这项工作是由来自杜克 - 新加坡国立大学医学研究生院新加坡吴作栋基础和新加坡NRF奖学金（NRF-NRFF2016-01），以Manvendra K.辛格的资金支持。

所有实验均在杜克 - 新加坡国立大学医学研究生院机构动物护理和使用委员会批准。 1.检索心室胚胎<ol><li>在祭祀用二氧化碳气体供应或其他认可的安乐死方法的安乐死室所需的萌芽阶段（E11.5 E12.5或）一个定时孕鼠。 </li><li>把鼠标放在其解剖台上。消毒用70％乙醇女性的腹部。提起在腹部皮肤，使一个小切口（1-2厘米），用刀片，然后用双手握住皮肤拉掉，露出腹壁。 </li><li>现在，切开腹壁，露出子宫角。使用无菌镊子抬起子宫角，小心切掉附着于子宫角的脂肪组织和血管。在宫颈切检索子宫。 </li><li>放置子宫角中含有无菌冷的1×磷酸盐的Buff培养皿<html> ERED液（PBS），轻轻冲洗。广场上冰的培养皿。 </li><li>用剪刀通过子宫角（相对到胎盘位于的站点）的中线切割以暴露仍然卵黄囊内，并通过胎盘附着于子宫壁的胚胎。 </li><li>使用无菌镊子，剖开胚胎卵黄囊释放胚胎。 </li><li>放入装有无菌冷1X PBS一个新的培养皿胚胎。斩首胚胎，并将其放置在它的后面。切开胸壁暴露心脏。 </li><li>使用无菌镊子解除心脏和切掉周围的血管从胸壁释放心脏。要特别注意不要用手术工具破坏心脏的外膜。 </li><li>剪掉流出道和两个心房。转移心室与冷1X PBS另一道菜。广场上冰的菜。 </li><li>重复步骤1.6-1.9，直到所有的心室检索。 </li></ol>乐"&gt; 2。心外膜外植体培养注意:这些文化心外膜外植体无论是在玻璃玻片或胶原凝胶下游应用。 <ol><li>玻璃商会幻灯片<ol><li>以制备培养基，10％胎牛血清（FBS），1％青霉素/链霉素和2纳克/ ml重组成纤维细胞生长因子2（FGF2）加入Dulbecco改良的Eagle培养基（DMEM）。执行在层流罩中的所有步骤。 </li><li>将500μl培养基添加到8孔腔室的每个孔中。 </li><li>将每个切心室在井的中心。定向心室保持解剖面朝下的阱的底表面上。 </li><li>轻轻的把盘子在37℃，5％ 的 CO 2细胞培养孵化器。 </li><li>保持与最小的干扰文化，让植坚持。心外膜单层的形成可后48-72小时进行观察。 </li><li>对于epicard分化胶质细胞分化成平滑肌细胞，培养的心外膜单层细胞在分化培养基中再6天。为了制备分化培养基，10％FBS，1％青霉素/链霉素和50ng / ml重组转化生长因子β1（TGF-β1）添加到DMEM中。 </li><li>更改隔日培养基。 </li></ol></li><li>胶原凝胶<ol><li>准备使用根据制造商的协议的商用3D胶原培养试剂盒在一个96孔板的胶原凝胶。 </li><li>为了制备胶原凝胶的所需体积，用DMEM的5倍和移液器上下混合的胶原溶液的适当的量。该解决方案应该变黄。 </li><li>加入中和液相应的体积和吹打向上和向下以及立即混合。该解决方案将变成粉红色。 </li><li>吸管100微升该混合物到96孔板的每个孔中。放置板在37℃，5％CO 2培养箱中30-60分钟，以人的低的凝胶聚合。 </li><li>添加培养基的200微升上面充分描述的每个。 </li><li>将每个切心室在井的中心。东方心室保持解剖面朝下放在胶原凝胶（心室心尖部应面对实验者）。轻轻把盘子放回细胞培养孵化器。 </li><li>在3天之后，取出心室。把盘子放回另外2天细胞培养孵化器。 </li><li>更改隔日培养基。 </li></ol></li></ol> 3.固定和可视化注意:心外膜细胞可以在任一玻璃玻片或胶原凝胶被可视化。 <ol><li>吸出培养基并加入无菌1×PBS中的等体积的洗细胞。 </li><li>加入足够的4％低聚甲醛（PFA），以覆盖细胞。定为10分钟，将细胞在4℃。 </li><li>删除PFA和1X PBST（1X PBS洗具植0.1％的Triton X-100），以透化细胞。 </li><li>具有所需的抗体（ 如的Alexa Fluor 488鬼笔环肽; ZO-1，α微管蛋白和α平滑肌肌动蛋白）免疫染色7。 </li></ol>

