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

This work was supported by funds from DUKE-NUS Graduate Medical School Singapore, Goh foundation and Singapore NRF fellowship (NRF-NRFF2016-01) to Manvendra K. Singh.

All experiments were approved by the Duke-NUS Graduate Medical School Institutional Animal Care and Use Committee.
1. Retrieving the Embryonic Ventricles
<ol>
	<li>Sacrifice a timed pregnant mouse at the desired embryonic stage (E11.5 or E12.5) using a euthanasia chamber with carbon dioxide gas supply or another approved euthanasia method.</li>
	<li>Place the mouse on its back on a dissecting table. Disinfect the abdomen of the female with 70% ethanol. Lift the skin over the belly and make a...

<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. M., Macias, D., Munoz-Chapuli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin,+formation+and+developmental+significance+of+the+epicardium:+a+review.">The origin, formation and developmental significance of the epicardium: a review.</a> Cells Tissues Organs. 169, 89-103 (2001).</li><li>Gittenberger-de Groot, A. C., Vrancken Peeters, M. P., Mentink, M. M., Gourdie, R. G., Poelmann, R. E. <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+cells+contribute+a+novel+population+to+the+myocardial+wall+and+the+atrioventricular+cushions.">Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions.</a> Circ Res. 82, 1043-1052 (1998).</li><li>von Gise, A., Pu, W. T. <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+epithelial+to+mesenchymal+transitions+in+heart+development+and+disease.">Endocardial and epicardial epithelial to mesenchymal transitions in heart development and disease.</a> Circ Res. 110, 1628-1645 (2012).</li><li>Katz, T. 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=Distinct+compartments+of+the+proepicardial+organ+give+rise+to+coronary+vascular+endothelial+cells.">Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells.</a> Dev Cell. 22, 639-650 (2012).</li><li>Singh, M. K., Lu, M. M., Massera, D., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-processing+enzyme+Dicer+is+required+in+epicardium+for+coronary+vasculature+development.">MicroRNA-processing enzyme Dicer is required in epicardium for coronary vasculature development.</a> J Biol Chem. 286, 41036-41045 (2011).</li><li>Degenhardt, K., Singh, M. K., Epstein, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+under+development:+cardiovascular+embryology+applied+to+heart+disease.">New approaches under development: cardiovascular embryology applied to heart disease.</a> J Clin Invest. 123, 71-74 (2013).</li><li>Singh, M. K., Epstein, J. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+outgrowth+inhibition+leads+to+compensatory+mesothelial+outflow+tract+collar+and+abnormal+cardiac+septation+and+coronary+formation.">Epicardial outgrowth inhibition leads to compensatory mesothelial outflow tract collar and abnormal cardiac septation and coronary formation.</a> Circ Res. 87, 969-971 (2000).</li><li>Lavine, K. 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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietin+and+retinoic+acid,+secreted+from+the+epicardium,+are+required+for+cardiac+myocyte+proliferation.">Erythropoietin and retinoic acid, secreted from the epicardium, are required for cardiac myocyte proliferation.</a> Dev Biol. 255, 334-349 (2003).</li><li>Huang, G. 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. M., 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=Tbx18+and+the+fate+of+epicardial+progenitors.">Tbx18 and the fate of epicardial progenitors.</a> Nature. 458, 8-9 (2009).</li><li>Rudat, C., Kispert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wt1+and+epicardial+fate+mapping.">Wt1 and epicardial fate mapping.</a> Circ Res. 111, 165-169 (2012).</li><li>Duim, S. N., Kurakula, K., Goumans, M. J., Kruithof, B. 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. 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=Generation+of+the+epicardial+lineage+from+human+pluripotent+stem+cells.">Generation of the epicardial lineage from human pluripotent stem cells.</a> Nat Biotechnol. 32, 1026-1035 (2014).</li></ol>

It is pivotal to develop techniques that facilitate the study of the epicardium to cater to the increasing significance of the epicardium in cardiac development and regeneration. The epicardial culture system poses significant advantages for epicardial research.
An alternative way to isolate epicardial cells is to use fluorescence-activated cell sorting (FACS). This method relies on the use of epicardial markers (or epicardium-cell-specific transgenic expression of a fluorescent protein) to se...

