Name: ex vivo culture of pharyngeal arches to study heart and muscle progenitors and their niche
Uploaded: 2015-07-20T15:00:00.000+00:00
Duration: 7 min 4 s
Description: Watch this Scientific Journal Video about Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche at JoVE.com

We thank Kwon laboratory members for helpful discussions. This work was supported by grants from NHLBI/NIH (R01HL111198) and Maryland Stem Cell Research Fund (MSCRF).

Tutti i topi sono stati mantenuti a una Associazione americana per l&#39;accreditamento di laboratorio Animal Care (AAALAC) -accredited stabulario dell&#39;Università Johns Hopkins e alloggiati in conformità con le procedure descritte nella Guida per la cura e l&#39;uso di animali da laboratorio. La cura e l&#39;uso degli animali Comitato Istituzionale (IACUC) ha approvato tutti i protocolli sperimentali. Preparazione 1. sperimentale <ol><li> Coat 12-pozzetti con 10% siero fetale bovino (FBS) in tampone fosfato Solution (PBS) per 60 min. </li><li> Rimuovere la soluzione di rivestimento e aggiungere 200 ml di media siero-free (SFM). Twirl piastra per assicurarsi che un film di supporto copre l&#39;intera superficie. </li></ol> 2. Procedure chirurgiche <ol><li> Euthanize topi in gravidanza che utilizzano il protocollo comitato approvato la cura degli animali dell&#39;istituzione seguita da dislocazione cervicale per garantire la completa eutanasia. </li><li> Spray e pulire la regione pancia del mouse con il 70%etanolo. Mantenere rigorose tecniche sterili per evitare la contaminazione. </li><li> Utilizzare pinze e forbici per rendere l&#39;0,5 cm 2 incisione alla regione ombelicale. </li><li> Usare le dita per pizzicare / afferrare la pelle sopra e sotto l&#39;incisione e tirare delicatamente la pelle in direzioni opposte (testa e coda). </li><li> Utilizzare pinze e forbici per fare un&#39;incisione iniziale nella membrana in corrispondenza della zona dell&#39;ombelico. Tagliare delicatamente la membrana a forma di v dall&#39;ombelico alle ovaie su ciascun lato (2 x 1,5 cm 2), rivelando così l&#39;utero. </li><li> Utilizzare pinze per pizzicare la ovidotto che collega l&#39;utero per l&#39;ovaio e forbici per pizzicare il ovidotto sul lato ovaio, liberando così l&#39;utero dall&#39;ovaio. </li><li> Tirare delicatamente l&#39;utero dal ovidotto con le pinze e tagliare l&#39;utero libera dalla regione della vescica con le forbici. </li><li> Estrarre l&#39;utero ulteriormente dalla oviduct con la pinza, e tagliare il oviduct con le forbici sull&#39;altro lato, liberando così l&#39;utero dal mouse. </li><li> Trasferire l&#39;utero di un tubo da 50 ml e aggiungere 20 ml di freddo PBS sterile con Ca 2 + e Mg 2+. PBS deve contenere Ca 2+ e Mg 2+ durante embrione dissezione. </li><li> Agitare delicatamente il tubo per 10 secondi per lavare l&#39;utero di sangue. </li><li> Trasferire l&#39;utero dal tubo con una pinza e posizionarlo in un piatto da 10 ml e aggiungere 5 ml di PBS freddo sulla parte superiore dell&#39;utero. </li><li> Porre la capsula con l&#39;utero allo stereomicroscopio. Nota: I passaggi 2,13-2,19 richiede uno stereomicroscopio. </li><li> Utilizzare due coppie di pinze per aprire delicatamente l&#39;utero e sezionare fuori cavità amniotiche con embrioni dall&#39;utero uno per uno. <ol><li> Utilizzare due coppie di pinze per aprire con attenzione il sacco amniotico che circonda l&#39;embrione, pizzicare la sacca con una pinza e delicatamente rimuoverlo dal embrione, utilizzare le altre pinze per tagliare e rilasciare il sacco amniotico dall&#39;embrione. Se un genotipo specifico necessario, tenere sacco amniotico per la genotipizzazione. </li></ol></li><li> Posizionare l&#39;embrione sul lato e utilizzare pinze per tagliare il 1 ° e 2 ° arco posteriormente al cuore tra l&#39;arco e la sacca faringea (Figura 1A &#39;&#39;). </li><li> Capovolgere delicatamente l&#39;embrione verso l&#39;altro lato e utilizzare pinze per tagliare il 1 ° e 2 ° arco posteriormente al cuore tra l&#39;arco e la sacca faringea. </li><li> Utilizzare pinze per tagliare il tratto di efflusso cardiaco che ancora collega il 1 ° e 2 ° faringei archi per l&#39;embrione. Rimuovere gli archi che sono ancora attaccati insieme nella forma di una farfalla (Figura 1B), dall&#39;embrione. </li><li> Utilizzare pinze per stringere e rilasciare il 1 ° archi faringei dal rimanente tratto di efflusso cardiaco e foregut endoderma (Figura 1B &#39;&#39; indicata con la linea tratteggiata e *). </li><li> Utilizzare pinze per pizzicare e rilasciare gli archi 2 ° faringei dal rimanente tratto di efflusso cardiacoe foregut endoderma (Figura 1B &#39;&#39; indicata con la linea tratteggiata e **). </li><li> Trasferire ogni coppia di archi separatamente utilizzando una pinza e inserire entrambe le arcate nel bel mezzo di un designato bene. Piastra 1 ° e 2 ° faringei archi in pozzetti separati. Assicurarsi che gli archi sono in contatto con la pellicola di supporto aggiunti nel passaggio 1.2. È fondamentale che gli archi non sono coperti da medium, in quanto hanno bisogno di entrare in contatto con la superficie del pozzetto per attacco durante questo periodo. </li></ol> 3. Incubazione e Imaging <ol><li> Incubare archi placcati in 12-pozzetti in 37 ° C / 5% di CO 2 per 2 ore per archi di allegare alla superficie bene. </li><li> Aggiungere delicatamente 200 ml di 37 ° C SFM lungo il lato del pozzo. Assicurarsi che gli archi rimangono attaccati e incubare O / N. </li><li> Il giorno dopo, controllare visivamente che archi faringei sono attaccati e aggiungere delicatamente 200 ml di 37 ° C SFM lungo il lato dellabene. Se archi restano distaccato e variabile, rimuovere delicatamente i media con una pipetta senza rimuovere archi fino a che solo un film di supporto è a sinistra. Utilizzare puntale per posizionare archi al centro del pozzo e ripetere il punto 3.1. </li><li> Monitorare quotidianamente; aggiungere ~ 100-200 ml di mezzi ogni 2 giorni per sostituire qualsiasi supporto evaporato. Nota: 24-48 ore dopo la migrazione delle cellule appaiono intorno l&#39;arco e si proliferare e migrare da arco per i prossimi 5-8 giorni. Dopo 36-72 ore la formazione di cardiomiociti battendo può essere osservato dal 2 faringeo arco (Figura 2B). Formazione myotube può osservare dal 1 ° arco faringeo 3-7 giorni dopo il collegamento (Figura 2A). </li></ol>

