Name: epicardial outgrowth culture assay and ex vivo assessment of epicardial-derived cell migration
Uploaded: 2016-03-18T14:00:00.000+00:00
Duration: 7 min 44 s
Description: Watch this Scientific Journal Video about Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration at JoVE.com

EMS是由来自卫生研究院（NIH）的国家机构的资金支持[授权号R01HL120919]。美国心脏协会[授权号10SDG4350046]。来自美国国立卫生研究院[授权号UL1 TR000042]罗切斯特CTSA奖的大学;从AAB CVRI启动资金。 MAT是由授予医学和牙科学院罗彻斯特来自霍华德休斯医学研究所医学成 - 格拉德倡议大学的部分资助;从NIH [授权号GM068411]一个机构露丝L. Kirschstein国家研究服务奖。

注意:用小白鼠试验全部由大学委员会动物资源在罗切斯特大学的认可。 1.心外膜生长培养试验（图1A） <ol><li>准备工作<ol><li>通过用5％胎牛血清（FBS）和1％青霉素/链霉素（青霉素/链霉素）补充培养基199（M199）准备5毫升原心外膜细胞的分离介质中的。 </li><li>预暖培养基A和100​​毫升的Hanks&#39;平衡盐溶液（HBSS）在37℃水浴中。 </li><li>允许胶原包被的玻片温热至室温30分钟。 </li><li>将100μl培养基A添加到腔室载玻片的每个孔中，轻轻地旋转，以均匀涂覆胶原表面。重复8-12井。涂装室后取出介质。 </li><li>等分试样预先温热的HBSS到含有各50毫升HBSS两个10cm 2的板。 </li></ol></li><li>从怀孕隔离坝心的胚胎在11.5天交配后（DPC）。三十 <ol><li>通过腹膜内注射0.5毫升的13毫克/毫升氯胺酮/ 0.88毫克/在1×Dulbecco氏磷酸盐缓冲盐水（DPBS）毫升甲苯噻嗪鸡尾酒麻醉小鼠。用脚趾捏测试确认正确麻醉。温和的捏就足以确定鼠标响应。任何运动或爪撤回指示动物不能充分麻醉做外科手术。颈椎脱位安乐死。 </li><li>打下鼠标朝向腹侧和喷雾腹部用70％乙醇由头发以减少污染。 </li><li>捏钳皮肤，并在手术剪腹部切一横向切口。握紧皮肤，然后拉开反对对头部和尾部的方向。 </li><li>捏钳腹膜并用剪刀剪以暴露腹腔。 </li><li>小心地沿系膜及切割去除蜕膜。在第一传送解剖蜕膜预先温热的HBSS10cm 2的板。 </li><li>通过胚胎切割之间的分离蜕膜。 </li><li>在解剖显微镜下转移胚胎到第二10cm 2的板带预热的HBSS。小心取出胚外组织和卵黄囊，然后斩首胚胎。 </li><li>捏钳胸壁和撕裂组织在中线，露出胸腔。 </li><li>位，仅次于心脏的镊子。抓住流出道拉的心脏从胚胎了。从心脏分离肺呼吸道和肺部，如果还连着。 </li></ol></li><li>的心脏转移到胶原包被的载玻片的单井，并将其放置背侧向下（ 图1B）。重复步骤1.2.7-1.3每个胚胎。 </li><li>一旦所有的心已被转移到各个孔中，温育30分钟腔室载玻片在37℃，5％的CO 2，以便有足够粘附于胶原基质。 </li><li>小心加入20-50微升围绕各个心脏预热植媒介A的。重要提示:不要添加介质过快或高于心脏水平，否则心可从胶原蛋白底物分离。 </li><li>培养在37℃，5％CO 2的心约24小时，以允许心外膜产物。注意:突的形态可以观察到早3-4小时培养的（ 图1C）和主要由显示鹅卵石形态，以最小的间充质细胞污染（ 图1D，E）的间皮细胞。使用装备有相机的解剖显微镜代表性结果分别获得的。 </li></ol> 2.遗传消融和EMT的诱导心外膜突的形态注意:根据批准的生物安全准则小心轻放腺病毒。 <ol><li>制备15毫升培养基B（M199补充有1％FBS和1％青霉素/链霉素）和预温至37℃的水浴中。 </li><li>对于切除术两侧装接loxP的等位基因，准备辅以表达Cre重组酶2.5×10 4 PFU / ml的腺病毒（广告/ CRE）或控制病毒外植体介质B。 注意:候选基因的腺病毒介导的过表达也可以进行，以评估功能表型的增益。病毒滴度应该由最终用户来确定。 </li><li>小心地从使用镊子生长的文化去除外膜耗尽的心。