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: Tutti gli esperimenti sono stati approvati dalla cura e uso degli animali Comitato Istituzionale presso l&#39;Università del North Carolina a Chapel Hill. 1 Excise della valvola aortica <ol><li> Utilizzando una tecnica di eutanasia approvato per la fase embrionale di interesse, eutanasia di un mouse incinta con cuccioli al giorno embrionale desiderata (E) dello sviluppo. </li><li> Utilizzare etanolo al 70% per sterilizzare l&#39;addome della femmina incinta. Sollevare la pelle ei muscoli del basso addome alto e lontano dagli organi interni, e sezionare cavità inferiore addominale open femminile per consentire l&#39;accesso al corno uterino. </li><li> Per recuperare il corno uterino, tenere il collo dell&#39;utero con una pinza e caudale con attenzione tagliare le pinze. Sollevare il corno uterino, tagliando qualsiasi tessuto connettivo che tiene il corno in luogo, e completare la rimozione tagliando la giunzione del corno uterino con l&#39;ovidotto. </li><li> Posizionare il corno uterino sezionato in una capsula di Petri contenente cold 0.1 M tampone Tris (pH 7.6) e sciacquare come necessario. Quindi, tagliare il corno uterino longitudinalmente per esporre i sacchi embrionali aperto. </li><li> Per esporre l&#39;embrione, tagliare aprire un sacco embrionale allo svincolo della placenta e l&#39;embrione e tagliare i vasi ombelicali per liberare l&#39;embrione. Posizionare l&#39;embrione sezionato in un secondo piatto di Petri con freddo 0.1 M tampone Tris. Ritorna il primo piatto di Petri con i rimanenti embrioni undissected per ghiaccio fino pronto per la prossima embrione. </li><li> Per migliorare l&#39;accesso della parete toracica all&#39;interno dell&#39;embrione, decapitare il embrione. Se genotipizzazione è necessario, rimuovere e salvare un pezzo di tessuto in più in una provetta Eppendorf posti su ghiaccio. </li><li> Inserire l&#39;embrione sul dorso, e tagliare la parete toracica verticalmente lungo il lato della gabbia toracica, vicino a una zampa anteriore, e orizzontalmente sopra il diaframma di visualizzare il cuore. Quindi, aprire la parete toracica per esporre il cuore. </li><li> Utilizzando pinze per tenere alto lungo i grossi vasi, sollevare il cuore con pinze e tagliare i vasi di seguito. Iln, tagliare sopra la pinza per liberare il cuore. Se i vasi polmonari rimangono non tagliata, i polmoni possono essere rimossi con il cuore e devono essere rimosse prima di continuare. </li><li> Tagliare e rimuovere l&#39;arteria polmonare di sopra del livello della valvola; nelle fasi embrionali qui descritte, l&#39;arteria polmonare è leggermente opaca, che aiuta nella sua discriminazione dalla regione valvola. Tagliare appena sotto la valvola polmonare, evitando il miocardio ventricolare trabeculated e raccogliere come descritto al punto 1.10, se questa regione è anche di interesse. Qui e nella fase successiva, utilizzare un&#39;azione capillare per disegnare tampone Tris eccesso di distanza dalla regione di interesse prima di raccogliere in tampone di lisi. </li><li> Rimuovere accuratamente il distale dell&#39;aorta alla valvola aortica, appena sopra il livello della valvola; come l&#39;arteria polmonare, l&#39;aorta rimane leggermente opaca in queste fasi embrionali, aiutando nella sua rimozione. Tagliare appena sotto la valvola aortica, ancora una volta evitando il miocardio ventricolare trabeculated, e trasferire ilvalvola con pinze ad una provetta Eppendorf con tampone di lisi 2 microlitri (descritto nella tabella materiali) contenente inibitori di proteasi e fosfatasi sul ghiaccio. </li><li> Ripetere i passaggi 1,6-1,10 finché le valvole sono stati rimossi da tutti gli embrioni, raccogliendo ogni valvola in un tubo Eppendorf separata. Se tutti gli embrioni hanno lo stesso genotipo, tutte le valvole possono essere raccolte in un unico tubo Eppendorf. </li><li> Se genotipizzazione deve essere eseguito per determinare quali valvole di riunire insieme, immediatamente mettere i campioni in tampone di lisi a -80 ° C fino a nuovo uso. I campioni possono quindi essere raggruppati dopo genotipizzazione (punto 2.1.1). </li></ol> Estrazione 2 Proteine <ol><li> Scongelare tessuti raccolti su ghiaccio e centrifugare a 13.100 xg per 1 min per raccogliere i tessuti in un tampone di lisi sul fondo della provetta. <ol><li> Se sono stati genotipizzati embrioni, utilizzare una pipetta per raccogliere delicatamente e piscina di buffer di lisi contenente le regioni valvole da embrioni con genotipi simili. Per garantire un forte be, riunendo 3-4 regioni valvole da embrioni E13.5 per si raccomanda campione. </li><li> Se tutti gli embrioni erano dello stesso genotipo e tutte le regioni valvole sono stati raccolti insieme nel passo 1.10, lisare l&#39;intero campione, come descritto al punto 2.2. </li></ol></li><li> Controllare il volume del campione composito risultante e aggiungere tampone di lisi per un volume totale di 40 microlitri; poi, interrompere e omogeneizzare campioni utilizzando perline in acciaio inox 5 mm provette Eppendorf. Azionare il Lyser per 2-4 min a 50 Hz, come da istruzioni del produttore. Tubi Spin brevemente, (~ 13.100 xg per 1 minuto), e il trasferimento dei campioni ai nuovi tubi, lasciando dietro di sé i detriti insolubile. </li><li> Preparare e campioni separati per SDS-PAGE e trasferimento per la successiva analisi Western Blot, a seguito di un protocollo come Eslami et al 9. Blotting per una proteina di controllo di carico è essenziale per la quantificazione delle proteine. </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 capacità di quantificare i livelli di proteine ​​nei primi mesi del topo embrionale e pulcino regioni della valvola cardiaca fornisce un ulteriore strumento per la comprensione degli eventi di segnalazione cellulari critici per lo sviluppo della valvola. Il nostro protocollo qui descritto non differisce molto da procedure di isolamento delle proteine ​​standard. Tuttavia, modificando alcuni passaggi chiave, abbiamo ottenuto con successo proteine ​​fosforilate da estremamente campioni di piccole dimension...

