Name: in vitro culture of epicardial cells from mouse embryonic heart
Uploaded: 2016-04-27T14:00:00.000+00:00
Duration: 6 min 31 s
Description: Watch this Scientific Journal Video about In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart at JoVE.com

Questo lavoro è stato sostenuto da fondi da Duke-NUS Graduate Medical School di Singapore, Goh fondazione e Singapore NRF borsa di studio (NRF-NRFF2016-01) per Manvendra K. Singh.

Tutti gli esperimenti sono stati approvati dal Comitato Istituzionale cura degli animali e Usa Duke-NUS Graduate Medical School. 1. Recupero embrionale Ventricoli <ol><li> Sacrifica un topo in stato di gravidanza a tempo allo stadio embrionale desiderato (E11.5 o E12.5) utilizzando una camera di eutanasia alla fornitura del gas di biossido di carbonio o un altro metodo di eutanasia approvato. </li><li> Posizionare il mouse sul dorso su un tavolo anatomico. Disinfettare l&#39;addome della femmina con il 70% di etanolo. Sollevare la pelle sopra la pancia e fare una piccola incisione (1-2 cm) con una lama, quindi tenere la pelle con entrambe le mani e tirare via per esporre la parete addominale. </li><li> Ora tagliare la parete addominale per esporre il corno uterino. Utilizzare pinza sterile per sollevare il corno uterino e con attenzione tagliare i tessuti grassi e dei vasi sanguigni collegati al corno uterino. Tagliare al collo dell&#39;utero per recuperare l&#39;utero. </li><li> Posizionare il corno uterino in una capsula di Petri contenente sterile freddo 1x fosfato Buff<html> ered Saline (PBS) e risciacquare delicatamente. Porre la capsula di Petri sul ghiaccio. </li><li> Usare le forbici per tagliare la linea mediana del corno uterino (di fronte al luogo in cui si trova la placenta) per esporre gli embrioni che sono ancora dentro il sacco vitellino e attaccati alla parete uterina attraverso la placenta. </li><li> Utilizzando pinze sterili, tagliare aperto il sacco vitellino embrionali per liberare l&#39;embrione. </li><li> Posizionare l&#39;embrione in un nuovo piatto di Petri contenente sterile freddo 1x PBS. Decapitare l&#39;embrione e posizionarlo sul dorso. Tagliare aperto la parete toracica per esporre il cuore. </li><li> Utilizzare pinze sterili per sollevare il cuore e tagliare via i vasi intorno ad esso per liberare il cuore dalla parete toracica. Prestare particolare attenzione a non danneggiare l&#39;epicardio del cuore con gli strumenti chirurgici. </li><li> Tagliare il tratto di efflusso ed entrambi atri. Trasferire il ventricolo di un altro piatto con 1x PBS freddo. Porre la capsula sul ghiaccio. </li><li> Ripetere i passaggi 1,6-1,9 fino a che tutti i ventricoli vengono recuperati. </li></ol>Le &quot;&gt; 2. epicardico espianto Nota: Culture questi espianti epicardici sia su vetrini da camera di vetro o su gel di collagene per applicazioni a valle. <ol><li> Vetro Camera Slides <ol><li> Per preparare il terreno di coltura, aggiungere il 10% di siero fetale bovino (FBS), 1% di penicillina / streptomicina e 2 ng / ml il fattore di crescita dei fibroblasti ricombinante 2 (FGF2) per mezzo di Dulbecco modificato Eagle (DMEM). Eseguire tutti i passaggi della cappa a flusso laminare. </li><li> Aggiungere 500 microlitri terreni di coltura a ciascun pozzetto di una camera 8 pozzetti. </li><li> Mettete ogni ventricolo asportato al centro di un pozzo. Orientare il ventricolo per mantenere il lato sezionato sulla superficie inferiore del pozzetto. </li><li> Delicatamente la piastra a 37 ° C, 5% CO 2 incubatore di coltura cellulare. </li><li> Mantenere la cultura con il minimo disturbo per permettere gli espianti di aderire. La formazione del monostrato epicardico può essere osservata dopo 48-72 ore. </li><li> Per la differenziazione di epicardcellule IAL in cellule muscolari lisce, la cultura le cellule monostrato epicardici in mezzi di differenziazione per altri 6 giorni. Per preparare i mezzi di differenziazione, aggiungere il 10% FBS, 1% di penicillina / streptomicina e 50 ng / ml ricombinante fattore di crescita trasformante beta 1 (TGF-β1) per DMEM. </li><li> Cambiare il terreno di coltura a giorni alterni. </li></ol></li><li> gel di collagene <ol><li> Preparare il gel di collagene in una piastra a 96 pozzetti utilizzando un kit commerciale 3D collagene cultura secondo il protocollo del produttore. </li><li> Per preparare il volume desiderato di gel di collagene, mescolare la quantità appropriata di soluzione di collagene con 5x di DMEM e pipetta su e giù. La soluzione dovrebbe diventare giallo. </li><li> Aggiungere un corrispondente volume di soluzione di neutralizzazione e miscelare bene immediatamente pipettando su e giù. La soluzione diventerà rosa. </li><li> Pipettare 100 ml di questa miscela in ciascun pozzetto di una piastra a 96 pozzetti. Posizionare la piastra a 37 ° C, 5% CO 2 incubatore per 30-60 min ad albasso il gel di polimerizzare. </li><li> Aggiungere 200 ml di terreni di coltura descritti sopra per ciascun pozzetto. </li><li> Mettete ogni ventricolo asportato al centro di un pozzo. Orient ventricolo per mantenere il lato sezionato giù sul gel di collagene (apice del ventricolo deve affrontare sperimentatore). Delicatamente la piastra posteriore in incubatrice coltura cellulare. </li><li> Dopo 3 giorni, rimuovere i ventricoli. Mettere la piastra posteriore nell&#39;incubatore coltura cellulare per altri 2 giorni. </li><li> Cambiare il terreno di coltura a giorni alterni. </li></ol></li></ol> 3. Fissazione e Visualizzazione Nota: le cellule epicardici possono essere visualizzati sia su vetrini da camera di vetro o gel di collagene. <ol><li> Aspirare i terreni di coltura e aggiungere un uguale volume di sterili 1x PBS per lavare le cellule. </li><li> Aggiungere appena sufficiente 4% paraformaldeide (PFA) per coprire le cellule. Fissare le cellule per 10 minuti a 4 ° C. </li><li> Rimuovere PFA e lavare espianti con 1x PBST (PBS 1x con0,1% triton X-100) per permeabilize cellule. </li><li> Immunostain con l&#39;anticorpo desiderato (ad esempio Alexa Fluor 488-falloidina; ZO-1, α-tubulina e α-actina del muscolo liscio) 7. </li></ol>