心中转移到1.5毫升管与800微升的Trizol并储存在-80℃供以后RNA分离和基因表达分析。 </li><li>用DPBS冲洗每个孔以除去血细胞和过量细胞碎片。 </li><li>加入500微升补充有腺病毒（ES）的外植体培养基B到每个生长培养，并在37℃，5％的CO 2孵育24小时。 </li><li>对于EMT诱导，取出媒体和补充了重组转化生长因子（TGF）37℃外植体介质B-β1[10毫微克/毫升]或车辆（4 mm高补充含ydrochloric酸（HCl）的1毫克/毫升的牛血清白蛋白（BSA））。培养外植另外24小时，在37℃，5％ 的 CO 2。与基因/蛋白表达的分析或为后续的详细协议进行免疫。 注意:心外膜EMT诱导已刺激用各种浓度的TGF-β1的范围从0.5纳克/毫升- 10纳克/毫升26,31-33。配位体的浓度较高，可能会导致非特异性结合到所有的TGF-β受体。 TGF-β1的浓度应该反映具体的实验目标。 </li></ol>心外膜突的形态3.基因表达分析<ol><li>解冻外膜贫心预先存储在Trizol试剂（2.2）。 </li><li>从EPDC培养吸媒体和DPBS洗两次。 </li><li>加入800微升室温的Trizol为外植体培养。 </li><li>心外膜孵育贫心和EPDC培养Trizol法中在室温temperatu 5分钟<html>回覆。吸管上下十倍至均质化裂解的样品。 注意:重​​点可能需要进一步吹打完全同质化。 </li><li>转移裂解物至1.5毫升管，并与每制造商的说明提取总RNA进行。加入10微克的糖原沉淀之前增加产量的RNA。一个给定的外植体将产生约0.5-1.0微克RNA​​。 </li><li>除去用DNase I污染DNA和反向转录每制造商的说明0.5微克的mRNA。 </li><li>验证外膜外植体和/或用定量逆转录EMT诱导的（定量RT）-PCR 34的纯度使用SYBR绿和表1中。规格化mRNA水平来描述甘油醛3-磷酸脱氢酶（GAPDH）的标志物，并使用计算相对表达2 - ΔΔCT方法35。典型的结果示于图2A。 </li></ol>心外膜突的形态的标题"&gt; 4。蛋白质分析<ol><li>从EPDC培养吸媒体和DPBS洗两次。 </li><li>添加10微升冰冷的蛋白质裂解缓冲液（50mM的Tris pH值7.5，150mM氯化钠，1mM的乙二胺四乙酸pH 8.0的，1％的Triton X-100，1×蛋白酶抑制剂鸡尾酒）到每个腔室孔，并放置在腔室滑动冰。大约四分之一的底部切断对P200的枪头，并用剩余的一块刮关闭单元的幻灯片。移液器上下裂解心外膜细胞和转移到1.5ml管中。 </li><li>超声处理对低（160瓦）5分钟裂解植与30秒​​ON和OFF周期的冰水浴中。 </li><li>离心裂解物在10,000 xg离心，4℃10分钟。转移上清液至新管，并确定使用蛋白测定36蛋白质浓度。 注:一个给定的外植体将产生高达1.5微克总蛋白。我们发现，从两心汇集的植每孔ð装载2微克蛋白是足以检测平滑肌α肌动蛋白（ACTA2）和GAPDH 9。其它靶蛋白的检测可能需要由最终用户来确定汇集多个植体样品。 </li></ol> 5.免疫细胞化学心外膜突的形态的<ol><li>从外植体培养取出媒体和DPBS洗两次。 </li><li>轻轻在-20加入500微升4％（体积/体积）低聚甲醛或冰冷的甲醇到每个孔中并固定的外植体在室温下5分钟或10分钟℃，分别。根据抗体规格制造商推荐的修复。 </li><li> 5分钟除去固定液和用DPBS冲洗各孔三次。 </li><li>透化的细胞用500μl的0.1％（体积/体积）的Triton X-100在DPBS 5分钟。 </li><li> 5分钟除去透化溶液并用DPBS冲洗三次。 </li><li>封锁在DPBS 10％血清的细胞在室温temperatu 30分钟回覆。 </li><li>每个制造商的建议，以及由最终用户确定最佳条件初级抗体染色进行。 </li><li> 5分钟除去抗体溶液并用DPBS冲洗三次。 </li><li>应用按照制造商的说明书和染液二级抗体与合适的核染料。 </li><li> 5分钟除去抗体溶液并用DPBS冲洗三次。 </li><li>按照制造商的说明卸下室滑动壁和直接添加水性安装介质到细胞中。放置盖玻片在细胞并用指甲油密封。使用倒置共焦扫描显微镜的二维 -典型结果示于图2B。 </li></ol> 6. 体外培养测定（图3A） <ol><li>准备工作<ol><li>制备12毫升体外用培养基:Dulbecco改良的Eagle培养基（DMEM）中的（1）的混合物:M199补充有10％FBS和1％青霉素/链霉素。 </li><li>在37℃水浴中，预温体外培养基和100ml的HBSS。 </li><li>等分试样1毫升预温热培养基成8-12孔24孔板的。 </li><li>转印预先温热的HBSS两个10cm 2的板，含有各50毫升。 </li></ol></li><li>分离小鼠胚胎心脏中的HBSS从怀孕水坝12.5 DPC（在1.2中描述）。 </li><li>每个心脏转移到含有体外培养基中的24孔平板的单个孔。在37℃，5％的CO 2与温和手动摇动或使用摇动平台以防止粘附孵育板。立即进行步骤7.1。 </li></ol> 7.心外膜标签和EMT诱导离体培养<ol><li>要标记的外膜，准备12毫升辅以3.0×10 6 PFU / ml的广告/ GFP的37℃ 体外培养液中。对于两侧装接loxP的等位基因有针对性的消融，A转移6毫升D / GFP补充培养基到一个新的15毫升锥形管中。添加广告/酶Cre或控制腺病毒为1.5×10 6 PFU / ml的终浓度。 注:功能增益分析也可以与合适的腺病毒介导的基因递送进行。 </li><li>小心地从使用P1,000枪头24孔板的每个孔中除去培养基。 </li><li>轻轻添加补充有腺病毒（ES）培养基到每个孔中。 </li><li>孵育所述板在37℃，5％CO 2的温和摇动24小时。 </li><li>为EMT诱导，制备12毫升37℃ 体外 （含有含有10纳克/ ml的TGF-β1和20毫微克/ ml血小板衍生的生长因子（PDGF）-BB或车辆培养基4mM的盐酸1毫克/毫升BSA的）。 </li><li>小心地从使用P1,000枪头24孔板的每个孔中除去培养基。轻轻冲洗用1毫升DPBS的心。 </li><li>取出DPBS，并补充与体外培养我的心含有TGF-β1和PDGF-BB或车辆dium。 </li><li>孵育在37℃下24小时，5％的CO 2与温和的摇摆。 </li></ol> 8.冷冻保存离体培养的免疫组化和<ol><li>准备心冷冻保存。 <ol><li>除去培养基并用冰冷的DPBS洗涤心两次。 </li><li>加入1ml 4％（体积/体积）低聚甲醛添加到每个心脏并固定在4℃下轻轻摇动2小时。 </li><li>取出固定液，用DPBS洗心的两倍。转移心中新鲜孔用1ml 10％的在DPBS制备（重量/体积）蔗糖。过夜孵育该板在4℃下轻轻摇动。 </li><li>仔细心转移到新井18％在DPBS制备（重量/体积）蔗糖。过夜孵育该板在4℃下轻轻摇动。 </li><li>每个心脏转移到含有冻结的组织介质的cryomold。在-80＆＃立即冻结使用液氮和存储块176℃。 </li></ol></li><li>对使用带正电荷的显微镜玻片上低温恒温器和地点组织切片在5微米节心。商店在幻灯片-80°C，直到染色。 </li><li>用于检测的子心外膜基底膜上执行部分免疫组织化学。 <ol><li>解冻载玻片在室温下至少30分钟。 </li><li>通过在DPBS 5分钟温和摇动洗涤两次再水合的部分。 </li><li>用0.1％（体积/体积）的Triton X-100的PBS通透部分，用于在室温下5分钟。 </li><li>洗净的幻灯片两次DPBS 5分钟用温和的摇摆。 </li><li>吸去多余的DPBS并用疏水性标记笔定义染色区域。块部分与在DPBS 10％血清，在室温下30分钟。 </li><li>去除块和应用IV型胶原抗体（ColIV，1:200），在方框稀释。在湿度受控室中于4℃孵育过夜。 </li><li>洗幻灯片三次为DPBS5分钟，温和的摇摆。 </li><li>在DPBS和应用于幻灯片稀释荧光标记的二抗（1:500）和DAPI（5000 1）。孵育在室温下1小时。选择与发射和激发波长从绿色荧光蛋白不同的适当的二级抗体。 </li><li>洗幻灯片三次PBS 5分钟用温和的摇摆。山滑带安装网上平台。 </li></ol></li></ol> 9.成像离体培养的定量分析和<ol><li>有蒙蔽用户图像和量化染色切片。图像至少五个非连续组织在沿使用荧光或共焦显微镜在40X放大率心外膜（心脏的边缘）4-5的任意位置从一个给定的心脏切片。典型的结果是使用倒共焦扫描显微镜， 如图3B所示 。 </li><li>手动计数给定图像中的GFP阳性细胞的总数。确定迁移EPDC如GFP婆位于之内或之外的ColIV外膜下基底膜sitive细胞。确定的GFP的迁移细胞作为一个给定图像中的迁移的GFP阳性细胞过度的GFP阳性细胞的总数的数量的百分比。典型的结果是使用反相共焦扫描显微镜如图3C-D。 </li></ol>

