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.

NOTA: Todos los experimentos fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Carolina del Norte en Chapel Hill. 1. Impuestos Especiales de la válvula aórtica <ol><li> Usando una técnica de eutanasia aprobadas para la etapa embrionaria de interés, la eutanasia a un ratón embarazada con cachorros en el día embrionario deseado (E) del desarrollo. </li><li> Utilice 70% de etanol para esterilizar el abdomen de la hembra embarazada. Levante la piel y los músculos de la parte inferior del abdomen hacia arriba y lejos de los órganos internos, y disecar la cavidad abdominal inferior Open femenino para permitir el acceso a la trompa uterina. </li><li> Para recuperar el cuerno uterino, mantenga el cuello del útero con una pinza y caudal cuidadosamente cortado al fórceps. Levante el cuerno uterino, cortar cualquier tejido conectivo que mantiene la bocina en su lugar, y completar la eliminación mediante la reducción de la unión de la trompa uterina con el oviducto. </li><li> Coloque el cuerno uterino diseccionado en una placa de Petri que contiene cold 0,1 M de tampón Tris (pH 7,6) y enjuague según sea necesario. A continuación, cortar el cuerno uterino en sentido longitudinal para exponer los sacos embrionarios abierta. </li><li> Para exponer el embrión, cortar y abrir un saco embrionario en la unión de la placenta y el embrión y cortar los vasos umbilicales para liberar el embrión. Coloque el embrión diseccionado en un segundo plato de Petri con frío tampón Tris 0,1 M. Devuelva la primera placa de Petri con los restantes embriones no seccionada a hielo hasta que esté listo para el próximo embrión. </li><li> Para mejorar el acceso de la pared torácica en el embrión, decapitar al embrión. Si genotipado es necesario, retire y guarde un pedazo de tejido extra en un tubo Eppendorf se colocó en hielo. </li><li> Coloque el embrión en su parte posterior, y cortar la pared torácica verticalmente a lo largo del lado de la caja torácica, cerca de una extremidad anterior, y horizontalmente por encima del diafragma para visualizar el corazón. Luego, abrir la pared torácica para exponer el corazón. </li><li> Con unas pinzas para mantener en alto a lo largo de los grandes vasos, levantar el corazón con unas pinzas y cortar los vasos debajo. Eln, corte por encima de la pinza para liberar el corazón. Si los vasos pulmonares permanecen sin cortar, los pulmones se pueden eliminar con el corazón y deben ser removidos antes de continuar. </li><li> Corte y retire la arteria pulmonar por encima del nivel de la válvula; en las etapas embrionarias descritos en este documento, la arteria pulmonar es ligeramente opaca, que ayuda en su discriminación de la región de la válvula. Corte justo debajo de la válvula pulmonar, evitando el miocardio ventricular trabeculado y recoger como se describe en el paso 1.10 si esta región es también de interés. Aquí y en la siguiente etapa, usar la acción capilar para extraer el exceso de tampón Tris lejos de la región de interés antes de la recogida en tampón de