<ol>
	<li>Kelly, 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=The+second+heart+field.">The second heart field.</a> Curr Top Dev Biol. 100, 33-65 (2012).</li><li>Bruneau, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+genetics+of+congenital+heart+disease.">The developmental genetics of congenital heart disease.</a> Nature. 451, 943-948 (2008).</li><li>Tzahor, E., Evans, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharyngeal+mesoderm+development+during+embryogenesis:+implications+for+both+heart+and+head+myogenesis.">Pharyngeal mesoderm development during embryogenesis: implications for both heart and head myogenesis.</a> Cardiovasc Res. 91, 196-202 (2011).</li><li>Uosaki, H., 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=Direct+Contact+with+Endoderm-like+Cells+Efficiently+Induces+Cardiac+Progenitors+from+Mouse+and+Human+Pluripotent+Stem+Cells.">Direct Contact with Endoderm-like Cells Efficiently Induces Cardiac Progenitors from Mouse and Human Pluripotent Stem Cells.</a> PLoS One. 7 (10), e46413 (2012).</li><li>Cheng, P., 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=Fibronectin+mediates+mesendodermal+cell+fate+decisions.">Fibronectin mediates mesendodermal cell fate decisions.</a> Development. 140, 2587-2596 (2013).</li><li>Kwon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+pathway+involving+Notch1/beta-catenin/Isl1+determines+cardiac+progenitor+cell+fate.">A regulatory pathway involving Notch1/beta-catenin/Isl1 determines cardiac progenitor cell fate.</a> Nature cell biology. 11, 951-957 (2009).</li><li>Shenje, L. T., 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=Precardiac+deletion+of+Numb+and+Numblike+reveals+renewal+of+cardiac+progenitors.">Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors.</a> Elife. 3, e02164 (2014).</li><li>Grevellec, A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharyngeal+pouches+and+clefts:+Development,+evolution,+structure+and+derivatives.">The pharyngeal pouches and clefts: Development, evolution, structure and derivatives.</a> Semin Cell Dev Biol. 21, 325-332 (2010).</li><li>Kwon, C., Cordes, K. R., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+acts+at+multiple+developmental+stages+to+promote+mammalian+cardiogenesis.">Wnt/beta-catenin signaling acts at multiple developmental stages to promote mammalian cardiogenesis.</a> Cell Cycle. 7, 3815-3818 (2008).</li><li>Cho, G. S., Fernandez, L., Kwon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+heart:+perspectives+on+stem-cell+therapy.">Regenerative medicine for the heart: perspectives on stem-cell therapy.</a> Antioxid Redox Signal. 21, 2018-2031 (2014).</li></ol>