作者什么都没有透露。

在这里，我们列出了详细的方法，通过产物分离主心外膜细胞和跟踪体外心脏文化EPDC迁移。对生长培养物，在适当的时间以除去外膜贫心脏实验之间略有不同。过夜温育后的外植体的定期监测建议来衡量心外膜增生的程度和成纤维细胞出现之前提取心脏。以确保纯度，心应explanting防止非心外膜谱系的积累的24小时内被除去。修剪掉流出道和之前的心脏转移到玻片还可以减少污染的细胞类型27,40两个心房。因为胎儿心是时间的窄窗期间从小鼠垫料分离并在相同条件下培养，为副产物出现的时间相对类似从一个心脏到另一个实验中。因此，所有的心应该bE当大多数植心外膜表现出副产物除去。在一些情况下，心外膜细胞不能有效地从正在比其他，在这种情况下，这植必须从实验中排除收缩更大力一个心脏迁移出来。 血细胞的积累常可掩盖心外膜细胞（箭头， 图1C）的平坦单层，从而难以可视化的副产物。为了避免过多的血细胞的贡献，心可以保持在HBSS用于在从胚胎解剖几分钟。这允许足够的时间使胎儿心脏在被转移到一个腔室滑动之前泵出残留血液在心脏的腔室。或者，第一孵育过夜后补充与植培养基A培养将有助于冲洗掉一些多余的血液，并揭示副产物;然而，介质的水平不应上升高于心脏作为表面张力可导致心脏拉远离衬底。强大的心外膜副产物使用I型胶原作为栽培基质观察;然而，其它基质已经根据不同的生物体和/或应用27,40-42被报道。使用该协议，主要胎儿心外膜细胞可以茁壮成长以下心脏切除和日常补水中等4天然而，需要培养的时间较长的实验设计应该由最终用户来确定。由于心外膜EMT是胚胎发育过程中强烈激活，这种生长培养技术仅限于胎儿心外膜细胞的富集。其他团体所描述的方法，分离成年心外膜细胞43,44。 对于体外迁移测定，心脏提取和心外膜细胞标记在E12.5推荐的原因有两个。首先，E12.5后分离的心都较大，因此需要更多的灌注，以支持可行性。营养的简单扩散s是不足以维持器官培养从较大心。二，心外膜EMT和移民EPDC周围出现E12.5。因此，心外膜应与心脏提取后立即表达荧光蛋白最大化EPDC的检测的腺病毒进行标记。另外，羧基双乙酸钠琥珀酰亚胺酯（CFSE）曾报道标签心脏的外观和微量元素EPDC迁移10,45。 相反，向外生长试验， 体外培养应定期摇晃，以免心脏依恋和心外膜细胞生长。培养也可以在配备有在最低速度设定一个摇杆的培养箱被连续搅拌。用户会发现在器官培养几天心脏形态的改变，并应避免超出心脏隔离72小时的分析。使用此测定，EPDC入侵是48至72小时培养的内观察到。而酶Cre-loxP系统在体内已经被用于EPDC的定位映射到冠状脉管，心肌间质，和室间隔7,22，这种离体技术被限于EPDC迁移子心外膜空间的几个细胞层内。因此，该方法更适合于确定EPDC运动和侵袭的调节剂，而不是限定迁移EPDC的最终目的地。 心外膜细胞隔离和体外迁移实验，与增益或功能丧失的方法相结合，已经被证明为评估候选基因可能影响EPDC生物学9,18,46有用。这些方法也为增益的功能或失功能的屏幕提供了一个平台，以确定EPDC迁移新监管机构。这一战略，在荧光活化细胞分选或单细胞RNA测序的同时，还可以提供一个机会，探索心外膜异质和EPDC分化更详细。最后，这些程序的修改可以用于开发屏幕为小分子影响心外膜细胞和EPDC生物学心脏再生医学的目的。