lisis. </li><li> Retire cuidadosamente la aorta distal a la válvula aórtica, justo por encima del nivel de la válvula; como la arteria pulmonar, la aorta se mantiene ligeramente opaca en estas etapas embrionarias, ayudar en su eliminación. Corte justo debajo de la válvula aórtica, evitando de nuevo el miocardio ventricular trabeculado, y transferir elválvula usando fórceps a un tubo Eppendorf con tampón de lisis 2 l (descrito en la Tabla de Materiales) que contiene inhibidores de la proteasa y fosfatasa en hielo. </li><li> Repetir los pasos 1.6 a 1.10 hasta que las válvulas han sido extirpados de todos los embriones, la recogida de cada válvula en un tubo Eppendorf separado. Si todos los embriones tienen el mismo genotipo, todas las válvulas pueden ser recogidos en un solo tubo Eppendorf. </li><li> Si el genotipado se va a realizar para determinar qué válvulas para agrupar juntos, colocar inmediatamente las muestras en tampón de lisis a -80 ° C hasta su uso posterior. Las muestras pueden luego ser agrupados después de genotipado (etapa 2.1.1). </li></ol> Extracción 2. Proteína <ol><li> Descongele tejidos recogidos en el hielo y se centrifuga a 13.100 xg durante 1 min para recoger los tejidos en tampón de lisis en la parte inferior del tubo. <ol><li> Si se genotipo embriones, utilizar una pipeta para recoger suavemente y tampón piscina lisis que contiene las regiones de la válvula a partir de embriones con genotipos similares. Para asegurar una fuerte by, la puesta en común 3-4 regiones de la válvula a partir de embriones E13.5 se recomienda por muestra. </li><li> Si todos los embriones eran de la misma genotipo y todas las regiones de la válvula se recogieron juntos en el paso 1.10, lisar toda la muestra, como se describe en el Paso 2.2. </li></ol></li><li> Compruebe el volumen de la muestra agrupada resultante y añadir tampón de lisis hasta un volumen total de 40 l; entonces, desarticular y homogeneizar muestras usando 5 mm bolas de acero inoxidable en tubos Eppendorf. Operar el Lyser durante 2-4 min a 50 Hz, según las instrucciones del fabricante. Centrifugar los tubos brevemente, (~ 13.100 xg durante 1 min), y transferencia de muestras a tubos nuevos, dejando atrás los restos insolubles. </li><li> Preparar y muestras separadas para SDS-PAGE y transferencia de Western blot posterior, siguiendo un protocolo como Eslami et al 9. Secante para una proteína de control de carga es esencial para la cuantificación de proteínas. </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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-ordinating+Notch,+BMP,+and+TGF-beta+signaling+during+heart+valve+development.">Co-ordinating Notch, BMP, and TGF-beta signaling during heart valve development.</a> Cell Mol Life Sci. 70, 2899-2917 (2013).</li><li>Jiang, X., Rowitch, D. H., Soriano, P., McMahon, A. P., Sucov, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+the+mammalian+cardiac+neural.">Fate of the mammalian cardiac neural.</a> Development. 127, 1607-1616 (2000).</li><li>Scherptong, R. 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.