<ol>
	<li>Mikawa, T., Fischman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+analysis+of+cardiac+morphogenesis:+discontinuous+formation+of+coronary+vessels.">Retroviral analysis of cardiac morphogenesis: discontinuous formation of coronary vessels.</a> Proc Natl Acad Sci U S A. 89, 9504-9508 (1992).</li><li>Mikawa, T., Gourdie, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericardial+mesoderm+generates+a+population+of+coronary+smooth+muscle+cells+migrating+into+the+heart+along+with+ingrowth+of+the+epicardial+organ.">Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ.</a> Dev Biol. 174, 221-232 (1996).</li><li>Manner, J., Perez-Pomares, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardium-derived+cardiac+mesenchymal+stem+cells:+expanding+the+outer+limit+of+heart+repair.">Epicardium-derived cardiac mesenchymal stem cells: expanding the outer limit of heart repair.</a> Circ Res. 110, 904-906 (2012).</li><li>Manner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+study+on+the+formation+of+the+epicardium+in+chick+embryos.">Experimental study on the formation of the epicardium in chick embryos.</a> Anat Embryol (Berl). 187, 281-289 (1993).</li><li>Manner, J., Schlueter, J., Brand, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+analyses+of+the+function+of+the+proepicardium+using+a+new+microsurgical+procedure+to+induce+loss-of-proepicardial-function+in+chick+embryos.">Experimental analyses of the function of the proepicardium using a new microsurgical procedure to induce loss-of-proepicardial-function in chick embryos.</a> Dev Dyn. 233, 1454-1463 (2005).</li><li>Pennisi, D. J., Ballard, V. L., Mikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardium+is+required+for+the+full+rate+of+myocyte+proliferation+and+levels+of+expression+of+myocyte+mitogenic+factors+FGF2+and+its+receptor,+FGFR-1,+but+not+for+transmural+myocardial+patterning+in+the+embryonic+chick+heart.">Epicardium is required for the full rate of myocyte proliferation and levels of expression of myocyte mitogenic factors FGF2 and its receptor, FGFR-1, but not for transmural myocardial patterning in the embryonic chick heart.</a> Dev Dyn. 228, 161-172 (2003).</li><li>Gittenberger-de Groot, A. C., Vrancken Peeters, M. P., Bergwerff, M., Mentink, M. M., Poelmann, R. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocardial+and+epicardial+derived+FGF+signals+regulate+myocardial+proliferation+and+differentiation+in+vivo.">Endocardial and epicardial derived FGF signals regulate myocardial proliferation and differentiation in vivo.</a> Dev Cell. 8, 85-95 (2005).</li><li>Merki, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+retinoid+X+receptor+alpha+is+required+for+myocardial+growth+and+coronary+artery+formation.">Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation.</a> Proc Natl Acad Sci U S A. 102, 18455-18460 (2005).</li><li>Stuckmann, I., Evans, S., Lassar, A. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C/EBP+transcription+factors+mediate+epicardial+activation+during+heart+development+and+injury.">C/EBP transcription factors mediate epicardial activation during heart development and injury.</a> Science. 338, 1599-1603 (2012).</li><li>Zhou, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mouse+epicardium+modulates+myocardial+injury+by+secreting+paracrine+factors.">Adult mouse epicardium modulates myocardial injury by secreting paracrine factors.</a> J Clin Invest. 121, 1894-1904 (2011).</li><li>Christoffels, V. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+endothelial+cells+express+Wilms'+tumor-1:+Wt1+expression+in+the+developing,+adult+and+infarcted+heart.">Cardiac endothelial cells express Wilms' tumor-1: Wt1 expression in the developing, adult and infarcted heart.</a> J Mol Cell Cardiol. 81, 127-135 (2015).</li><li>Iyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+derivation+of+epicardium+and+its+differentiated+smooth+muscle+cell+progeny+from+human+pluripotent+stem+cells.">Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells.</a> Development. 142, 1528-1541 (2015).</li><li>Witty, A. 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The authors declare that they have no competing financial interests.

