制备培养皿用15mL PBS中，并将其存储在冰上。通过把它们放在干燥的玻璃珠灭菌消毒2镊子和小剪刀。 </LI&gt; <li>断头牺牲新生转基因小鼠（CAG-EGFP-anillin / MYH6-H2B-MCH）。打开胸腔，解剖心脏出来，如Ehler 等人。 ，2013（J显示精通;（79）:50154 DOI:10.3791 / 50154），并转移到冰冷的PBS。移动到下一个鼠标。 </li><li>如果使用非纯合子繁殖对，确定与荧光显微镜转基因心。放入PBS的心在荧光显微镜下。检查EGFP-anillin表达式（例:488纳米; EM:509纳米），H2B-MCH表达（例:587纳米; EM:610纳米）用适当的过滤器设置。 </li></ol></li><li>心肌隔离<ol><li>通过新生儿心脏分离试剂盒分离选定的心。孵育酶混合物1在37℃下5分钟。要获得1毫升最终酶混合物，加酶混合物1 945μL到酶混合物2的55微升。 </li><li>传送到从P1 4心 - P3小鼠或多达从P4 2的心 - P6小鼠的1.5mlř含1毫升酶混合物反应的影响管切的心脏切成小块，用剪刀。转移溶液加到15mL反应管中。 </li><li>孵育在37℃下15分钟。为了最大限度地提高组织与酶之间的接触，把15毫升的管几乎水平在孵化器。用5毫升吸管10倍上下 - 吹打5混合。重复此步骤三次。 </li><li>添加7.5毫升介质2（20％FCS）中的以停止酶反应。通过一个70微米的细胞滤网过滤细胞悬浮液。冲洗用3mL介质2的细胞过滤网。 </li><li>离心在300×g的15分钟的细胞悬浮液并丢弃上清液。悬浮在介质1的500微升沉淀;不需要进一步的实验红细胞裂解。吸管10细胞悬液到细胞计数室的微升和确定细胞的数量。 </li><li>对于一个96孔板:种子，每孔10,000个细胞的120微升培养基1（2％FCS）中。地点将细胞成培养箱中（37℃和5％的CO 2），并立即进行转染。 </li></ol></li></ol></li><li>乳鼠心肌细胞的转染的siRNA或miRNA的<ol><li>请用核糖核酸酶净化解决方案的长凳去除RNA酶。请用核糖核酸酶净化解决方案如下材料:10μL，100μL和200μL的移液器和冰盒。解冻的siRNA股票等份（100μM）在冰上。 </li><li>准备SI / miRNA的2μM工作库存降低血清培养基（血清培养基减少+ 2的siRNAμL100微米股票的98微升）。工作股价应在当天使用。不建议冻结。 </li><li>至用血清培养基减少6微升加入4微升的2微米的库存，以0.5毫升的反应管（量对于一个96孔;最后SI / miRNA的浓度在140微升:〜57纳米）。选择为井的数量适当体积，以具有一定的SI / miRNA的被转染。 </li><li>准备转染试剂的主结构。使用10μL（0.6μL转染试剂+ 9.4μL降低血清培养基的），每口井孔（96个）。 </li><li>添加SI /混合的miRNA的转染试剂混合。孵育在冰上5分钟。吸管20微升到每个孔中。孵育所述细胞在37℃下48小时和5％的CO 2，然后与介质1的120微升替换介质中的每个井24小时或更长时间后，继续执行固定和免疫荧光染色。 </li></ol></li><li>固定和免疫荧光染色<ol><li>除去培养基并用PBS洗一次细胞。覆盖20分钟4％甲醛溶液中的细胞。脱下甲醛溶液，并用PBS洗一次细胞，然后用PBS覆盖它们用于存储或进行免疫染色。 </li><li> （根据制造商的说明浓度）在0.2％的Triton-X和5％驴血清为至少2小时，用第一抗体孵育。如果染色α辅肌动蛋白（稀释1:400;单克隆抗α辅肌动蛋白），染色过夜，在4℃。 </li><li>除去第一抗体并用PBS洗涤3次。稀释二级抗体在Hoechst的溶液1:400和在室温下孵育1小时避光。除去抗体，用PBS洗涤3次，并覆盖用PBS将细胞储存于4℃。 </li></ol></li><li>共聚焦显微镜的视频<ol><li>使用共聚焦显微镜用于图像采集。打开图像处理软件。打开"A1plus设置"，然后检查是否有CH1，CH2，CH3和箱子。通过单击下拉菜单上的CH1设置为DAPI，CH2到绿色荧光蛋白，CH3为Cy3的，和CH4为CY4。 <ol><li>对于每个通道，点击HV和通过使用滑动条其设置为80。通过使用滑动条设置偏移量为0，然后点击"主页"按钮来设置针孔位置。通过单击下拉菜单中的扫描尺寸为1024×1024。 </li><li>点击优化开"XYZ尺寸设置"窗口。检查下"建议的步骤（Z）""完美体素"框。增加激光强度，直到画面既不是曝光不足，也不过度。 注:例如， CH1（DAPI）:16％，CH 2（EGFP）:12％，CH 3（Cy3标记）:100％，CH 4（Cy5的）:1％。设置偏移所有通道为0。 </li></ol></li><li>以使用20倍的目标（200倍）的照片。扫描一个大的图像，包括平铺图像（ 例如，3×3）。 <ol><li>在成像软件，进入"获取"，然后从下拉菜单中选择"扫描大的图像"。在"区域"，选择"当前位置是左上角的"从下拉菜单中，并设置为3×3，点击"在X和Y字段数"，"扫描"。 </li></ol></li></ol></li><li>图像分析<ol><li>通过计数H2B-CH信号和心肌细胞的通过计数α辅肌动信号的数目限定心肌细胞核的数目。 <ol> &lt;李&gt;点击"测量"，然后从下拉菜单中选择"手动测量"。选择从下一个下拉菜单"计数"。点击与H2B-MCH信号核，由此标记和计数它们。执行相同的α辅肌动蛋白阳性细胞以获得心肌细胞的数目。 </li></ol></li><li>算上S /核EGFP-anillin信号（EGFP-anillin + / H2BmCh +核）G2期心肌细胞。点击"测量"，然后从下拉菜单中选择"手动测量"。选择从下一个下拉菜单"计数"。标记通过点击与EGFP-anillin信号和H2B-MCH信号核。 </li><li>计数具有特定的有丝分裂-EGFP-anillin信号（ 例如， 图1F和G），例如胞质信号，收缩环和midbodies的心肌细胞。点击"测量"，然后从下拉菜单中选择"手动测量"。从下一个PUL选择"计数"ldown菜单。马克通过点击特定的有丝分裂-EGFP-anillin信号（ 例如， 图1F和G），细胞质信号，收缩环和midbodies心肌细胞。 </li></ol></li></ol> 2.确定核成人的心肌Langendorff离被解离和厚组织切片<ol><li>通过分离的Langendorff成人的心肌细胞的分离<ol><li>心脏解剖前的准备工作<ol><li>填充胰岛素注射器（30G针）与肝素（20单位/ g体重）。由颈背把握鼠标。提起并打开鼠标向后揭露注射腹部。 </li><li>执行腹膜内注射。把肝素鼠标背部入笼，并开始清扫心脏前等待至少15分钟。冲洗并用5通风Langendorff装置上 - 灌注缓冲液的10毫升（灌注缓冲液:135毫摩尔NaCl，4mM的氯化钾，1mM的氯化镁2，2.5毫米的HEPES，5毫米的葡萄糖和25毫米BDM在DDH 2 O，调节pH至7.4用NaOH）。 </li></ol></li><li>心脏解剖<ol><li>制备一个10厘米的培养皿用冷（4℃）PBS中并放置在冰上。填充和通风用PBS 1毫升注射器连接到套管（20号）。修正了在培养皿的边框造型粘土套管。放置插管尖端略低于PBS表面。 </li><li>颈椎脱位牺牲鼠标。擦拭用70％乙醇溶液中的腹部。建立从中期腹部手术剪胸骨切口。取出膜片，用手术剪剪双侧胸腔。 </li><li>抬起并用20号针头固定胸骨，打开胸腔，并与解剖钳轻轻抓住心脏。提起心脏和切割流出道的血管从肺部和血管取出。 注意:要识别主动脉，心脏与定位其腹侧。这减少两个心房之间的距离，因此与它的分支主动脉可以心房之间找到。取决于个体心脏结构，分支可以如主动脉的主要分支仍然足够长被移除。 </li><li>的心脏转移到含冷冻PBS 10厘米的培养皿（见步骤2.1.2.1）。通过拉动一个G20x1半注射插管，这是由一个振荡的多工具钝化，以避免组织损伤升主动脉导管插入心脏。修复主动脉与上套管的线程。轻轻地用PBS冲洗心脏推注射器活塞，去除多余血液。的心脏有轻微的扩大应该是可见的。 </li></ol></li><li>的Langendorff心脏分离<ol><li>快速连接心脏插管来在的Langendorff灌流露儿锁适配器。灌注用30mL充氧的灌注缓冲液中的心脏，在37℃进行5分钟，1滴/秒的流速。钍È流率可以通过使用减压阀（0和200毫巴之间的压力）调节的Langendorff装置内的O 2流量进行调整。 </li><li> 6ü胶原酶B，10000台胰蛋白酶，和50μM 氯化钙灌注缓冲液:使用前立刻准备消化缓冲液。除去灌注缓冲液，并通过添加30毫升温水（37℃）和含氧消化缓冲液开始酶消化。 注意:需要胶原酶B的量取决于不同批次的活性被滴定。 </li><li>调节流量以1滴/秒的速率和消化心脏为10 - 13分钟。为了避免污染，不要再使用外排。的心脏转移到一个10厘米的培养皿用5ml消化缓冲液中。用一对镊子举行的心脏，并使用另一对到心脏撕裂成小块，用钳子手动解剖组织。 注意:在这一步，心房可以分离，切成小件（与虹膜剪），并且为了获得分离的心房细胞消化在培养箱消化缓冲液750微升30分钟，在37℃。要使用1毫升吸管得到纯净的心房心肌细胞，吸管上下几次。停止加入终止液750微升的酶反应。 </li><li>加入5毫升的终止溶液（含5％FBS和50μM的氯化钙灌注缓冲液）停止消化。使用血清吸管，通过100微米的细胞滤网过滤细胞悬浮液收集细胞，在50-mL离心管中。离心过滤的细胞悬浮液在80 XG在室温下1分钟。 </li><li>弃去上清液，并用10毫升血清吸管轻轻重悬细胞沉淀在10ml的终止溶液。离心细胞悬浮液，在80×g离心在室温下1分钟。 </li><li>离心后，弃上清，重悬细胞在1mL培养基（IMDM的用20％的FCS，0.1mM非必需氨基酸，50微克/毫升每青霉素和链霉素和0.1mMβ巯基乙醇）用于培养。另外，继续执行步骤2.2，"免疫荧光染色"，并修复细胞。 </li><li>在37℃下固定于24孔板，每孔8微克/毫升层粘连蛋白涂层的盖玻片分离的细胞的板10,000用培养基的500微升24孔板和5％CO 2的至少3小时。 </li></ol></li></ol></li><li>在悬挂的Langendorff隔离心肌细胞免疫荧光染色<ol><li>固定的Langendorff隔离心肌在1mL 4％PFA的PBS，pH为7.4，在室温下15分钟。离心细胞悬浮液以800×g离心在室温下2分钟，除去固定剂，并用PBS洗细胞两次。 </li><li>重悬在固定心肌在1mL PBS中，要么与免疫荧光染色进行或将它们存储在PBS 500微升每孔上在4℃的24孔板中。细胞悬液转移的一部分（〜100 - 200微升）到微量离心管中。离心细胞悬浮液以800×g离心在室温下2分钟，弃上清。 </li><li>通透，块，并通过加入稀释的1初级抗体的200μL的染色用抗α辅肌动蛋白一抗细胞:在PBS 400与0.2％的Triton X-100和5％驴血清，在室温下1小时。 </li><li>离心以800 xg离心在室温下2分钟，弃上清。用PBS洗细胞沉淀和离心以800 xg离心在室温下2分钟。弃去上清液，并在200微升的Alexa-氟结合的第二抗体（抗小鼠IgG1）孵育稀释1:在室温下1小时后，避光:在Hoechst的染料400（1微克/毫升工作溶液）。 </li><li>离心以800 xg离心在室温下2分钟，弃上清。重复此步骤。避光样品。 - [Resuspend将细胞在200 - 的PBS500μL，并传送100 - 细胞悬浮液的300μL到一个显微腔室的孔中。关闭室的盖子，将细胞在4℃下保存，直到成像。避光。 </li></ol></li><li>的Langendorff隔离心肌细胞nuclearity分析<ol><li>取从α辅肌动蛋白染色的心肌细胞的图像用25X物镜，对应于250X放大倍数。计数仅在经常由α辅肌动蛋白染色，并显示出典型的棒状的形态，因此被认为是完整的那些心肌H2B-MCH +核的数目。 </li><li>算从三个不同心的至少100心肌细胞。计算单核，双核，三核和心肌细胞的百分比。 注意:通常情况下，双核细胞应该弥补约80 - 90％，而三核细胞和那些与原子核的更大数目是罕见的（在的Langendorff隔离心肌&lt;1％，&lt;在厚片3％）。心房心肌比心室肌显著较小。通常情况下，单核细胞占90％，占总人口的。 </li></ol></li><li>隔离，固定，和成人的心冷冻保存为厚片准备<ol><li>用50毫升的注射器连接到带有一个海德堡延伸管和一个第二的三通旋塞3路阀组装的固定用灌注装置。固定在使得通流通过重力使能的方式在一个支架上的注射器。填充系统15毫升的PBS。 </li><li>按照步骤2.1.2.1-2.1.2.4，"心脏的准备。"在G20x1½注射导管与心脏连接到无气泡-PBS填充灌注设备的鲁尔锁适配器，用PBS灌注它。填充系统10 - 15毫升的4％甲醛的PBS中，然后通过心脏灌注。浸没在4％甲醛溶液固定心脏过夜。 </li><li>更换4％甲醛用PBS溶液，并在150rpm下洗在PBS的心脏在水平摇动器在室温下8小时。交换的PBS，用20％的蔗糖溶液，以在4℃下过夜脱水心脏。 </li><li>冻结冷冻包埋剂的心脏在一个小型模具。 <ol><li>设置填充有2-甲基丁烷的烧杯中，并将其放置在与干冰的盒子。把心脏到以前一半装满冷冻包埋剂的冷冻模具和低温包埋剂完全覆盖。小心地将模具放入冷水的烧杯中，防止与2-甲基丁烷的冷冻包埋剂的接触。在-80°C，直到使用存储冰冻组织。 </li></ol></li></ol></li><li>厚片心脏的制备<ol><li>使用在一个cryotome以下设置:-18至-20℃的物体的温度，-21至-23℃的室中的温度，对4°刀架结算角，和羽毛R35切片机刀片。 </li><li>就拿冻存Ø从冷冻集装箱rgan并修复它使用速冻架样品盘与冷冻嵌入媒体上。的心脏在切片开始在心尖这样的位置。 </li><li>直到均匀切割平面是实现和器官变得可见修剪掉50微米的薄片。通过使用在手动模式下cryotome用慢速度，并与放置在刀防卷板切50微米的组织切片。在盐水处理显微镜载玻片山片。让切片干燥，在室温下30分钟，并在载玻片在-80直接存储℃或污点。 </li></ol></li><li>厚心脏切片染色（细胞核和细胞膜） <ol><li>在37℃下处理在洗涤缓冲液（0.5M氯化钠，0.1M的Tris pH值7.5，和50mM EDTA）中与RNA酶A（20微克/毫升）心脏切片的反应杯1小时。孵育在洗涤缓冲液中的0.2％的Triton-X的切片在室温下30分钟。洗片两次，每次5分钟，在洗涤缓冲液中在比色皿在室温下水平摇床。 </li><li>染色过夜用1μM的TO-PRO3碘（661分之642）和荧光素偶联麦胚凝集素（WGA，1:100），在洗涤缓冲液中于4℃。洗切片三次，每次用洗涤缓冲液在试管5分钟在水平摇动器并用聚乙烯醇安装介质与抗褪色试剂和玻璃盖玻片覆盖。 </li></ol></li><li>图像采集<ol><li>理想情况下，使用配有40X / 1.15 NA水浸渍目标倒置激光共聚焦显微镜。通过眼睛搜索，由纵向内衬心肌细胞的切片内的区域;为此目的，荧光素WGA信道（例如:488纳米）是最适合。 注意:此步骤是必不可少的，作为心肌细胞的上限和下限应的Z堆叠购买分析中可见。作为成像深度被限制到〜30纳米和切片的厚度为50μm，它是不可能的即时这些细胞的年龄截面（单元长度:〜120微米）。 </li><li>调整成像软件中的设置。打开图像处理软件。打开A1plus的设置和检查CH2，CH3和CH4的箱子。设置通道2通过单击下拉菜单上的EGFP，CH3为Alx546和CH4为Alx647。 <ol><li>对于每个通道，点击HV和通过使用滑动条其设置为80。设置使用滑动条偏移量为0。点击"主页"按钮设置针孔的位置。通过单击下拉菜单中的扫描尺寸为1024×1024，单击优化，打开"XYZ尺寸设置"窗口。检查下"建议的步骤（Z）"中的"完美体素"框。 </li></ol></li><li>点击"扫描直播"，并通过点击单个通道滑动条调整激光的强度。检查的"收购ND"窗口中的"Z系列"复选框，然后单击"按上面的按钮定义"对话框。限定的顶部和通过点击相应的按钮调整焦点显微镜后的z堆栈按钮。的z步长设定为0.5微米。 注意:与z堆栈的深度由组织和信噪比内的光穿透的限制。 </li><li>进入"A1plus设置"，然后单击下拉菜单上设置的线路平均/积分为2-4。微调激光强度;针孔应，以便尽可能小，以图像的焦平面。 </li></ol></li><li>核分析<ol><li>打开图像分析软件和加载的兴趣的z栈。 </li><li>通过叠层的不同的层，以便确定该堆栈内完全位于细胞滚动手动确定在Z堆叠核;是这种情况时，WGA染色是在所有维度中可见。算核的数量（其中大部分将是双核），并通过在软件的注释标记分析细胞。 </li> &lt;利&gt;确定的CM核（H2B-MCH +）和在3D重建的单个信道（ 图2D）总核（TO-PRO3 +）的数量。在成像软件，点击"二进制"，然后从下拉菜单中选择"定义阈值3D"。选择从信道下拉菜单或Alx546，CM为原子核或Alx647的总核数目的数目。通过单击相应的箭头，设置程序"平滑4倍"，清洁1x和ON填充孔。检查"大小"框，并使用滑块将其设置为5微米。通过使用滑块，直到单独的对象出现设置阈值。 注:施加破碎后，结果窗口显示量化事件和图像的着色的碎片的表。如一些细胞核彼此直接接触，手动校正双峰，这可能不是由软件正确地分离出的自动测量结果。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