心外膜是间皮细胞单层的所有行心脏和影响心脏发育，成熟和修理。通过旁分泌信号的高度协调交流，心肌和心外膜之间的亲密对话，是心脏的生长和非心源性心肌细胞谱系1的形成是必不可少的。心外膜衍生细胞（EPDC）从心外膜细胞的子集出现通过上皮至间质转变（EMT）2，侵入外膜下空间和潜在的心肌，并分化主要成冠状血管壁细胞和心脏成纤维细胞，并以一其次，内皮细胞和心肌细胞3-9。 调节外膜EMT和EPDC入侵机制已被归因于各种分子，包括分泌配体10-13，细胞表面受体和粘附分子14,15，单组的协调动作心尖基极性16,17，小GTP酶18,19，和转录因子lators 2,20,21。虽然许多EPDC迁移的分子效应已经确定，更好地理解激发调动EPDC在胚胎可能会加速发展战略的生理信号来操纵在成年改善心脏修复这个过程。 旨在查明EPDC动员新的监管机构研究依赖于这种细胞群的净化或追踪个人心外膜细胞的迁移。一个或标记基因，包括Wt1的 ，TCF21，Tbx18，和/或ALDH1A2的组合，通常用于确定胎儿心外膜1。然而，使用这些标记来跟踪迁移EPDC不是最佳的，因为外膜标志物的表达减弱在心脏发育的过程中，常常失去时心外膜细胞进行transdifferentiation到间质细胞。 在的Cre / loxP系统的应用，在与谱系追踪记者一起已经在永久性标记心脏发育过程中的心外膜和其后代，并在成人7,22,23以下缺血性损伤是有用的。几个心外膜限制性酶Cre线已经产生，并且广泛用于标记EPDC和有条件基因缺失策略1。这些研究已经导致各种心外膜谱系的表征，和EPDC运动和分化的关键介体的鉴定。然而，越来越多的证据也表明心外膜是祖细胞4,24,25的异质群体。因此，仅心外膜细胞的一个子集将使用一个给定的Cre司机进行定位。 为了标签的整个外膜的无论酶Cre特异性，许多实验室已经利用生长尽头TURE系统或体外器官培养的方法来隔离忠实或标签心外膜细胞而不取决于遗传谱系的标志物表达26-28。对于先体外后体内的迁移研究，胚胎的心前心外膜EMT并在补充有绿色荧光蛋白（GFP）-expressing腺病毒（广告/ GFP）9,18培养基中培养分离的。这种方法允许对整个心外膜的有效的标记，而不是利用Cre介导的重组的细胞的子集。心脏培养物暴露于EMT的已知诱导剂，刺激EPDC 28,29的动员。 体外 和体内分析通过体外心外膜外植体培养，这是为探索驱动EPDC迁移的详细机制的特别有用的方法是补充。 在这里，我们描述了用于分离主心外膜细胞在体外 epicardi研究方法人EMT，以及用于离体的器官培养系统中分析EPDC蠕动。我们最近通过遗传调制信号轴操纵EPDC 9的基于肌动蛋白的迁移的心肌素相关转录因子（MRTF）/血清应答因子（SRF）证明了该方法的实用性和鲁棒性。虽然我们的结果强调必要调节EPDC迁移一​​个信令通路，这些方法适用于解开，它们共同协调EPDC迁移和分化的机制。此外，外植体和体外培养系统能够在功能屏幕来实现，以确定EPDC动员新的监管机构在心脏再生治疗应用。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Hyclone&amp;nbsp;</td><td>SH3002201</td><td>DMEM</td></tr><tr><td>Hanks&#039; Balanced Salt Solution</td><td>Hyclone</td><td>SH3003102</td><td>HBSS</td></tr><tr><td>Medium 199</td><td>Hyclone</td><td>SH3025301</td><td>M199</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline&amp;nbsp;</td><td>Hyclone</td><td>SH3002802</td><td>DPBS</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gemini&amp;nbsp;</td><td>100-106</td><td>FBS</td></tr><tr><td>Penicillin/Streptomycin Solution</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>Corning Biocoat Collagen I, 4-Well Culture Slides</td><td>Corning</td><td>354557</td><td></td></tr><tr><td>Multiwell 24-well plates</td><td>Falcon</td><td>08-772-1</td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>M50</td><td>Equipped with Leica KL300 LED might source</td></tr><tr><td>Confocal miscroscope</td><td>Olympus</td><td>1X81</td><td>Equipped with muli-argon laser 405/488/515, FV10-MCPSU laser 405/440/635, 559 laser, and mercury lamp</td></tr><tr><td>Bioruptor</td><td>Diagenode&amp;nbsp;</td><td>UCD-200</td><td></td></tr><tr><td>Transforming growth factor beta 1</td><td>R&amp;amp;D Systems</td><td>100-B-001</td><td>TGF-&amp;beta;1</td></tr><tr><td>Platelet-derived growth factor BB</td><td>R&amp;amp;D Systems</td><td>220-BB-010</td><td>PDGF-BB</td></tr><tr><td>Trizol