We would like to thank Andrea Portbury and Davin Townley-Tilson for critical reading of the manuscript and the NIH (grant # R01HL061656) for funding support.

注:所有实验均批准的机构动物照顾及使用委员会在北卡罗莱纳大学教堂山分校。 1，消费税主动脉瓣<ol><li>使用经批准的安乐死技术感兴趣的萌芽阶段，安乐死怀孕小鼠幼崽的发展所需要的胚胎一天（五）。 </li><li>用70％乙醇消毒的怀孕雌性的腹部。抬起小腹向上并远离内脏器官的皮肤和肌肉，并解剖开女性的下腹腔内，以允许访问的子宫角。 </li><li>要检索的子宫角，保持宫颈钳，小心切尾的镊子。提起子宫角，切割任何结缔组织，保持号角到位，并通过切割角子宫交界处的输卵管完成删除。 </li><li>将解剖子宫角中含有培养皿山坳ð的0.1M Tris缓冲液（pH7.6）中，并根据需要冲洗。然后，切开子宫角开口长度明智揭露胚胎囊。 </li><li>揭露胚胎，剖开胚胎囊，在胎盘和胎儿的交界处剪断脐带的船只来释放胚胎。放置在第二个培养皿用冷的0.1M Tris缓冲液中的解剖胚胎。返回第一个培养皿与其余undissected胚胎至冰上直至准备进行下一次的胚胎。 </li><li>为了提高胚胎内胸壁访问，杀头的胚胎。如果基因分型是必要的，删除和保存额外的组织片在Eppendorf管置于冰上。 </li><li>将胚胎在它的后面，切断胸壁垂直沿左肋侧，靠近前肢和水平横膈膜以上的可视化的心脏。然后，打开胸壁暴露心脏。 </li><li>使用镊子举行沿大血管高，使用产钳提起心脏，减少血管下方。该N，上面剪钳释放的心脏。如果肺血管保持完整无缺，肺部可能与心脏取出，然后再继续应予删除。 </li><li>切割并去除肺动脉上述阀的电平;在本文所描述的胚胎阶段，肺动脉稍微不透明的，这有助于从阀地区的歧视。切略低于肺动脉瓣，避免了小梁心室肌，并收集在步骤1.10中描述，如果这个地区的利益也。这里和在下一步骤中，使用毛细作用收集在裂解缓冲液中之前绘制过量Tris缓冲远离感兴趣的区域。 </li><li>小心地取出主动脉远端的主动脉瓣，正上方的阀的水平;像肺动脉，主动脉保持略微不透明在这些胚胎阶段，协助在其去除。切略低于主动脉瓣，再次避免了小梁心肌，并传送阀用钳子向含有蛋白酶和磷酸酶抑制剂在冰上的Eppendorf管中加入2μl裂解缓冲液（在材料表中所述）。 </li><li>重复步骤1.6-1.10，直到阀门已切除所有的胚胎，收集在一个单独的Eppendorf管中各阀门。如果所有的胚胎具有相同的基因型，所有的阀可被收集在一个单一的Eppendorf管中。 </li><li>如果基因分型将被执行以确定哪些阀门汇集在一起​​，立即将样品在裂解缓冲液中于-80℃直至进一步使用。样品可以进行基因分型（步骤2.1.1）后汇集。 </li></ol> 2，蛋白提取<ol><li>在冰上融化并离心收集组织在13,100 xg离心1分钟以收集在裂解缓冲液中的组织在管的底部。 <ol><li>如果胚胎进行基因分型，用吸管轻轻收集和含游泳池裂解液阀地区的胚胎具有类似的基因型。为了确保一个强大的b并且，汇集来自E13.5胚胎3-4阀的区域，每个样品建议。 </li><li>如果所有的胚胎是相同的基因型和所有阀门区域收集一起在步骤1.10，裂解整个样品，如在步骤2.2中所描述。 </li></ol></li><li>检查所生成的合并的样品的体积并加入裂解缓冲液中，以40微升的总体积;然后，破坏和均匀化，使用5毫米的不锈钢珠在Eppendorf管中的样品。操作lyser为2-4分钟，在50赫兹，按照制造商的说明。旋管短暂地（〜13,100 xg离心1分钟），并转移样品至新管中，留下的不溶性碎片之后。 </li><li>准备和SDS-PAGE和转移的后续Western blot分析不同的样品，下面的协议，如伊斯拉米等[9]。印迹为上样对照蛋白为蛋白定量是必不可少的。 </li></ol>