È fondamentale per sviluppare tecniche che facilitano lo studio della epicardio per soddisfare la crescente importanza della epicardio in sviluppo cardiaco e rigenerazione. Il sistema di coltura epicardico pone notevoli vantaggi per la ricerca epicardico. Un modo alternativo per isolare le cellule epicardial è usare fluorescenza attivato cell sorting (FACS). Questo metodo si basa sull&#39;utilizzo di marcatori epicardici (o-specifica epicardio cellule transgenici espressione di una protein...

Un patrimonio di dati sperimentali hanno mostrato che l&#39;epicardio influenza passaggi critici nello sviluppo cardiaco. Durante lo sviluppo, la transversum setto dà luogo ad un gruppo di cellule mesoteliali noto come proepicardium 1-4. Le cellule del proepicardium poi migrano e la busta miocardio formare il epicardio. In seguito, un sottogruppo di cellule epicardici subiscono EMT dando luogo ad una popolazione di cellule migratoria epicardio-derivato (EPDCs) che poi invadono il miocardio. Genetica così come retrovirale lignaggio tracciando esperimenti hanno dimostrato che EPDCs differenziarsi in vari lignaggi comprese le cellule muscolari lisce, i fibroblasti, cellule endoteliali e cardiomiociti (se presenti). Pertanto EPDCs contribuiscono significativamente allo sviluppo del sistema vascolare coronarico e architettura miocardica 1,2,4-9. Inoltre, l&#39;epicardio è essenziale per lo sviluppo dello strato compatto ventricolare 10-12. per postaxample Gittenberger-de Groot et al. hanno dimostrato che inibendo la conseguenza del proepicardium porta ad una serie di difetti come un miocardio sottile, loop carente del cuore e anormale formazione setto interventricolare e, di conseguenza, embrionali letalità 13. fattori paracrini secreti dal dell&#39;epicardio embrionale modulano la proliferazione dei cardiomiociti e differenziazione. Coerentemente con questo, specifici per epicardium cancellazione dei percorsi di segnalazione come l&#39;acido retinoico (RA), i fattori di crescita dei fibroblasti (FGF) e Wnt / β-catenina ha provocato la crescita del miocardio difettoso e letalità embrionale 14-16. Anche se il epicardio è stato creduto di essere quiescente nei cuori adulti, recenti studi hanno dimostrato che il programma di sviluppo si riattiva nel epicardio seguente lesione cardiaca 17,18. Dopo l&#39;attivazione, le cellule subiscono una rapida proliferazione e EMT che si traducono in formazione EPDC. Queste cellule presentano la capacity di differenziarsi in fibroblasti e cellule muscolari lisce, ma non cardiomiociti e cellule endoteliali 18. Inoltre, i EPDCs secernono fattori pro-angiogenici che gli aiuti nella vascolarizzazione della zona danneggiata e quindi facilitare il miglioramento della funzione cardiaca, riducendo la dimensione dell&#39;infarto. A causa di questi risultati, l&#39;epicardio ha guadagnato interesse per lo studio dello sviluppo cardiovascolari, malattie e rigenerazione. tecnologia transgenica ha rivoluzionato la ricerca medica nel 21 ° secolo. Con l&#39;aiuto di tecnologie transgeniche, modelli di malati di topo che mimano la condizione umana metabolicamente e fisiopatologico sono stati sviluppati con successo. Tuttavia, lo studio del comportamento delle cellule epicardico in questi mutanti è stata una sfida dovuta principalmente ai primi di letalità embrionale. Considerando il ruolo significativo che la epicardio gioca nello sviluppo cardiaco e la rigenerazione, abbiamo istituito un sistema di coltura in vitro per epicardia del mouseL cellule. Questo metodo permette la coltura a lungo termine delle cellule epicardici e facilita lo studio dettagliato delle due proprietà importanti dell&#39;epicardio: la sua capacità di migrare e differenziarsi. I ventricoli escisse dal mouse potrebbero essere coltivati ​​in gel di collagene che possono essere utilizzati per condurre saggi di migrazione. Essendo coltivate in una matrice 3D che replica la matrice extracellulare ricca di collagene dello strato subepicardico meglio riassume il vivo fisiologia cellulare in. In alternativa possono essere coltivate su vetrini da camera per stabilire un monostrato epicardico che può quindi essere utilizzato per una varietà di applicazioni a valle. Questo monostrato può essere utilizzato per colorare per proteine ​​di giunzione stretti che forniranno approfondimenti sulla capacità del epicardio sottoporsi EMT che è cruciale per la migrazione. Inoltre, esperimenti differenziazione possono essere effettuati anche su queste cellule. Inoltre, profilo di espressione genica può essere analizzata per estrarre l&#39;RNA dalle cellule el&#39;esecuzione di reazione a catena della polimerasi quantitativa (qPCR). Infine, i monostrati potrebbero anche essere trattati con agenti seguita da un&#39;analisi molecolare per verificare le potenziali terapie. Mettere insieme, questo sistema di coltura epicardico ci offre la possibilità di visualizzare e raccogliere dati molecolare che favorisce la nostra comprensione dello sviluppo epicardico. Un&#39;altra caratteristica desiderabile di questo metodo è che è semplice e non richiede alcuna configurazione elaborata. In breve, gli embrioni vengono raccolte al E11.5 E12.5 o in seguito alla quale il cuore è asportato. I ventricoli sono poi coltivate su entrambi i gel di collagene o diapositive da camera. Successivamente, queste cellule possono essere usate per condurre esperimenti a valle.