Reagent</td><td>Applied Biosystems</td><td>15596-026</td><td>Caution, hazardous material</td></tr><tr><td>TURBO DNA-free Kit</td><td>Life Technologies</td><td>AM1907</td><td>DNaseI</td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>BioRad</td><td>1708891</td><td></td></tr><tr><td>UltraPure Glycogen</td><td>Life Technologies</td><td>10814-010</td><td></td></tr><tr><td>SYBR Green</td><td>BioRad</td><td>170-8880</td><td></td></tr><tr><td>Protease inhibtor cocktail tablets</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>Alexa Goat-anti-rabbit 488</td><td>Life Technologies</td><td>A11008</td><td>Secondary antibody</td></tr><tr><td>Alexa Goat anti-rabbit 594</td><td>Life Technologies</td><td>A-11012</td><td>Secondary antibody</td></tr><tr><td>Alexa Goat anti-mouse 594</td><td>Life Technologies</td><td>A-11005</td><td>Secondary antibody</td></tr><tr><td>Mouse anti-smooth muscle alpha actin, Cy3-conjugated&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C6198</td><td>monoclonal antibody, clone 1A4</td></tr><tr><td>Mouse anti-Wilms tumor 1 (WT1)</td><td>Life Technologies</td><td>MA1-46028</td><td>monoclonal antibody, clone 6F-H2</td></tr><tr><td>Rabbit anti-ZO1</td><td>Life Technologies</td><td>40-2200</td><td>polyclonal antibody</td></tr><tr><td>Rabbit anti-Collagen Type 4</td><td>Millipore</td><td>AB756P</td><td>polyclonal antibody</td></tr><tr><td>4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride&amp;nbsp;</td><td>Life Technologies</td><td>D1306</td><td>DAPI</td></tr><tr><td>Fluorescent mounting medium&amp;nbsp;</td><td>DAKO</td><td>S3023</td><td></td></tr><tr><td>16% Paraformaldehyde solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td>PFA diluted to 4% in DPBS. Caution, hazardous material</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Tissue Freezing Medium</td><td>Triangle Biomedical Sciences</td><td>TFM-B</td><td></td></tr><tr><td>Pap pen</td><td>DAKO</td><td>S2002</td><td></td></tr><tr><td>Disposable mictrotome blades&amp;nbsp;</td><td>Sakura Finetek</td><td>4689</td><td></td></tr><tr><td>Microscope slides</td><td>Globe Scientific</td><td>1358W</td><td>positively charged, 25 mm x 75 mm x 1 mm</td></tr><tr><td>Cryostat&amp;nbsp;</td><td>Leica</td><td>CM1950</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11295-00</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>91460-11</td><td></td></tr><tr><td>Disposable base molds</td><td>Fisher Scientific</td><td>22-363-553</td><td></td></tr><tr><td>Ketamine</td><td>Hospira</td><td>NDC 0409-2051-05</td><td></td></tr><tr><td>Xylazine</td><td>Akorn</td><td>NADA 139-236</td><td></td></tr></tbody></table>