<ol>
	<li>Mikawa, T., Fischman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+analysis+of+cardiac+morphogenesis:+discontinuous+formation+of+coronary+vessels.">Retroviral analysis of cardiac morphogenesis: discontinuous formation of coronary vessels.</a> Proc Natl Acad Sci U S A. 89, 9504-9508 (1992).</li><li>Mikawa, T., Gourdie, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericardial+mesoderm+generates+a+population+of+coronary+smooth+muscle+cells+migrating+into+the+heart+along+with+ingrowth+of+the+epicardial+organ.">Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ.</a> Dev Biol. 174, 221-232 (1996).</li><li>Manner, J., Perez-Pomares, J. M., Macias, D., Munoz-Chapuli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin,+formation+and+developmental+significance+of+the+epicardium:+a+review.">The origin, formation and developmental significance of the epicardium: a review.</a> Cells Tissues Organs. 169, 89-103 (2001).</li><li>Gittenberger-de Groot, A. C., Vrancken Peeters, M. P., Mentink, M. M., Gourdie, R. G., Poelmann, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardium-derived+cells+contribute+a+novel+population+to+the+myocardial+wall+and+the+atrioventricular+cushions.">Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions.</a> Circ Res. 82, 1043-1052 (1998).</li><li>von Gise, A., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocardial+and+epicardial+epithelial+to+mesenchymal+transitions+in+heart+development+and+disease.">Endocardial and epicardial epithelial to mesenchymal transitions in heart development and disease.</a> Circ Res. 110, 1628-1645 (2012).</li><li>Katz, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+compartments+of+the+proepicardial+organ+give+rise+to+coronary+vascular+endothelial+cells.">Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells.</a> Dev Cell. 22, 639-650 (2012).</li><li>Singh, M. K., Lu, M. M., Massera, D., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-processing+enzyme+Dicer+is+required+in+epicardium+for+coronary+vasculature+development.">MicroRNA-processing enzyme Dicer is required in epicardium for coronary vasculature development.</a> J Biol Chem. 286, 41036-41045 (2011).</li><li>Degenhardt, K., Singh, M. K., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+under+development:+cardiovascular+embryology+applied+to+heart+disease.">New approaches under development: cardiovascular embryology applied to heart disease.</a> J Clin Invest. 123, 71-74 (2013).</li><li>Singh, M. K., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardium-derived+cardiac+mesenchymal+stem+cells:+expanding+the+outer+limit+of+heart+repair.">Epicardium-derived cardiac mesenchymal stem cells: expanding the outer limit of heart repair.</a> Circ Res. 110, 904-906 (2012).</li><li>Manner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+study+on+the+formation+of+the+epicardium+in+chick+embryos.">Experimental study on the formation of the epicardium in chick embryos.</a> Anat Embryol (Berl). 187, 281-289 (1993).</li><li>Manner, J., Schlueter, J., Brand, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+analyses+of+the+function+of+the+proepicardium+using+a+new+microsurgical+procedure+to+induce+loss-of-proepicardial-function+in+chick+embryos.">Experimental analyses of the function of the proepicardium using a new microsurgical procedure to induce loss-of-proepicardial-function in chick embryos.</a> Dev Dyn. 233, 1454-1463 (2005).</li><li>Pennisi, D. J., Ballard, V. L., Mikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardium+is+required+for+the+full+rate+of+myocyte+proliferation+and+levels+of+expression+of+myocyte+mitogenic+factors+FGF2+and+its+receptor,+FGFR-1,+but+not+for+transmural+myocardial+patterning+in+the+embryonic+chick+heart.">Epicardium is required for the full rate of myocyte proliferation and levels of expression of myocyte mitogenic factors FGF2 and its receptor, FGFR-1, but not for transmural myocardial patterning in the embryonic chick heart.</a> Dev Dyn. 228, 161-172 (2003).</li><li>Gittenberger-de Groot, A. C., Vrancken Peeters, M. P., Bergwerff, M., Mentink, M. M., Poelmann, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+outgrowth+inhibition+leads+to+compensatory+mesothelial+outflow+tract+collar+and+abnormal+cardiac+septation+and+coronary+formation.">Epicardial outgrowth inhibition leads to compensatory mesothelial outflow tract collar and abnormal cardiac septation and coronary formation.</a> Circ Res. 87, 969-971 (2000).</li><li>Lavine, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocardial+and+epicardial+derived+FGF+signals+regulate+myocardial+proliferation+and+differentiation+in+vivo.">Endocardial and epicardial derived FGF signals regulate myocardial proliferation and differentiation in vivo.</a> Dev Cell. 8, 85-95 (2005).</li><li>Merki, E., 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=Epicardial+retinoid+X+receptor+alpha+is+required+for+myocardial+growth+and+coronary+artery+formation.">Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation.</a> Proc Natl Acad Sci U S A. 102, 18455-18460 (2005).</li><li>Stuckmann, I., Evans, S., Lassar, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietin+and+retinoic+acid,+secreted+from+the+epicardium,+are+required+for+cardiac+myocyte+proliferation.">Erythropoietin and retinoic acid, secreted from the epicardium, are required for cardiac myocyte proliferation.</a> Dev Biol. 255, 334-349 (2003).</li><li>Huang, G. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C/EBP+transcription+factors+mediate+epicardial+activation+during+heart+development+and+injury.">C/EBP transcription factors mediate epicardial activation during heart development and injury.</a> Science. 338, 1599-1603 (2012).</li><li>Zhou, B., 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=Adult+mouse+epicardium+modulates+myocardial+injury+by+secreting+paracrine+factors.">Adult mouse epicardium modulates myocardial injury by secreting paracrine factors.</a> J Clin Invest. 121, 1894-1904 (2011).</li><li>Christoffels, V. 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=Tbx18+and+the+fate+of+epicardial+progenitors.">Tbx18 and the fate of epicardial progenitors.</a> Nature. 458, 8-9 (2009).</li><li>Rudat, C., Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wt1+and+epicardial+fate+mapping.">Wt1 and epicardial fate mapping.</a> Circ Res. 111, 165-169 (2012).</li><li>Duim, S. N., Kurakula, K., Goumans, M. J., Kruithof, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+endothelial+cells+express+Wilms'+tumor-1:+Wt1+expression+in+the+developing,+adult+and+infarcted+heart.">Cardiac endothelial cells express Wilms' tumor-1: Wt1 expression in the developing, adult and infarcted heart.</a> J Mol Cell Cardiol. 81, 127-135 (2015).</li><li>Iyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+derivation+of+epicardium+and+its+differentiated+smooth+muscle+cell+progeny+from+human+pluripotent+stem+cells.">Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells.</a> Development. 142, 1528-1541 (2015).</li><li>Witty, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+the+epicardial+lineage+from+human+pluripotent+stem+cells.">Generation of the epicardial lineage from human pluripotent stem cells.</a> Nat Biotechnol. 32, 1026-1035 (2014).</li></ol>

