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>

<ol>
	<li>Bergmann, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+cardiomyocyte+renewal+in+humans.">Evidence for cardiomyocyte renewal in humans.</a> Science. 324 (5923), 98-102 (2009).</li><li>Raulf, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+systems+for+unequivocal+identification+of+cardiac+myocyte+nuclei+and+analysis+of+cardiomyocyte+cell+cycle+status.">Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status.</a> Basic Res.Cardiol. 110 (3), 33 (2015).</li><li>Field, C. M., Alberts, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin,+a+contractile+ring+protein+that+cycles+from+the+nucleus+to+the+cell+cortex.">Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex.</a> J.Cell Biol. 131 (1), 165-178 (1995).</li><li>Hesse, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+cell+division+using+high-resolution+imaging+of+M-phase+of+the+cell+cycle.">Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle.</a> Nat.Commun. 3, 1076 (2012).</li><li>Eulalio, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+screening+identifies+miRNAs+inducing+cardiac+regeneration.">Functional screening identifies miRNAs inducing cardiac regeneration.</a> Nature. 492 (7429), 376-381 (2012).</li><li>Di, S. V., Giacca, M., Capogrossi, M. C., Crescenzi, M., Martelli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+cyclin-dependent+kinase+inhibitors+induces+cardiomyocyte+re-entry+in+the+cell+cycle.">Knockdown of cyclin-dependent kinase inhibitors induces cardiomyocyte re-entry in the cell cycle.</a> J.Biol.Chem. 286 (10), 8644-8654 (2011).</li><li>Hosoda, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonality+of+mouse+and+human+cardiomyogenesis+in+vivo.">Clonality of mouse and human cardiomyogenesis in vivo.</a> Proc.Natl.Acad.Sci.U.S.A. 106 (40), 17169-17174 (2009).</li><li>Soonpaa, M. H., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cardiomyocyte+DNA+synthesis+in+normal+and+injured+adult+mouse+hearts.">Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts.</a> Am.J.Physiol. 272 (1 Pt 2), H220-H226 (1997).</li><li>Ang, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+conventional+approaches+to+identify+myocyte+nuclei+in+histologic+sections+of+the+heart.">Limitations of conventional approaches to identify myocyte nuclei in histologic sections of the heart.</a> Am.J.Physiol Cell Physiol. 298 (6), C1603-C1609 (2010).</li><li>Engel, F. B., Schebesta, M., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin+localization+defect+in+cardiomyocyte+binucleation.">Anillin localization defect in cardiomyocyte binucleation.</a> J Mol Cell Cardiol. 41 (4), 601-612 (2006).</li><li>Bergmann, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+Cell+Generation+and+Turnover+in+the+Human+Heart.">Dynamics of Cell Generation and Turnover in the Human Heart.</a> Cell. 161 (7), 1566-1575 (2015).</li></ol>

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，...