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

in vitro culture of epicardial cells from mouse embryonic heart

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

Usando questo protocollo la cultura, le cellule epicardial primarie possono essere isolate con elevata purezza per le applicazioni a valle. Le cellule coltivate sono in grado di subire EMT, migrare e differenziarsi come cellule epicardial fare in vivo. Per determinare la purezza della nostra cultura di cellule epicardico primarie, abbiamo analizzato gli espianti epicardici generati da Sema3d GFPCre /...

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

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

Coltura in vitro di cellule provenienti da epicardici Cuore embrionali di topo

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

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

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Research

JoVE Journal

Engineering

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

7.3K Views.  Duke-NUS Graduate Medical School. The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells. 

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

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

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

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

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

Epicardico escrescenza Cultura Assay e<em&gt; Ex Vivo</em&gt; La valutazione della migrazione delle cellule di derivazione epicardico

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

Ricerca

Biologia dello sviluppo

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

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

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

L'isolamento e coltura di cellule primarie dell'epicardio ricavate da campioni di cuore umano adulto e fetale

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

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

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

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

Generazione in vitro di cellule progenitrici cardiache specifiche del campo del cuore del mouse

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

Transgenesi basata sull'elettroporazione di cellule cardiache in cuori di topo

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

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

Autore in primo piano: Transgenesi delle cellule cardiache per lo screening genico rapido

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

Un approccio efficiente alla transgenesi per la consegna genica nel cuore embrionale di topo

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

Isolating Primary Melanocyte-like Cells from the Mouse Heart

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

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

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 ...

Biologia

Isolation and Culture of Neonatal Mouse Cardiomyocytes

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

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

Isolamento e Cultura della neonatali mouse Cardiomyocytes

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

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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

A Novel<em&gt; Ex vivo</em&gt; Metodo di Cultura per il mouse embrionale del cuore

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

Isolation of Embryonic Ventricular Endothelial Cells

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

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

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 ...

Coltura in vitro di cellule provenienti da epicardici Cuore embrionali di topo

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Epicardico escrescenza Cultura Assay e

L'isolamento e coltura di cellule primarie dell'epicardio ricavate da campioni di cuore umano adulto e fetale

Generazione in vitro di cellule progenitrici cardiache specifiche del campo del cuore del mouse

Un approccio efficiente alla transgenesi per la consegna genica nel cuore embrionale di topo

Un approccio efficiente alla transgenesi per la consegna genica nel cuore embrionale di topo

Un approccio efficiente alla transgenesi per la consegna genica nel cuore embrionale di topo

Isolamento di cellule melanociti-come primario dal Cuore del mouse

Isolamento e Cultura della neonatali mouse Cardiomyocytes

A Novel

Isolamento della embrionali ventricolari cellule endoteliali