epicardial outgrowth culture assay and ex vivo assessment of epicardial-derived cell migration

心外膜细胞系单层的心脏，提供刺激心肌细胞增殖旁分泌因子和发育和疾病过程中直接​​促进心血管祖细胞。而许多因素已在外膜的细胞（EPDC）动员牵连，管理其后续的迁移和分化的机制知之甚少。这里，我们目前在体外和体外的战略研究EPDC运动和分化。首先，我们描述了从胚胎小鼠心脏由生长的文化获得初级心外膜细胞的方法。我们还介绍了一个详细的协议，以评估在器官培养系统标记EPDC的三维迁移。我们提供了使用这些技术，在心外膜心肌素相关的转录因子基因缺失削弱EPDC迁移的证据。这种方法作为一个平台来评估EPDC候选修饰符生物学和可用于开发遗传或化学筛选，以确定EPDC动员的新颖调节可能是对心脏修复有用。

心外膜可以通过采取其外观的位置优势，剥削发育可塑性和EPDC内在的迁移行为采用生长培养法进行有效隔离。小鼠胚胎的心都在E11.5心外膜EMT和培养的背面朝下放在胶原涂层玻片前隔离26（ 图1A，B）。植心将继续萎缩;然而，心外膜将坚持胶原基质和从心肌培养24小时（ 图1C）内迁移关闭。可以用鹅卵石样的形态，表明身份间皮（ 图1C-E）的发出远离心脏观察到心外膜细胞。在培养24小时，心外膜耗尽心中的基因或蛋白质表达分析中删除。 除了观察epitheli人的形态，心外膜细胞培养物的纯度，可以进一步通过心外膜受限基因和蛋白标志物的表达进行验证。心外膜细胞外植体显示的心外膜和间皮标记（ALDH1A2，TCF21，以及Wt1的 ）并与心外膜贫心（ 图2A）的心肌细胞的基因（NKX2-5和MYH7）的表达低稳健表达。为了进一步核实身份，心外膜细胞培养固定48小时后心脏切除，并与抗肾母细胞瘤1（WT1）和透明occludens蛋白1（ZO1）抗体标记间皮细胞和细胞间紧密连接，分别为37,38（合染色图2B）。这种生长培养方法产生的间皮细胞的相对高纯度然而，用户可能观察细胞污染的一小部分。成纤维细胞和平滑肌细胞表现出与一个明显缺少nucle的细长的间充质表型的芳WT1和组织紧密连接（ 图2C）。故障排除技术来限制非心外膜细胞污染的量在讨论被扩展。 外植体长出测定法是在体外询问外膜生物学有用的培养系统。为了证明外膜细胞的可塑性，外植体与TGF-β1[10毫微克/毫升]刺激以诱导转成间充质细胞26,28。刺激后的24小时，EPDC采用具有降低的ZO1染色和平滑肌α肌动蛋白阳性应力纤维（ACTA2或SMA）的健壮从头形成一个间充质表型，特别是在培养39（ 图2D，红）的周边。相反强调纤维装配在植体的外周，汇合心外膜细胞在monolaye的中心更定义间的接触（ZO1，绿色）在皮层肌动蛋白r显示ACTA2蛋白的表达。 除了经历EMT，EPDC会侵入心肌和分化中，构成非心肌细胞群体的相当大的比例。 EPDC的运动可以用先前建立的离 ​​体器官培养系统，由此E12.5心的外标以表达GFP 9,18（ 图3A）的腺病毒进行评估。标记心是在TGF-β1的[10毫微克/毫升]和PDGF-BB [20毫微克/毫升]，促进EPDC迁移的存在3天进一步培养。器官培养物周期性地转动，以防止对培养板的表面上离体心的粘合。该系统允许在一个三维模型，整个心外膜和EPDC蠕动评估有效的标记，而不是定向迁移的体外划痕测定。 EPDC的迁移，安装前后是VED作为GFP的侵袭阳性心外膜细胞进入或超出ColIV（红色）外膜基底膜16（ 图3B，C）。为了说明该系统的效用，我们最近所示MRTF / SRF信号轴的那个操纵可以影响EPDC 9的蠕动。由两侧装接loxP的等位基因的Cre介导的切除（MRTFdKO）Mrtfa和Mrtfb缺失衰减EPDC的迁移进入心外膜下空间。相反，被迫在增加EPDC迁移和ColIV基底膜（ 图3C，D）的破坏C57BL / 6心中结果MRTF-A（广告/ MRTF-A）的表达。 <table fo:keep-together.within-page="1"><tbody><tr><td> 基因 </td><td> 前锋 </td><td> 相反 </td><td> NCBI登录 </td></tr><tr><td> ALDH1A2 </td><td> GGCACTGTGTGGATCAACTG </td><td> TCACTTCTGTGTACGCCTGC </td&gt; <td> NM_009656 </td></tr><tr><td> GAPDH </td><td> CGTGCCGCCTGGAGAAAC </td><td> TGGGAGTTGCTGTTGAAGTCG </td><td> NM_001289726 </td></tr><tr><td> MYH7 </td><td> GTGGCTCCGAGAAAGGAAG </td><td> GAGCCTTGGATTCTCAAACG </td><td> NM_080728 </td></tr><tr><td> NKX2-5 </td><td> GACGTAGCCTGGTGTCTCG </td><td> GTGTGGAATCCGTCGAAAGT </td><td> NM_008700 </td></tr><tr><td> TCF21 </td><td> CATTCACCCAGTCAACCTGA </td><td> CCACTTCCTTCAGGTCATTCTC </td><td> NM_011545 </td></tr><tr><td> WT1 </td><td> ATCCGCAACCAAGGATACAG </td><td> GGTCCTCGTGTTTGAAGGAA </td><td> NM_144783 </td></tr></tbody></table> 表1:正向序列和反向引物由心外膜的分析和限制性心肌基因的基因表达由定量RT-PCR测定的Wi 表达用于心外膜产物纯度文化的验证。个下列参数:变性95℃，10秒;退火在60℃下30秒;重复50X周期。 <img alt="图1" src="/files/ftp_upload/53750/53750fig1.jpg" /> 图1:使用一个突出文化含量 （A）与外膜调制和分析建议的时间表心外膜生长植法的示意图原心外膜细胞的分离 。 （B - E）外植体试验过程中各个时间点的代表性明图像。 （B）胚胎一天E11.5小鼠的心都放在背面向下胶原基质上。 （C）心外膜细胞（白色箭头）将开始培养3-4小时内心脏移植了。血细胞（黑色箭头），可能会对附近积聚或心外膜生长过程中覆盖外植体。 （D，E）后约24小时的文化，心灵被删除，心外膜单层仍然坚持以玻片。蜂窝副产物显示与细胞的鹅卵石状排列的上皮形态。比例尺= 250微米（BD）或50微米（C，E）。 