<ol>
	<li>Wirrig, E. E., Hinton, R. B., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+cartilage+and+bone-related+proteins+in+pediatric+and+adult+diseased+aortic+valves.">Differential expression of cartilage and bone-related proteins in pediatric and adult diseased aortic valves.</a> J Mol Cell Cardiol. 50, 561-569 (2011).</li><li>Chakraborty, S., 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=Twist1+promotes+heart+valve+cell+proliferation+and+extracellular+matrix+gene+expression+during+development+in+vivo+and+is+expressed+in+human+diseased+aortic+valves.">Twist1 promotes heart valve cell proliferation and extracellular matrix gene expression during development in vivo and is expressed in human diseased aortic valves.</a> Dev Biol. 347, 167-179 (2010).</li><li>Lee, M. P., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twist1+directly+regulates+genes+that+promote+cell+proliferation+and+migration+in+developing+heart+valves.">Twist1 directly regulates genes that promote cell proliferation and migration in developing heart valves.</a> PLoS One. 6 (e29758), (2011).</li><li>Peacock, J. D., Huk, D. J., Ediriweera, H. N., Lincoln, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox9+transcriptionally+represses+Spp1+to+prevent+matrix+mineralization+in+maturing+heart+valves+and+chondrocytes.">Sox9 transcriptionally represses Spp1 to prevent matrix mineralization in maturing heart valves and chondrocytes.</a> PLoS One. 6 (e26769), (2011).</li><li>Chakraborty, S., Cheek, J., Sakthivel, B., Aronow, B. J., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shared+gene+expression+profiles+in+developing+heart+valves+and+osteoblast+progenitor+cells.">Shared gene expression profiles in developing heart valves and osteoblast progenitor cells.</a> Physiol Genomics. 35, 75-85 (2008).</li><li>Vogel, C., Marcotte, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+regulation+of+protein+abundance+from+proteomic+and+transcriptomic+analyses.">Insights into the regulation of protein abundance from proteomic and transcriptomic analyses.</a> Nat Rev Genet. 13, 227-232 (2012).</li><li>Meng, X., 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=Expression+of+functional+Toll-like+receptors+2+and+4+in+human+aortic+valve+interstitial+cells:+potential+roles+in+aortic+valve+inflammation+and+stenosis.">Expression of functional Toll-like receptors 2 and 4 in human aortic valve interstitial cells: potential roles in aortic valve inflammation and stenosis.</a> Am J Physiol Cell Physiol. 294, 29-35 (2008).</li><li>Dyer, L. A., Wu, Y., Moser, M., Patterson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMPER-induced+BMP+signaling+promotes+coronary+artery+remodeling.">BMPER-induced BMP signaling promotes coronary artery remodeling.</a> Developmental Biology. 386, 385-394 (2014).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J Vis Exp. (44), (2010).</li><li>Garside, V. C., Chang, A. C., Karsan, A., Hoodless, P. 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W., 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=Morphogenesis+of+outflow+tract+rotation+during+cardiac+development:+the+pulmonary+push+concept.">Morphogenesis of outflow tract rotation during cardiac development: the pulmonary push concept.</a> Dev Dyn. 241, 1413-1422 (2012).</li><li>Mohler, E. R., Adam 3rd, L. P., McClelland, P., Graham, L., Hathaway, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+osteopontin+in+calcified+human+aortic+valves.">Detection of osteopontin in calcified human aortic valves.</a> Arterioscler Thromb Vasc Biol. 17, 547-552 (1997).</li></ol>

The authors have nothing to disclose.

量化蛋白质水平在早期胚胎小鼠和鸡胚心脏瓣膜区域的能力提供了理解的关键细胞信号传递活动阀发展的额外工具。我们这里所描述的协议不从标准蛋白分离方法有很大的不同。然而，通过修改某些关键步骤，我们已经成功地从非常小的样品尺寸相加得到的磷酸化蛋白。为实现这一结果，下面的步骤是特别重要的。以确保得到的优质蛋白，关键的是要保持样品冷却，不论在冰上或用冰冷却的缓冲?...