The authors have nothing to disclose.

In questo video, dimostriamo come isolare e cultura prima e seconda faringei archi di 9,5 giorni antichi embrioni di topo. Archi branchiali sono transitori, rigonfiamenti che appaiono sul lato craniolateral di embrioni in via di sviluppo 9, che contengono blocchi cardiaco progenitrici delle cellule di costruzione multi-potenti per rendere il cuore durante l&#39;embriogenesi 10,11 -in secondo arco e progenitori testa muscolari prime arcate 8 segmentati . La dissezione di archi faringei richiede competenze avanzate di dissezione del mouse embrione. Una volta che gli archi sono stati sezionati con successo utilizzando questo protocollo graduale e trasferito pozzi di pre-rivestite, è fondamentale che si inarca attribuiscono alla zona inferiore. In questo protocollo, abbiamo pozzi cappotto con il 10% FBS seguiti da cultura in SFM con conseguente attaccamento e la crescita del&gt; 75% di archi, ma altri materiali di rivestimento possono essere utilizzati (matrigel, gelatina, fibronectina ecc) così come conta sierosupporti ining. Gli archi faringei serve come primordi per una moltitudine di strutture durante lo sviluppo. Dopo la formazione iniziale degli archi faringei (8 giorni dopo la fecondazione nei topi), CPC espandono nella PA2 e migrano nel tratto di efflusso cardiaco dove si differenziano in cellule cardiache, e rappresenta pertanto una fonte rinnovabile di CPC che fornisce cellule necessarie per sostenere il cuore la crescita. Come gli archi faringei sono strutture temporali dello sviluppo (E8-11.5) e la migrazione del CPC nel tratto di efflusso avviene da circa E8-E10) questo comporta alcune limitazioni alla tecnica. Per gli studi di CPC, si consiglia di utilizzare archi faringei sezionati tra E9-10, in questa fase ogni PA2 contenere circa 5-800 RFP + cellule (Mesp1 traccia linage). Con marcatura fluorescente, questa cultura ex vivo ci permette di monitorare progenitori in via di sviluppo e la loro differenziazione in cardiomiociti battendo o miotubi in tempo reale. Inoltre, questo sistema di coltura ex vivo può essere usato per studiare i ruoli di fattori cellula-autonomi e microambientali influenzano la proliferazione cardiaca / progenitore muscolo cellulare, la migrazione e la differenziazione utilizzando linee Cre / loxP esistenti. Come tale, questo sistema dovrebbe facilitare gli studi progenitore-nicchia cardiaci / muscolari, che possono condurre ad una migliore comprensione del cuore formazione / muscolare e malattie.