有一个争论的心肌细胞是否能够重新进入细胞周期和损伤后和组织稳态期间分割。对于心肌细胞的基本成交值已被赋予在范围为1％1和80％的7之间。还心脏损伤后，细胞周期活性的诱导和新的心肌细胞的产生已经报道在边界区，具有值0.0083％8和25之间- 40％7。这些差异可部分通过不同的实验方法进行说明，以确定心肌细胞核，这一过程是在组织切片9和细胞分裂期间非常具有挑战性。作为心肌细胞很容易在细胞周期的变化，这是极其重要的，从核内复制和acytokinetic有丝分裂，这导致多倍体和多核细胞区分开正宗细胞分裂。对于细胞分裂的识别，有必要以可视化的细胞分裂的标志，如收缩环和midbodies，以及确定双核心肌细胞的百分比。我们开发的技术来分析在详细的心肌细胞周期活动，并确定在分离的细胞或在厚的部分其nuclearity的程度。 值得注意的是，EGFP-anillin系统提供细胞分裂的直接证明，通过对称收缩环（ 图1F）和中间体（ 图1G），以及用于核内复制和有丝分裂acytokinetic间接证明的可视化是很重要的。如前所述，收缩环的一个单方面侵入的发生是binucleation 10的指示，并且这可以与EGFP-anillin系统中观察到。 内复制建议只要不收缩环或心肌梗死dbody观察，这使得检测这些本地化强制性的概率的计算。假设25小时的细胞周期的持续时间，在20分钟收缩环能见度的平均持续时间，和1小时的中间体持久性，应该有1收缩环100分割的eGFP-anillin阳性细胞和4 midbodies。统计上讲，至少25 EGFP-anillin阳性细胞将需要进行分析，以从细胞周期的变化区分细胞分裂。这个数量相应地改变为细胞周期的持续时间（这常常是未知）增加或减少。这也意味着，大多数的eGFP-anillin信号将核（ 图1E），为M-相，非核本地化的唯一阶段，仅持续约1小时。 在出生后的发育，binucleation发生在小鼠心脏的地方，在心室肌细胞增加了90％（〜人类25％）11。佛中的R心脏再生的分析，重要的是要确定binucleation的程度。我们描述了解决这一重要的形态特征的两种方法:即，的Langendorff解离和心脏组织的厚切片的创建。同时的Langendorff隔离是更容易和更快，厚切片的形态是更费时也更准确。有趣的是，我们已发现的Langendorff隔离高估双核心肌细胞的百分比。这可能这个相当刚性的过程中，是由于单核双核和细胞的不同的存活率。 作为增殖的产后心肌细胞诱导为心肌再生一个新兴的方法，该协议描述了通过结合两个转基因小鼠线，MYH6-H2BmCh和CAG-EGFP-anillin一个筛选系统。从产后天P1心脏分离的心肌细胞 - P6可以很容易地培养和转染的miRNA或的siRNA或小分子库处理。心肌细胞核可以通过软件算法来手动或自动检测，并且潜在的"点击"可以通过增大对MCH +细胞核数目来确定。从核内复制和有丝分裂acytokinetic细胞分裂的歧视可以通过EGFP-anillin信号的不同本地化的量化来完成。该系统应该摆脱一些新的光线进入监管机制基本产后心肌细胞增殖，并可能导致新的治疗药物的发现心脏疾病的治疗。