The authors declare that they have no competing financial interests.

这是关键的发展促进了外膜的研究，以满足在心脏发育和再生心外膜的日益重要的技术。心外膜培养体系带来显著优势心外膜的研究。 分离心外膜细胞的另一种方法是使用荧光激活细胞分选（FACS）。此方法依赖于使用心外膜标记（或荧光蛋白的外膜细胞特异性的转基因表达）的心外膜细胞从其他谱系分离。然而，越来越的证据量表明心外膜标记不是仅仅在外膜表达。例如，T...

实验数据的财富已经说明了外膜影响心脏发育的关键步骤。在开发过程中，所述横隔引起称为proepicardium 1-4间皮细胞丛。从proepicardium细胞然后迁移和信封心肌形成心外膜。在此之后，心外膜细胞的子集经历EMT引起心外膜衍生细胞（EPDCs），该随后侵入心肌的迁徙人口。遗传以及逆转录病毒谱系追踪实验已经证明，EPDCs分化成各种细胞谱系，包括平滑肌细胞，成纤维细胞，内皮细胞和心肌细胞（如有的话）。因此EPDCs显著向冠状脉管和心肌架构1,2,4-9的发展。此外，心外膜是心室致密层10-12的发展至关重要。前面xample Gittenberger德Groot 等人证明了抑制proepicardium的生长导致的缺陷的阵列例如薄心肌，心脏和室间隔形成异常的缺陷循环，其结果，胚胎致死13。从胚胎外膜分泌的旁分泌因子调节心肌细胞的增殖和分化。与此相一致，信号传导途径，如视黄酸（RA），成纤维细胞生长因子（的FGFs）和Wnt /β-catenin的特异性外膜-缺失导致有缺陷的心肌的生长和胚胎致死14-16。 虽然心外膜被认为是在成年心脏静止的，最近的研究已经表明，在发育程序跟随的心脏损伤17,18激活的心外膜。在激活时，将细胞进行快速增殖和EMT导致在EPDC形成。这些细胞表现出capacity以分化成成纤维细胞和平滑肌细胞，但不心肌或血管内皮细胞18。此外，EPDCs分泌促血管生成因子，援助在受伤部位的血管形成，从而通过减小梗塞尺寸促进改善心脏功能。由于这些发现，心外膜已经获得了在心血管发育，疾病和再生的研究兴趣。 转基因技术在21世纪革命性的医学研究。随着转基因技术的帮助下，患病小鼠模型代谢和病理生理模仿人类状况已开发成功。然而，在研究这些突变体的外膜细胞行为一直主要是由于早期胚胎死亡的一个挑战。考虑到心外膜在心脏发育和再生发挥的作用显著，我们已经建立了鼠标epicardia 体外培养体系L细胞。这种方法允许心外膜细胞的长期培养和便于心外膜的两个重要的属性的详细研究:其迁移和分化的能力。从小鼠切下的心室可以在其可以被用来进行迁移测定胶原凝胶中培养。在复制层外膜的富含胶原蛋白的细胞外基质更好地概括了体内细胞生理学3D矩阵待培养。或者，它们可以在腔室玻片，以建立一个心外膜单层然后可以用于各种下游应用进行培养。此单层可用于染色为紧密连接蛋白，这将提供在外膜的经历EMT的能力是用于迁移至关重要的见解。此外，分化实验也可以在这些细胞中进行。此外，基因表达模式可以通过从细胞中提取的RNA进行分析，并执行定量聚合酶链反应（qPCR的）。最后，单层也可以与代理后跟一个分子分析来测试潜在治疗处理。放在一起，这个心外膜培养体系为我们提供了机会，可视化和收集这进一步加强了我们对心外膜发展的理解分子数据。 这种方法的另一个期望的特征是，它是简单的，并且不需要复杂的设置。简言之，将胚胎在其中心脏切除E11.5或E12.5以下收获。脑室然后在任胶原凝胶或玻片培养。随后，这些细胞可以被用来进行下行实验。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Life tech invitrogen&amp;nbsp;</td><td>11995065</td><td></td></tr><tr><td>Penicillin/streptomycin solution</td><td>Life tech invitrogen&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Life tech invitrogen&amp;nbsp;</td><td>10500064</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148-5KG</td><td></td></tr><tr><td>Recombinant fibroblast growth factor 2 (FGF2)</td><td>PeproTech</td><td>450-33</td><td></td></tr><tr><td>Recombinant transforming growth factor beta 1 (TGF-&amp;beta;1)</td><td>PeproTech</td><td>100-21</td><td></td></tr><tr><td>ZO-1 antibody</td><td>Life tech invitrogen&amp;nbsp;</td><td>40-2200</td><td></td></tr><tr><td>&amp;alpha;-Tubulin antibody</td><td>Sigma</td><td>T 6074</td><td></td></tr><tr><td>&amp;alpha;-smooth muscle actin (SMA) antibody</td><td>Sigma</td><td>A 2547</td><td></td></tr><tr><td>Phalloidin