Cellule mesoderma faringei danno origine a parti del cuore e dei muscoli faringei. Durante lo sviluppo embrionale, le cellule progenitrici multipotenti cardiache dal secondo campo cuore migrano dal mesoderma faringeo e popolano il tratto di efflusso cardiaco e ventricolo destro, e il loro sviluppo anormale è strettamente associata con la malattia-cuore congenita principale causa di difetti alla nascita e nascita-difetto decessi correlati nell&#39;uomo 1-3. Recenti studi hanno dimostrato che faringea mesoderma contribuisce a testa muscoli, oltre al cuore, rendendo il mesoderma una parte critica della cardio-craniofacciale sviluppo 4. Così, i processi di sviluppo, tra cui l&#39;induzione, la proliferazione e la differenziazione di testa e del cuore progenitori del mesoderma faringea sono sotto inchiesta attivi 5-7. Fino a poco tempo fa, è rimasto sconosciuto se le cellule progenitrici cardiache subiscono espansione without differenziazione, in parte a causa della mancanza di informazioni sul loro ambiente cellulare. Il nostro recente studio suggerisce che archi branchiali fungono da microambiente per il rinnovo dei progenitori cardiaci e muscolari e possono essere coltivate ex vivo per diverse settimane 8. Questo metodo espianto offre un&#39;opportunità nuova e unica per studiare lo sviluppo dei progenitori cardio-cranio-facciale ex vivo.

<table><tbody><tr><td>FBS</td><td>Hyclone</td><td>SH30071.03</td><td></td></tr><tr><td>PBS + Ca&lt;sub&gt;2&lt;/sub&gt; + Mg&lt;sub&gt;2&lt;/sub&gt;</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>10 cm Petri plates</td><td>Fishersci</td><td>FB0875712</td><td></td></tr><tr><td>Scissor</td><td></td><td></td><td></td></tr><tr><td>Dissection forceps</td><td></td><td></td><td></td></tr><tr><td>Serum Free Media:</td><td></td><td></td><td></td></tr><tr><td>IMDM</td><td>Cellgro</td><td>15-016-CV</td><td></td></tr><tr><td>Ham&amp;rsquo;s F12</td><td>Cellgro</td><td>10-080-CV</td><td></td></tr><tr><td>N2-SUPPLEMENT</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>B27-retinoic acid</td><td>Gibco</td><td>12587-010</td><td></td></tr><tr><td>10% BSA (in PBS) (Invitrogen Cat#. P2489)</td><td>Life Sciences</td><td>P2489</td><td></td></tr><tr><td>100x Glutamine (Gibco Cat.# 25030-081)</td><td>Gibco</td><td>35050-61</td><td></td></tr><tr><td>100x Pen/Strep (Gibco Cat#. 15070-063)</td><td>Gibco</td><td>15140-122</td><td></td></tr></tbody></table>

ex vivo culture of pharyngeal arches to study heart and muscle progenitors and their niche

Il mesoderma faringeo di embrioni in via di sviluppo contribuisce ad ampie regioni della testa e la muscolatura cardiaca. Abbiamo sviluppato un nuovo metodo per studiare la testa e il cuore lo sviluppo delle cellule progenitrici con archi faringei (noto anche come archi branchiali) ex vivo. Utilizzando questo metodo, abbiamo recentemente descritto che il secondo arco faringeo contiene progenitori cardiaci auto-rinnovamento e serve come un microambiente per l&#39;espansione dei progenitori durante lo sviluppo del cuore del mouse. Le cellule progenitrici rimangono indifferenziate ed espansivo dentro l&#39;arco, ma rapidamente diventano cardiomiociti funzionali mentre migrano fuori dell&#39;arco. Abbiamo anche segnalato che il primo arco faringeo contiene progenitori muscolari che danno luogo a miotubi dopo aver lasciato l&#39;arco. Qui, dimostriamo la procedura per la dissezione e ex vivo cultura del primo e del secondo archi faringei da embrioni di topo in via di sviluppo. Il metodo permette di studiare la testa e il cuore progenitore / dev muscolareelopment, compresi cardiomiociti e myotube formazione in dettaglio ex vivo.