Essere in grado di identificare e quantificare i livelli di espressione della proteina è una tecnica standard per ragioni sanitarie e di esperimenti basati su celle. Tuttavia, nonostante una lunga interesse per lo sviluppo della valvola cardiaca embrionale precoce, valutando l&#39;espressione della proteina in questo tessuto specifico durante lo sviluppo è attualmente limitato a immunoistochimica sia il pulcino e il mouse 1,2. Parte della difficoltà di quantificare l&#39;espressione della proteina nelle valvole di sviluppo della maggior parte degli organismi modello (ad esempio, ceci e topo) è la piccola dimensione delle valvole, che limita la quantità di proteine ​​che si possono ottenere. Così, per analisi quantitative, i ricercatori in genere si basano su l&#39;estrazione dell&#39;RNA e l&#39;amplificazione per la successiva PCR quantitativa o analisi microarray 2-5. Tuttavia, i livelli di espressione di RNA e proteine ​​non sono del tutto correlativo 6, in modo da concentrarsi su espressione di RNA non è in grado di fornire una rigorosa conto dei numerosi cambiamenti che si verificano in una determinata segnalazionepercorso in diversi momenti durante l&#39;evoluzione. Sulla base di questo limite nella metodologia attualmente disponibili, l&#39;obiettivo di questa procedura è stato quello di sviluppare un protocollo per ottenere in modo affidabile una quantità sufficiente di proteine ​​dalle embrionali di topo in via di sviluppo le regioni valvola cardiaca per l&#39;analisi quantitativa dei cambiamenti che si verificano in diverse vie di segnalazione che sono importanti in la maturazione di questo tessuto. Le valvole embrionali sono già comunemente dissezionati da topi per l&#39;isolamento di RNA e la successiva analisi di espressione genica 2-5. Tuttavia, questi studi si sono limitati a usare l&#39;espressione genica come out lettura di segnalare l&#39;attivazione della via, che non consente il rilevamento di rilevare modificazioni post-traduzionali di proteine ​​che possono influenzare la segnalazione a valle. Utilizzando le tecniche di isolamento dell&#39;RNA come punto di partenza, abbiamo iniziato con dissezione delle regioni di interesse. Perché il nostro interesse è stato rilevando proteine ​​fosforilate che erano indicativi di pat segnalazioneattività HWAY durante un periodo specifico di sviluppo della valvola aortica (E13.5-14.5), abbiamo effettuato tutte le dissezioni in tampone Tris senza fosfati e raccolto le valvole in un tampone di lisi a base di Tris con inibitori della proteasi e fosfatasi. Nel nostro caso specifico, solo le regioni valvola aortica sono stati raccolti, ma la regione valvola polmonare possono essere facilmente ottenuti allo stesso tempo. Le regioni valvole erano poi omogeneizzato e combinato con un tampone che viene attualmente utilizzato per studiare l&#39;espressione della proteina in valvole cardiache adulte 7. Utilizzando piccoli volumi di campione (ad esempio, 2 microlitri) e la messa in comune delle regioni valvole da embrioni con lo stesso genotipo, siamo stati in grado di rilevare le proteine ​​fosforilate e nonphosphorylated al giorno embrionale 13.5 8. Poiché le regioni di valvole possono essere congelati e conservati in tampone di lisi, gli embrioni possono essere genotipizzati se necessario prima di pooling. Questa tecnica amplia il set di strumenti che sono disponibili per la valutazione signalin cellulareg percorsi durante lo sviluppo e fornisce un complimento quantitativo di immunoistochimica, specificatamente per le valvole cardiache in via di sviluppo. Questa tecnica dovrebbe essere di beneficio non solo ai cardiologi di sviluppo, ma anche a tutti i biologi dello sviluppo che lavorano con gli embrioni in fase iniziale sono interessati nelle regioni che contengono tessuti limitata.