心肌细胞核和细胞周期状态的正确识别是对心肌的营业额和再生的决心至关重要。这是一种使用核标记物，如pHH3，Ki-67的，或胸苷类似物，用于识别细胞周期活动的情况尤其如此。如成年哺乳动物的心肌细胞的增殖能力是非常小的图1中 ，核阳性心肌细胞核的增殖标志物可以使在一个增殖试验的结果的一个关键差的误识别。此外，心肌细胞很容易在细胞周期的变化，如核内复制和acytokinetic有丝分裂，这导致多倍体和多核细胞，而不是在细胞分裂。为此，对常见的细胞周期标记物的抗体染色的解释不是在所有情况下定论。 在这里，我们提出了直forwa方法<html>小鼠心肌细胞的RD承认和其核的明确的身份标识在产后和成年阶段的本土分离出的细胞和厚厚的组织切片的nuclearity。为此目的，与MYH6启动子（MYH6-H2B-MCH）的控制下，由人类组蛋白H2B和mCherry融合蛋白的特异性心肌表达的转基因小鼠品系使用2。杂交育种用的转基因增殖指示剂鼠标线这个鼠标线，其中一个的eGFP-anillin融合蛋白的表达是与CMV增强无处不鸡肌动蛋白启动子的控制下（CAG-EGFP-anillin），允许测定的细胞周期状态。支架蛋白anillin在细胞周期特异性表达的活性细胞3，和在细胞周期中其差的亚细胞定位允许活跟踪细胞周期进程用高分辨率的M-相EF"&gt; 4，因此，双转基因小鼠可用于增殖心肌细胞和那些经历细胞周期的变化来区分。这证明在筛选用于体外增殖诱导物质特别有用。