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 border="0" cellpadding="0" cellspacing="0" width="999">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">10 cm petri dish</td>
			<td style="width:331px;">Sarstedt</td>
			<td style="width:119px;">821472</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">100 &#181;m cell strainer</td>
			<td style="width:331px;">Becton Dickinson GmbH/Falcon</td>
			<td style="width:119px;">352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">2,3-Butanedione monoxime (BDM)</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G20x1 &#189; injection cannula, Sterican</td>
			<td style="width:331px;">Braun, Melsungen</td>
			<td>4657519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">20 gauge needle</td>
			<td>Becton Dickinson GmbH</td>
			<td style="width:119px;">301300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">24-well plates</td>
			<td style="width:331px;">Becton Dickinson GmbH/Falcon</td>
			<td style="width:119px;">353047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">2-Methyl-butane</td>
			<td style="width:331px;">Carl Roth GmbH + Co. KG</td>
			<td style="width:119px;">3927.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">37% formaldehyde solution</td>
			<td style="width:331px;">AppliChem GmbH&#160;</td>
			<td>A0936,1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">3-way stopcock</td>
			<td style="width:331px;">B. Braun Medical Inc.</td>
			<td style="width:119px;">16494C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">50 ml syringe</td>
			<td style="width:331px;">B. Braun Medical Inc.</td>
			<td style="width:119px;">8728810F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">70% ethanol</td>
			<td style="width:331px;">Otto Fischar GmbH</td>
			<td style="width:119px;">27669</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Alexa-Fluor-conjugated secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td style="width:119px;">115-605-205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Alpha-Aktinin EA-53, Mouse IgG</td>
			<td style="width:331px;">Sigma-Aldrich, Steinheim</td>
			<td style="width:119px;">A7811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">CaCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Cell Culture Microplate, 96 Well, Half Area</td>
			<td style="width:331px;">Greiner bio-one</td>
			<td style="width:119px;">675986</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Collagenase B</td>
			<td style="width:331px;">Roche</td>
			<td style="width:119px;">11088815001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">confocal microscope Eclipse Ti-E</td>
			<td style="width:331px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">cryostat CM 3050S</td>
			<td style="width:331px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">donkey serum</td>
			<td>Jackson Immuno Research, Suffolk, GB</td>
			<td style="width:119px;">017-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:383px;">EDTA</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal calf serum</td>
			<td>PromoCell, Heidelberg</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde solution (4%)</td>
			<td>PanReac AppliChem</td>
			<td style="width:119px;">A3697</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Gelatine from porcine skin, Type A</td>
			<td>Sigma-Aldrich, Steinheim</td>
			<td style="width:119px;">G2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">glass coverslips</td>
			<td style="width:331px;">VWR</td>
			<td style="width:119px;">631-0146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Glucose</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Heidelberger extension tube</td>
			<td>IMPROMEDIFORM GmbH</td>
			<td style="width:119px;">MF 1833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Heparin-Natrium</td>
			<td style="width:331px;">Ratiopharm</td>
			<td style="width:119px;">5394.02.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">HEPES</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">HistoBond microscope slides</td>
			<td style="width:331px;">Marienfeld</td>
			<td style="width:119px;">0810000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 (1mg/ml)</td>
			<td>Sigma Aldrich, Taufkirchen</td>
			<td style="width:119px;">B2261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Insulin syringe</td>
			<td>Becton Dickinson GmbH</td>
			<td style="width:119px;">300334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscove&#8217;s ModifiedDulbecco&#8217;s Medium (IMDM)</td>
			<td>Gibco/Life Technologies, Darmstadt</td>
			<td>21980-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">KCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Laminin</td>
			<td style="width:331px;">Corning</td>
			<td style="width:119px;">354221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser Scanning Mikroskop Eclipse Ti</td>
			<td>Nikoninstruments, D&#252;sseldorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine RNAiMAX</td>
			<td>Invitrogen/Life Technologies, Darmstadt</td>
			<td>13778075</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:383px;">Mouse IgG Cy5 (donkey)</td>
			<td style="width:331px;">Jackson ImmunoResearch</td>
			<td>715-175-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">MGCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">microcentrifuge tube</td>
			<td style="width:331px;">Sarstedt</td>
			<td style="width:119px;">72690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Mini shaker</td>
			<td style="width:331px;">VWR</td>
			<td>12620-940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">mirVana miRNA mimic, hsa-miR199a-3p</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4464066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Biopsy Mold</td>
			<td style="width:331px;">Sakura Finetek/ VWR</td>
			<td>4565</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">M-slide 8-well ibiTreat</td>
			<td style="width:331px;">ibidi</td>
			<td style="width:119px;">80826</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">NaCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">NaOH</td>
			<td style="width:331px;">Merck Millipore</td>
			<td style="width:119px;">567530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">negative control(scrambled RNA)</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>AM4611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neonatal Heart Dissociation Kit</td>
			<td>Miltenyi Biotech, Bergisch Gladbach</td>
			<td style="width:119px;">130-098-373</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS Elements AR 4.12.01-4.30.02-64bit</td>
			<td>Nikoninstruments, D&#252;sseldorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non essential amino acids, NEAA</td>
			<td>Gibco/Life Technologies, Darmstad</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Opti-MEM, Reduced Serum Medium</td>
			<td style="width:331px;">Gibco</td>
			<td style="width:119px;">51985-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">P21-siRNA</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4390771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">P27-siRNA</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4390771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco/Life Technologies, Darmstadt</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich, Steinheim</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:383px;">Polyvinyl alcohol mounting medium with DABCO&#174;, antifading</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">10981</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">RNase A</td>
			<td style="width:331px;">Qiagen</td>
			<td>1007885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNaseZap</td>
			<td>Invitrogen/Life Technologies, Darmstadt</td>
			<td style="width:119px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">sample containers</td>
			<td style="width:331px;">Vitlab</td>
			<td style="width:119px;">80731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Serological pipette</td>
			<td style="width:331px;">Greiner</td>
			<td style="width:119px;">607180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">software NIS Elements</td>
			<td style="width:331px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Sucrose</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Tissue-Tek O.C.T. Compound</td>
			<td style="width:331px;">Sakura Finetek/ VWR</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">ToPro3 iodide (642/661)</td>
			<td style="width:331px;">Molecular probes/ThermoFisher Scientific</td>
			<td style="width:119px;">T3605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Tris</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Triton X</td>
			<td style="width:331px;">Fluka</td>
			<td style="width:119px;">93418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Triton X-100</td>
			<td style="width:331px;">Fluka</td>
			<td style="width:119px;">93418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Trypsin</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Wheat germ agglutinine (WGA) Fluorescein labeled</td>
			<td>Vector Laboratories</td>
			<td>VEC-FL-1021-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">&#945;-actinin antibody</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-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-anil...

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

Research

JoVE Journal

Developmental Biology

8.6K 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

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

科研

发育生物学

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

从人类诱导多能干细胞使用小分子调制分化和随后的葡萄糖饥饿高度纯化心肌推导

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

隔离和乳鼠心肌细胞冷冻保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

通过在胚胎梅克尔软骨Zebrabow克隆细胞分析软骨插层和定向扩散的可视化

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

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

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

从多能干细胞使用BMP拮抗剂Grem2心房心肌细胞分化

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

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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, ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

使用电刺激人干细胞衍生的心肌细胞的成熟中Biowires

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

使用多电极阵列（MEA）的人多能干细胞来源的心肌细胞（hPSC-CM）的电生理分析

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...