antibody</td><td>Life tech invitrogen&amp;nbsp;</td><td>A12379</td><td></td></tr><tr><td>3D Collagen Culture kit&amp;nbsp;</td><td>Millipore&amp;nbsp;</td><td>ECM 675</td><td></td></tr><tr><td>8-well chamber slide</td><td>Fisher Scientific</td><td>NNU 154534-PK</td><td></td></tr><tr><td>Trizol reagent</td><td>Life Technologies</td><td>15596-018</td><td></td></tr><tr><td>ViiA 7 Real-Time PCR System</td><td>Life Technologies</td><td>4453536</td><td></td></tr><tr><td>Superscript First Strand Synthesis kit</td><td>Life Technologies</td><td>11904-018</td><td></td></tr></tbody></table>

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.

使用该培养协议，主心外膜细胞可以用高纯度的下游应用中分离。培养的细胞能够经历EMT，迁移和分化，正如心外膜细胞在体内做。 为了确定我们的主要心外膜细胞培养的纯度，我们分析Sema3d GFPCre / +生成的心外膜外植体; R26 汤姆/ +胚胎。 Sema3d是早期心脏发育过程中的心外膜?...

Watch this Scientific Journal Video about In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart at JoVE.com

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

心外膜细胞小鼠胚胎心脏的体外培养

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.

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

in-vitro-culture-of-epicardial-cells-from-mouse-embryonic-heart

Research

JoVE Journal

Engineering

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

7.4K 次观看.  Duke-NUS Graduate Medical School. 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. 

视频: In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

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

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

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

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.

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

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

科研

发育生物学

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 ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

小鼠心脏场特定心脏祖细胞的体外生成

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

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, ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

从小鼠心脏中分离主黑素细胞样细胞

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

生物学

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

心外膜细胞小鼠胚胎心脏的体外培养

摘要

探索更多视频

心外膜生长培养测定和

人成体和胎心标本中原发性心外膜细胞的分离培养

小鼠心脏场特定心脏祖细胞的体外生成

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

从小鼠心脏中分离主黑素细胞样细胞

分离新生小鼠心肌文化和

一种新型的

胚胎心室血管内皮细胞的分离