<table><tbody><tr><td>10 cm Petri dish</td><td>Sarstedt</td><td>821472</td><td></td></tr><tr><td>100 &amp;micro;m cell strainer</td><td>Becton Dickinson GmbH/Falcon</td><td>352360</td><td></td></tr><tr><td>2,3-Butanedione monoxime (BDM)</td><td>Sigma-Aldrich</td><td>B0753</td><td></td></tr><tr><td>G20x1 &amp;frac12; injection cannula, Sterican</td><td>Braun, Melsungen</td><td>4657519</td><td></td></tr><tr><td>20 gauge needle</td><td>Becton Dickinson GmbH</td><td>301300</td><td></td></tr><tr><td>24-well plates</td><td>Becton Dickinson GmbH/Falcon</td><td>353047</td><td></td></tr><tr><td>2-Methyl-butane</td><td>Carl Roth GmbH + Co. KG</td><td>3927.1</td><td></td></tr><tr><td>37% formaldehyde solution</td><td>AppliChem GmbH&amp;nbsp;</td><td>A0936,1000</td><td></td></tr><tr><td>3-way stopcock</td><td>B. Braun Medical Inc.</td><td>16494C</td><td></td></tr><tr><td>50 mL syringe</td><td>B. Braun Medical Inc.</td><td>8728810F</td><td></td></tr><tr><td>70% ethanol</td><td>Otto Fischar GmbH</td><td>27669</td><td></td></tr><tr><td>Alexa-Fluor-conjugated secondary antibody</td><td>Jackson ImmunoResearch</td><td>115-605-205</td><td></td></tr><tr><td>Alpha-Aktinin EA-53, Mouse IgG</td><td>Sigma-Aldrich, Steinheim</td><td>A7811</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>Cell Culture Microplate, 96 Well, Half Area</td><td>Greiner bio-one</td><td>675986</td><td></td></tr><tr><td>Collagenase B</td><td>Roche</td><td>11088815001</td><td></td></tr><tr><td>confocal microscope Eclipse Ti-E</td><td>Nikon</td><td></td><td></td></tr><tr><td>cryostat CM 3050S</td><td>Leica</td><td></td><td></td></tr><tr><td>donkey serum</td><td>Jackson Immuno Research, Suffolk, GB</td><td>017-000-121</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E4884</td><td></td></tr><tr><td>fetal calf serum</td><td>PromoCell, Heidelberg</td><td></td><td></td></tr><tr><td>Formaldehyde solution (4%)</td><td>PanReac AppliChem</td><td>A3697</td><td></td></tr><tr><td>Gelatine from porcine skin, Type A</td><td>Sigma-Aldrich, Steinheim</td><td>G2500</td><td></td></tr><tr><td>glass coverslips</td><td>VWR</td><td>631-0146</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Heidelberger extension tube</td><td>IMPROMEDIFORM GmbH</td><td>MF 1833</td><td></td></tr><tr><td>Heparin-Natrium</td><td>Ratiopharm</td><td>5394.02.00</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>HistoBond microscope slides</td><td>Marienfeld</td><td>0810000</td><td></td></tr><tr><td>Hoechst 33342 (1&amp;nbsp;mg/mL)</td><td>Sigma Aldrich, Taufkirchen</td><td>B2261</td><td></td></tr><tr><td>Insulin syringe</td><td>Becton Dickinson GmbH</td><td>300334</td><td></td></tr><tr><td>Iscove&amp;rsquo;s ModifiedDulbecco&amp;rsquo;s Medium (IMDM)</td><td>Gibco/Life Technologies, Darmstadt</td><td>21980-032</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Laminin</td><td>Corning</td><td>354221</td><td></td></tr><tr><td>Laser Scanning Mikroskop Eclipse Ti</td><td>Nikoninstruments, D&amp;uuml;sseldorf</td><td></td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen/Life Technologies, Darmstadt</td><td>13778075</td><td></td></tr><tr><td>Mouse IgG Cy5 (donkey)</td><td>Jackson ImmunoResearch</td><td>715-175-151</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>microcentrifuge tube</td><td>Sarstedt</td><td>72690</td><td></td></tr><tr><td>Mini shaker</td><td>VWR</td><td>12620-940</td><td></td></tr><tr><td>mirVana miRNA mimic, hsa-miR199a-3p</td><td>Ambion/Thermo Fischer Scientific</td><td>4464066</td><td></td></tr><tr><td>Biopsy Mold</td><td>Sakura Finetek/ VWR</td><td>4565</td><td></td></tr><tr><td>M-slide 8-well ibiTreat</td><td>ibidi</td><td>80826</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>NaOH</td><td>Merck Millipore</td><td>567530</td><td></td></tr><tr><td>negative control(scrambled RNA)</td><td>Ambion/Thermo Fischer Scientific</td><td>AM4611</td><td></td></tr><tr><td>Neonatal Heart Dissociation Kit</td><td>Miltenyi Biotech, Bergisch Gladbach</td><td>130-098-373</td><td></td></tr><tr><td>NIS Elements AR 4.12.01-4.30.02-64bit</td><td>Nikoninstruments, D&amp;uuml;sseldorf</td><td></td><td></td></tr><tr><td>Non essential amino acids, NEAA</td><td>Gibco/Life Technologies, Darmstad</td><td>11140-035</td><td></td></tr><tr><td>Opti-MEM, Reduced Serum Medium</td><td>Gibco</td><td>51985-026</td><td></td></tr><tr><td>P21-siRNA</td><td>Ambion/Thermo Fischer Scientific</td><td>4390771</td><td></td></tr><tr><td>P27-siRNA</td><td>Ambion/Thermo Fischer Scientific</td><td>4390771</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco/Life Technologies, Darmstadt</td><td>15140-122</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich, Steinheim</td><td>14190-094</td><td></td></tr><tr><td>Polyvinyl alcohol mounting medium with DABCO&amp;reg;, antifading</td><td>Sigma-Aldrich</td><td>10981</td><td></td></tr><tr><td>RNase A</td><td>Qiagen</td><td>1007885</td><td></td></tr><tr><td>RNaseZap</td><td>Invitrogen/Life Technologies, Darmstadt</td><td>AM9780</td><td></td></tr><tr><td>sample containers</td><td>Vitlab</td><td>80731</td><td></td></tr><tr><td>Serological pipette</td><td>Greiner</td><td>607180</td><td></td></tr><tr><td>software NIS Elements</td><td>Nikon</td><td></td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura Finetek/ VWR</td><td>25608-930</td><td></td></tr><tr><td>ToPro3 iodide (642/661)</td><td>Molecular probes/ThermoFisher Scientific</td><td>T3605</td><td></td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Triton X</td><td>Fluka</td><td>93418</td><td></td></tr><tr><td>Triton X-100</td><td>Fluka</td><td>93418</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T1426</td><td></td></tr><tr><td>Wheat germ agglutinin (WGA) Fluorescein labeled</td><td>Vector Laboratories</td><td>VEC-FL-1021-5</td><td></td></tr><tr><td>&amp;alpha;-actinin antibody</td><td>Sigma-Aldrich</td><td>A7811</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Sigma-Aldrich, Steinheim</td><td>M3148</td><td></td></tr></tbody></table>