La capacidad de cuantificar los niveles de proteína a principios de embriones de ratón y Chick regiones válvula cardíaca proporciona una herramienta adicional para la comprensión de los eventos de señalización celular críticas para el desarrollo de la válvula. Nuestro protocolo descrito en este documento no difiere en gran medida de los procedimientos de aislamiento de proteínas convencionales. Sin embargo, mediante la modificación de algunas medidas clave, que hemos obtenido con éxito proteínas fosforilada...

Ser capaz de identificar y cuantificar los niveles de expresión de proteínas es una técnica estándar para animal- y los experimentos basados ​​en células. Sin embargo, a pesar de un interés de larga data en el desarrollo de la válvula cardíaca embrionaria temprana, la evaluación de la expresión de proteínas en este tejido específico durante el desarrollo se limita actualmente a la inmunohistoquímica en tanto el pollo y 1,2 ratón. Parte de la dificultad de cuantificar la expresión de proteínas en las válvulas de desarrollo de la mayoría de organismos modelo (por ejemplo, de pollo y de ratón) es el pequeño tamaño de las válvulas, lo que limita la cantidad de proteína que se puede obtener. Por lo tanto, para los análisis cuantitativos, los investigadores normalmente se basan en la extracción de ARN y amplificación por PCR cuantitativa posterior o el análisis de microarrays 2-5. Sin embargo, los niveles de expresión de ARN y proteínas no son del todo correlativa 6, por lo que se centra en la expresión del ARN no pueden dar una explicación rigurosa de los numerosos cambios que se producen en cualquier señalización dadavía en varias ocasiones durante la evolución. Sobre la base de este límite en la metodología actualmente disponible, el objetivo de este procedimiento fue desarrollar un protocolo para la obtención fiable de cantidades suficientes de proteína a partir de las regiones de la válvula cardiaca de embriones de ratón en desarrollo para el análisis cuantitativo de los cambios que se producen en diversas vías de señalización que son importantes en la maduración de este tejido. Válvulas embrionarias son ya comúnmente disecados de los ratones para el aislamiento de ARN y análisis de expresión génica posterior 2-5. Sin embargo, estos estudios se han limitado a la utilización de la expresión génica como una lectura de salida de activación de la vía de señalización, que no permite la detección de detectar proteínas modificaciones post-traduccionales que pueden afectar a la señalización aguas abajo. Utilizando las técnicas de aislamiento de ARN como punto de partida, comenzamos con la disección de las regiones de interés. Debido a que nuestro interés se detecta proteínas fosforiladas que eran indicativos de pat señalizaciónhway actividad durante un período específico de desarrollo de la válvula aórtica (E13.5-14.5), que realiza todas las disecciones en tampón Tris-fosfato libre y se recogió las válvulas en un tampón de lisis a base de Tris con inhibidores de fosfatasa y proteasa. En nuestro caso específico, sólo se recogieron las regiones de la válvula aórtica, pero la región de la válvula pulmonar podrían obtenerse fácilmente al mismo tiempo. Las regiones de la válvula fueron entonces homogeneizada y combinado con un tampón de muestra que se utiliza actualmente para estudiar la expresión de proteínas en las válvulas cardiacas adultas 7. Mediante el uso de pequeños volúmenes de muestra (por ejemplo, 2 l) y puesta en común de las regiones de la válvula a partir de embriones con el mismo genotipo, hemos sido capaces de detectar las proteínas fosforiladas y no fosforilada en el día embrionario 13.5 8. Debido a que las regiones de válvula pueden ser congelados y almacenados en tampón de lisis, los embriones pueden ser genotipados si es necesario antes de la puesta en común. Esta técnica amplía el conjunto de herramientas que están disponibles para evaluar signalin celularg vías durante el desarrollo y ofrece un cumplido cuantitativa de inmunohistoquímica, específicamente para las válvulas cardíacas en desarrollo. Esta técnica debe ser de beneficio no sólo para los cardiólogos de desarrollo, sino también a todos los biólogos del desarrollo que trabajan con embriones en fase inicial están interesados ​​en regiones que contienen tejido limitado.

<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.

Utilizando esta técnica de preparación, hemos sido capaces de detectar fosforilados Smad 1,5,8 (pSmad) en las regiones de la válvula aórtica individuales de E13.5 embriones. Como se muestra en la Figura 1A, la proteína aislada de incluso una sola región de la válvula es suficiente para detectar una banda pSmad débil. Intensidad de la señal aumenta proporcionalmente con el número de regiones de la válvula que se agruparon. Es importante destacar que la relación pSmad / β-actina se...

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.

Proteína Aislamiento del desarrollo embrionario del corazón del ratón Válvula Región

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 vistas.  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.

Video: 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.

Inducción de la diferenciación endotelial en las células cardiacas progenitoras en suero bajo condiciones

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

Investigación

Biología del Desarrollo

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.

Aislamiento y cultivo de células embrionarias progenitoras aviar valvular

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

Biología

Aislamiento de núcleos de células progenitoras cardíacas de ratón con resolución de una sola célula

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.

Autor destacado: Aislamiento de núcleos de células progenitoras cardíacas de ratón para el perfil de epigenoma y expresión génica a resolución de una sola célula  

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.

Aislamiento de núcleos de células progenitoras cardíacas de ratón para epigenoma y perfil de expresión génica a resolución de una sola célula

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;

Aislar las células melanocitos como primarios del corazón alfombrillas

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.

Aislamiento, cultivo y caracterización funcional de ratón adulto 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 ...

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.

Aislamiento de células endoteliales embrionarias ventriculares

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.

Modelo In Vitro de Angiogénesis Coronaria

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

Medicina

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.

Aislamiento de las células murinas de la válvula endoteliales

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

Proteína Aislamiento del desarrollo embrionario del corazón del ratón Válvula Región

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