Durante lo sviluppo, muscolo e cuore progenitori facciali possono essere rintracciati in quanto proliferano e migrano dal 1 ° e 2 ° arco faringeo, per diventare capo e la muscolatura del cuore, rispettivamente (Figura 1A, A &#39;e A&#39; &#39;). Coltura archi faringei offre un modo unico per lo studio del cuore e sviluppo muscolare in dettaglio ex vivo. Dopo la dissezione e l&#39;attaccamento di archi faringei, cellule che migrano da archi collegati possono essere osservati entro 24-48 ore della cultura. Entro 3 giorni di formazione della cultura myotube si può osservare dal 1 ° archi faringei e formazione cardiomiociti dagli archi faringei 2 nd (Figura 2a e 2b). Formazione Cardiomyocyte può essere confermata visivamente dalle amministrazioni spontaneamente gruppi di cellule e / o analisi di marcatori specifici cardiomiociti migrazione (ad esempio, cardiaca Troponina T). Myotuessere formazione / facciale muscolare può essere osservato visivamente lungo allungato (fino a 500 micron di lunghezza) cellule e / o analisi di marcatori muscolari specifici (ad esempio, miogenina) spontaneamente contrazioni. Usando il sistema di cre-lox nei topi, mesoderma progenie può essere fatta dai giornalisti fluorescenti su cre-espressione durante ex vivo cultura (Figura 2A &#39;e 2B&#39;). Allo stesso modo, con questo metodo è possibile studiare il destino della progenie in cui uno specifico gene di interesse viene condizionatamente eliminato o overexpressed, che altrimenti provocherebbe letalità embrionale precoce come precedentemente descritto 8. <img alt="Figura 1" src="/files/ftp_upload/52876/52876fig1.jpg" /> Figura 1: embrione Mouse (Mesp1 cre, AI9) 9,5 giorni dopo la fecondazione (E.9.5 </strong<html> &gt;). (A) Lato sinistro di embrione. (PA: faringeo Arco, OT: tratto di efflusso, LV:.. Ventricolo sinistro (A &#39;) Mesp1 linage-traccia; RFP segna + cellule Mesp1 e la loro progenie freccia indica RFP + Mesp1-progenie in PA2 che è in continuità con la OT. (A &#39;&#39;) La linea tratteggiata indica dove tagliare PA1 e PA2 posteriormente al cuore (B) Butterfly-come destra e sinistra PA1 e PA2 dopo dissezione dall&#39;embrione FE:.. endoderma Foregut (B. &#39;) segna RFP Mesp1 progenie . in PA1 e PA2 (B &#39;&#39;) * e ** insieme con le linee tratteggiate indica dove tagliare e rilasciare PA1 e PA2 dal OT e FE bar Scala:.. 500 micron <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52876/52876fig1large.jpg" target="_blank">Cliccate qui per visualizzare una versione più grande di questo figura.</a> caricare / 52.876 / 52876fig2.jpg &quot;/&gt; Figura 2:. Archi faringei coltivate (A) PA1 coltivate (3 giorni di cultura). (A &#39;) RFP segna Mesp1 progenie in PA1. Frecce e * indicano formazione myotube presto. (A &#39;&#39;) Sovrapposizione di espressione RFP in PA1. (B) PA2 coltivata (8 giorni di cultura). (B &#39;) segna RFP Mesp1 progenie in PA2. Frecce e ** indicano un&#39;area di cardiomiociti (modificato da Shenje et al. 8) di battere. (B &#39;&#39;) Sovrapposizione di espressione RFP in PA2. Scala bar:. 250 micron <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52876/52876fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche at JoVE.com

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

Ex Cultura Vivo del faringea Archi allo Studio cuore e muscoli Progenitori e la loro nicchia

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

ex-vivo-culture-pharyngeal-arches-to-study-heart-muscle-progenitors

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

7.8K Views.  Johns Hopkins University School of Medicine. Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

Video: Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Imaging Azzerato embrionale e postnatale Cuori a risoluzione di una singola cella

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Ricerca

Biologia dello sviluppo

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.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

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.

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

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

Ingegneria

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

Analisi quantitativa intero-monta immunofluorescenza delle popolazioni di cellule progenitrici cardiache in embrioni di topo

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Sistema basato su microiniezione per In Vivo l'impianto di cardiomiociti embrionali nell'embrione aviaria

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

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 ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.

Visualizzazione e analisi delle arterie dell'arco faringo utilizzando immunohistochimica a monte intero e ricostruzione 3D

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

Generazione di organoidi cardiaci umani auto-assemblanti derivati da cellule staminali pluripotenti

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

Biochimica

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

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

Imaging dal vivo dei primi progenitori cardiaci nell'embrione di topo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...