visualization of cell cycle variations and determination of nucleation in postnatal cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

为了分析的siRNAs / miRNA的体外产后心肌细胞，双转基因MYH6-H2B-MCH / CAG-EGFP-anillin小鼠心肌细胞的细胞周期活动的影响被隔绝在3日龄（P3）和与转细胞周期活动诱导miR199 5，P27的siRNA，和siRNA Fzr1。相比于阴性对照（ 图1A），miR199-（ 图1B）和siRNA p27-（ 图1C）的照片转染心肌细胞表现出细胞周期活性的诱导。在EGFP-anillin小鼠模型中，针对Fzr1的siRNA可用作转染对照，作为Fzr1的抑制导致的eGFP-anillin融合蛋白在转染细胞的细胞核中积累和Fzr1的损失导致的APC的抑制Fzr1。 Fzr1是后期促进复合物的E3连接酶的辅因子，其中用于龙眼靶向anillinOMAL降解。 图1D示出的siRNA Fzr1转染心肌细胞转染后3天，指示〜45％的转染效率的一个共焦概述图像。执行核内复制（ 例如，的p27蛋白6击倒后）的心肌细胞表现出完全核的eGFP-anillin表达（ 图1E）或是EGFP-anillin阴性（见讨论）。它们不表达在M相-典型本地化（ 图1F和G）的eGFP-anillin，作为核内复制只由一个内切S期（EGFP-anillin阳性）和内切-G相（EGFP-anillin阴性）。在桥-G相，在APC是活动的，从而导致的eGFP-anillin在蛋白酶的泛素化和降解。 可在兰后单细胞水平被执行在成年阶段的单 - 和双核心肌部分的量化任的MYH6-H2B-MCH转基因心或转基因心厚cryoslices gendorff解离。心脏组织的在Langendorff装置上，心房和心室的酶消化后可以机械分离，并且相互独立地进行分析。 图2A示出后的Langendorff隔离具有高度双核心肌非固定H2B-MCH转基因心室心肌细胞的代表性图像，由核H2B-MCH表达所指示的。与此相反，大多数心房心肌被单个核（ 图2B）。作为酶促消化不100％的单细胞产生，用α辅肌动蛋白染色显示横条纹的图案便于和细胞双峰双核心肌区分（交叉条纹的连续图案， 图2C）。 图2D示出了双核心肌在鉴定的一例乡巴佬片。 成人MYH6-H2B-MCH转心厚片的三维重建可被用于确定心肌细胞核的组织内的生理条件下的比例。利用成像软件的三维模块，赫斯特-染色的细胞核，并如在图2E中所示的H2B-MCH阳性细胞核可自动检测并计数。最终的结果应手动校正双峰，即直接相互接触，在这种情况下，核。为了分析在厚片心肌细胞的核指数，有必要通过堆栈手动滚动，作为晶核一个Z平面内不必要谎言。 WGA染色允许检测单元格边框。 <img alt="图1" src="/files/ftp_upload/55204/55204fig1.jpg" /> 图1: 在 实施例在产后心肌细胞周期活动的体外可视化的siRNA转染后。 （AD）从染色α辅肌动蛋白（白）EGFP-anillin / MYH6-H2B-MCH心中P3心肌细胞。心肌细胞核由H2B-MCH信号（红），细胞周期活动由EGFP-anillin信号（绿色），和核由Hoechst核染料（蓝色）标识。与扰频转染（A）的 P3心肌作为阴性对照。酒吧是100微米。用的miRNA-199显示显著更EGFP-anillin信号比对照（A）转（B）P3心肌细胞。酒吧是100微米。 （C）与p27蛋白的siRNA转染后只核的eGFP-anillin信号，表示核内复制的实施例。酒吧是80微米。 （D）转染的siRNA对Fzr1的转染效率的决心。作为EGFP-anillin交流累加，EGFP-anillin +心肌细胞的数量表示转染效率。酒吧是80微米。 （EG）在细胞周期期间心肌的eGFP-anillin（绿色）的不同的本地化:核定位（于E的箭头），收缩环（F中的箭头），和中间体本地化（G中箭头）。酒吧是在20微米（E）和10微米（F和G）。 <a href="http://ecsource.jove.com/files/ftp_upload/55204/55204fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55204/55204fig2.jpg" /> 图2:多核由心肌细胞从H2B-MCH小鼠的Langendorff隔离评估和厚片的3D分析实例。 （A，B）心室（A）和心房（B）从成年H2心肌心通过分离的Langendorff后隔离B-MCH转基因小鼠。该酒吧是50微米。 （C）从心肌染色α辅肌动蛋白（绿色）MYH6-H2B-MCH心。心肌细胞核由H2B-MCH信号（红色）来识别。酒吧是10微米。 （D）双核心肌细胞的厚片（箭头）。心肌细胞核由H2B-MCH信号（红色），由WGA染色细胞边界（绿），和核通过ToPro3（白色）标识。酒吧是50微米。 （五）工作流程binucleation的厚片的三维分析。 <a href="http://ecsource.jove.com/files/ftp_upload/55204/55204fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes at JoVE.com

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

细胞周期变化的可视化和核测定心肌出生后

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

visualization-cell-cycle-variations-determination-nucleation

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.7K 次观看.  University of Bonn. To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

视频: Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

科研

发育生物学

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4&#8242;,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

心肌细胞DNA的合成和倍性的同时评价:一个方法来帮助心肌细胞再生和营业额的量化

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

成像清除胚胎和出生后心脏在单细胞分辨率

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

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

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

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

在微孔聚集尺寸优化人类多能干细胞的基于悬浮心脏分化

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

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

验尸报告组织心肌细胞核分离

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

医学

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

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

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

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

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

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

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

使用成熟的人诱导多能干细胞来源心肌细胞单层进行高通量心脏毒性筛选

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

生物工程

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

使用hiPSC衍生心肌细胞的缺血性心脏病模型和基于视频的心肌细胞收缩比较

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

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 ...