<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.

Utilizzando questa tecnica di preparazione, siamo stati in grado di rilevare fosforilata Smad 1,5,8 (pSmad) nelle singole regioni della valvola aortica da E13.5 embrioni. Come mostrato nella Figura 1A, la proteina isolata da anche una sola regione valvola è sufficiente per rilevare una banda pSmad debole. Intensità del segnale aumenta in proporzione al numero di regioni valvole che vengono pool. È importante sottolineare che il / rapporto β-actina pSmad rimane pressoché costante tra le d...

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.

Proteine ​​Isolamento dal Sviluppare embrionali di topo Cuore Valve Regione

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 Views.  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.

Induzione di differenziazione endoteliale in cellule progenitrici cardiache in condizioni di basso del siero

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

Ricerca

Biologia dello sviluppo

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.

Isolamento e la coltura di cellule embrionali aviaria progenitrici valvolare

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

Biologia

Isolamento dei nuclei da cellule progenitrici cardiache di topo con risoluzione a singola cellula

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.

Autore in primo piano: Isolamento di nuclei da cellule progenitrici cardiache di topo per la profilazione dell'epigenoma e dell'espressione genica con risoluzione di una singola cellula  

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.

Isolamento di nuclei da cellule progenitrici cardiache di topo per il profilo dell'epigenoma e dell'espressione genica a risoluzione di singole cellule

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;

Isolamento di cellule melanociti-come primario dal Cuore del mouse

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.

Isolamento, Cultura e caratterizzazione funzionale di topo 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.

Isolamento della embrionali ventricolari cellule endoteliali

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.

Modello in vitro dell'angiogenesi coronarica

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.

Isolamento di cellule murine endoteliali Valve

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

Proteine Isolamento dal Sviluppare embrionali di topo Cuore Valve Regione

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Induzione di differenziazione endoteliale in cellule progenitrici cardiache in condizioni di basso del siero

Isolamento e la coltura di cellule embrionali aviaria progenitrici valvolare

Isolamento di nuclei da cellule progenitrici cardiache di topo per il profilo dell'epigenoma e dell'espressione genica a risoluzione di singole cellule

Isolamento di nuclei da cellule progenitrici cardiache di topo per il profilo dell'epigenoma e dell'espressione genica a risoluzione di singole cellule

Isolamento di nuclei da cellule progenitrici cardiache di topo per il profilo dell'epigenoma e dell'espressione genica a risoluzione di singole cellule

Isolamento di cellule melanociti-come primario dal Cuore del mouse

Isolamento, Cultura e caratterizzazione funzionale di topo adulto Cardiomyoctyes

Isolamento della embrionali ventricolari cellule endoteliali

Modello in vitro dell'angiogenesi coronarica

Isolamento di cellule murine endoteliali Valve

Proteine ​​Isolamento dal Sviluppare embrionali di topo Cuore Valve Regione

Riepilogo

Esplora Altri Video

Capitoli in questo video

Induzione di differenziazione endoteliale in cellule progenitrici cardiache in condizioni di basso del siero

Isolamento e la coltura di cellule embrionali aviaria progenitrici valvolare

Isolamento di nuclei da cellule progenitrici cardiache di topo per il profilo dell'epigenoma e dell'espressione genica a risoluzione di singole cellule

Isolamento di nuclei da cellule progenitrici cardiache di topo per il profilo dell'epigenoma e dell'espressione genica a risoluzione di singole cellule

Isolamento di nuclei da cellule progenitrici cardiache di topo per il profilo dell'epigenoma e dell'espressione genica a risoluzione di singole cellule

Isolamento di cellule melanociti-come primario dal Cuore del mouse

Isolamento, Cultura e caratterizzazione funzionale di topo adulto Cardiomyoctyes

Isolamento della embrionali ventricolari cellule endoteliali

Modello in vitro dell'angiogenesi coronarica

Isolamento di cellule murine endoteliali Valve

Proteine Isolamento dal Sviluppare embrionali di topo Cuore Valve Regione