能够识别并量化蛋白表达水平是一个标准技术，动物 - 和基于细胞的实验。然而，尽管一个长期的兴趣在早期胚胎心脏瓣膜的开发，在开发过程中评估蛋白表达在这个特定的组织目前仅限于免疫组化在两个小鸡和鼠标1,2。在大多数模型生物体（ 如小鸡和小鼠）的显影阀定量蛋白表达的难度的部分是阀，从而限制了蛋白质，可以得到的数量的小尺寸。因此，为了定量分析，研究人员通常依靠RNA提取和扩增用于随后定量PCR或微阵列分析2-5。然而，RNA和蛋白的表达水平是不完全相关6，所以着眼于RNA的表达不能提供一个严格的帐户中发生的任何给定的信号的许多改变在发展途径，在不同的时间。基于此限制，在目前可用的方法，该方法的目标是开发一种协议，用于可靠地获得足够量的蛋白质从显影小鼠胚胎心脏瓣膜的区域为所发生的各种信号通路是在重要的变化的定量分析这个组织的成熟。 胚胎瓣膜已普遍从老鼠RNA分离和后续基因表达分析2-5解剖。然而，这些研究已不限于使用基因表达作为一个读出信号通路的激活，它不允许检测翻译后蛋白修饰，其可能会影响下游信号的检测。利用RNA分离技术为出发点，我们开始与解剖感兴趣的区域。因为我们关心的是检测磷酸化蛋白质，都说明信号拍拍在主动脉瓣发展（E13.5-14.5）的一个特定时期hway活性，我们进行的所有解剖在无磷酸盐的Tris缓冲液和收集的阀门与磷酸酶和蛋白酶抑制剂的Tris-基的裂解缓冲液。在我们的具体情况下，仅收集主动脉瓣区域，但肺动脉瓣区域可以很容易地在相同的时间来获得。该阀的区域分别然后均质化并结合当前用于研究在成年心脏瓣膜7蛋白表达的样品缓冲液。通过使用小样品体积（ 例如 ，2微升），并从胚胎中具有相同的基因型汇集阀的区域，我们能够检测磷酸化和未磷酸化的蛋白质在13.5 8天胚胎。因为阀的区域可以被冻结并存储在裂解缓冲液中，胚胎可以在需要集中之前进行基因分型。 这种技术拓宽了一套工具，可用于评估细胞signalin在开发过程中克途径，并提供了定量的恭维免疫组化，专门为开发心脏瓣膜。这种技术应该是有益的，不仅要发展心脏病，也给所有谁与早期胚胎的工作有兴趣，包含有限的组织区域发育生物学家。

<table><tbody><tr><td>Timed-pregnant mouse</td><td></td><td></td><td>To be dissected at the embryonic stage of interest</td></tr><tr><td>Stereoscopic microscope</td><td>Nikon</td><td>SMZ645</td><td></td></tr><tr><td>0.1 M Tris, pH 7.6</td><td></td><td></td><td></td></tr><tr><td>Microscissors</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Fine forceps, #5</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Dissecting needle holders</td><td>Ted Pella Inc.</td><td>13560</td><td></td></tr><tr><td>Dissecting needles</td><td>Ted Pella Inc.</td><td>13561-10</td><td></td></tr><tr><td>Micropipette, 20 &amp;mu;l, with tips</td><td></td><td></td><td></td></tr><tr><td>Lysis buffer</td><td></td><td></td><td>50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton</td></tr><tr><td>PhosSTOP</td><td>Roche</td><td>4906845001</td><td>Add 1 tablet to 10 ml lysis buffer</td></tr><tr><td>TissueLyser LT</td><td>Qiagen</td><td>85600</td><td></td></tr><tr><td>Stainless steel beads</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td></td></tr></tbody></table>

protein isolation from the developing embryonic mouse heart valve region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

采用这种制备技术，我们能够从E13.5胚胎检测磷酸化的Smad 1,5,8（pSmad）在单一主动脉瓣区域。 如图1A所示 ，从甚至一个单一的阀区域分离的蛋白是足以检测微弱pSmad带。信号强度成比例地增大到汇集阀的区域的数量。重要的是，pSmad /β-肌动蛋白的比值保持在不同的样本大小（ 图1C）几乎恒定。由于可用的蛋白质的水平低，同样的印迹无法剥离，并再次探测总Smad蛋...

Watch this Scientific Journal Video about Protein Isolation from the Developing Embryonic Mouse Heart Valve Region at JoVE.com

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

从发展胚胎小鼠心脏瓣膜区蛋白分离

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

protein-isolation-from-developing-embryonic-mouse-heart-valve

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.3K 次观看.  University of North Carolina at Chapel Hill. The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

视频: Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

低血清条件下心脏祖细胞内皮分化的诱导作用

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

科研

发育生物学

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

禽流感胚胎瓣膜祖细胞的分离和培养

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

生物学

以单细胞分辨率从小鼠心脏祖细胞中分离细胞核

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

作者聚焦:从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率下的表观基因组和基因表达分析  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

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 ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

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 ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

冠状血管生成体模型

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

医学

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

从发展胚胎小鼠心脏瓣膜区蛋白分离

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低血清条件下心脏祖细胞内皮分化的诱导作用

禽流感胚胎瓣膜祖细胞的分离和培养

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

从小鼠心脏祖细胞中分离细胞核，用于单细胞分辨率的表观基因组和基因表达谱分析

从小鼠心脏中分离主黑素细胞样细胞

分离，培养和成年小鼠Cardiomyoctyes的功能分析

胚胎心室血管内皮细胞的分离

冠状血管生成体模型

鼠阀门内皮细胞的分离