H =心脏，LA =左心房，左心室=左心室，OFT =流出道，RA =右心房，RV =右心室。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53750/53750fig1large.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/53750/53750fig2.jpg" /> 图2:心外膜细胞生长纯度的评价 （A）相比，心外膜耗尽E11心肌基因表达（NKX2-5和MYH7）的生长培养心外膜限制标记（TCF21，ALDH1A2，以及Wt1的）的定量RT-PCR验证。 5心。数据represent是平均值±SEM（ 每组n = 8）。星号使用Student T检验与**** P &lt;0.0001表示统计显着性。 （B）的未刺激的副产物为心外膜表达（WT1，红），细胞间紧密连接（ZO1，绿色），和核的代表性共免疫荧光染色（DAPI，蓝色）。 （C）在生长培养细胞污染的一个例子。心外膜细胞（星号）观察到与特性间皮身份（核WT1，红色）和组织上皮紧密连接（ZO1，绿色）的面板的右侧。在中心（白色箭头）的相邻小区呈现出一个细长的间充质的形态，通过微分干涉对比（DIC）成像示出，用最少的核WT1蛋白和无组织ZO1染色。 （D）的心外膜细胞培养物（在1mg / ml的牛血清白蛋白的4mM HCl中）与车辆刺激或TGF-β1的[10毫微克/毫升]以诱导心外膜EMT。 TGF-β1的刺激促进了在植体或细胞以单层的边缘或周迁移的应力纤维的中心在汇合细胞的皮层区域ACTA2（红色）的稳健表达。细胞粘连被ZO1（绿色）标记。比例尺= 25微米（BD）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53750/53750fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53750/53750fig3.jpg" /> 图3:EPDC能动性体外评估 （ 一 ） 体外心脏培养法的示意图时间表。 （B）一C57BL / 6 E12.5心脏与Ad / GFP（绿色）转24小时的一名代表5微米的部分标注心脏的外观。 离体心脏随后再培养48小时与TGF-β β1[10毫微克/毫升]和PDGF-BB [20毫微克/毫升]，刺激EPDC迁移。切片共同染色ColIV（红色）标注外膜下基底膜和细胞核（DAPI，蓝色）。 体外心脏文化形态是由DIC中。 LV =左心室，RV =右心室，RA =右心房。 （C，D）通过调制MRTF / SRF调节轴体外迁移测定的一个实例应用。这个数字已经从[9]修改。 E12.5心从Mrtfa隔离- / - 。Mrtfb FL / FL胚胎，并与Ad / GFP和控制病毒或表达Cre重组酶24小时的腺病毒转导然后体外培养与TGF-β1刺激[10毫微克/毫升]和PDGF-BB [20毫微克/毫升]另外48小时。 EPDC蠕动（白色箭头）被观察为GFP阳性细胞（绿色）的迁移进入或超出ColIV（红色）。的Cre介导的Mrtfb的缺失在&lt;EM&gt; Mrtfa - / -背景（MRTFdKO）的结果相比，控制心脏移植EPDC衰减。相反，被迫在E12.5 C57BL / 6心MRTF-A（广告/ MRTF-A）的表达增强EPDC迁移。 （D）％GFP阳性细胞迁移的定量分析。数据表示为平均值±SEM（n = 4 时的心为每个条件）。使用单向ANOVA与星号由杜克事后检验表示统计显着性与**** P &lt;0.0001数据进行了分析。比例尺=200μm的（B）或25微米（℃）。所有图像用倒置激光共聚焦显微镜获得的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53750/53750fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration at JoVE.com

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

心外膜生长培养测定和<em&gt;离体</em&gt;心外膜源性细胞迁移的评估

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

epicardial-outgrowth-culture-assay-ex-vivo-assessment-epicardial

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

8.2K 次观看.  University of Rochester School of Medicine and Dentistry. Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility. 

视频: Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

内皮细胞迁移评估暴露于有毒化学品后，

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

科研

发育生物学

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

心外膜细胞小鼠胚胎心脏的体外培养

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

工程学

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

En Face 用于小鼠胚胎平面形态发生分析的心内膜垫制备

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

基因小鼠胚胎干细胞的基础含量为心源性诱导效率量化

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

生物学

小鼠心脏中基于电穿孔的心肌细胞转基因

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

作者聚焦:用于快速基因筛选的心肌细胞转基因

This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

心外膜生长培养测定和

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为外植体含量的颅间充质细胞行为评定