A wealth of experimental data have illustrated that the epicardium influences critical steps in cardiac development. During development, the septum transversum gives rise to a clump of mesothelial cells known as the proepicardium1-4. Cells from the proepicardium then migrate and envelope the myocardium forming the epicardium. Following this, a subset of epicardial cells undergo EMT giving rise to a migratory population of epicardium-derived cells (EPDCs) that subsequently invade the myocardium. Genetic as well as retroviral lineage tracing experiments have demonstrated that EPDCs differentiate into various lineages including smooth muscle cells, fibroblasts, endothelial cells and cardiomyocytes (if any). Therefore EPDCs contribute significantly to the development of the coronary vasculature and myocardial architecture1,2,4-9. Furthermore, the epicardium is essential for the development of the ventricular compact layer10-12. For example Gittenberger-de Groot et al. demonstrated that inhibiting the outgrowth of the proepicardium leads to an array of defects such as a thin myocardium, deficient looping of the heart and abnormal interventricular septum formation and as a result, embryonic lethality13. Paracrine factors secreted from the embryonic epicardium modulate cardiomyocyte proliferation and differentiation. Consistent with this, epicardium-specific deletion of signaling pathways such as retinoic acid (RA), fibroblast growth factors (FGFs) and Wnt/&#946;-catenin resulted in defective myocardial growth and embryonic lethality14-16.
Though the epicardium was believed to be quiescent in adult hearts, recent studies have shown that the developmental program is reactivated in the epicardium following cardiac injury17,18. Upon activation, the cells undergo rapid proliferation and EMT that result in EPDC formation. These cells exhibit the capacity to differentiate into fibroblast and smooth muscle cells but not cardiomyocytes or endothelial cells18. In addition, the EPDCs secrete proangiogenic factors that aid in the vascularization of the injured area and thus facilitate improved cardiac function by reducing the infarct size. Due to these findings, the epicardium has gained interest in the study of cardiovascular development, disease and regeneration.
Transgenic technology has revolutionized medical research in the 21st century. With the aid of transgenic technologies, diseased mouse models mimicking the human condition metabolically and pathophysiologically have been successfully developed. However, studying the epicardial cell behavior in these mutants has been a challenge mainly due to early embryonic lethality. Considering the significant role that the epicardium plays in cardiac development and regeneration, we have established an in vitro culture system for mouse epicardial cells. This method allows the long term culture of the epicardial cells and facilitates the detailed study of the two important properties of the epicardium: its ability to migrate and differentiate. The excised ventricles from the mouse could be cultured on collagen gels which can be used to conduct migration assays. Being cultured in a 3D matrix that replicates the collagen-rich extracellular matrix of the subepicardial layer better recapitulates the in vivo cell physiology. Alternatively they can be cultured on chamber slides in order to establish an epicardial monolayer which can then be used for a variety of downstream applications. This monolayer can be used to stain for tight junction proteins which will provide insights on the ability of the epicardium to undergo EMT which is crucial for migration. In addition, differentiation experiments can also be carried out on these cells. Furthermore, gene expression profile can be analyzed by extracting RNA from the cells and performing quantitative polymerase chain reaction (qPCR). Lastly, the monolayers could also be treated with agents followed by a molecular analysis to test for potential therapeutics. Put together, this epicardial culture system provides us with the opportunity to visualize and gather molecular data which furthers our understanding about epicardial development.
Another desirable feature of this method is that it is straightforward and no elaborate setup is required. In brief, the embryos are harvested at E11.5 or E12.5 following which the heart is excised. The ventricles are then cultured on either collagen gel or chamber slides. Subsequently, these cells can be used to conduct downstream experiments.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:131px;">Life tech invitrogen&#160;</td>
			<td style="width:131px;">11995065</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin solution</td>
			<td>Life tech invitrogen&#160;</td>
			<td>15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)&#160;</td>
			<td>Life tech invitrogen&#160;</td>
			<td>10500064</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148-5KG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant fibroblast growth factor 2 (FGF2)</td>
			<td style="width:131px;">PeproTech</td>
			<td style="width:131px;">450-33</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant transforming growth factor beta 1 (TGF-&#946;1)</td>
			<td style="width:131px;">PeproTech</td>
			<td style="width:131px;">100-21</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">ZO-1 antibody</td>
			<td>Life tech invitrogen&#160;</td>
			<td style="width:131px;">40-2200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-Tubulin antibody</td>
			<td>Sigma</td>
			<td style="width:131px;">T 6074</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">&#945;-smooth muscle actin (SMA) antibody</td>
			<td>Sigma</td>
			<td style="width:131px;">A 2547</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Phalloidin antibody</td>
			<td>Life tech invitrogen&#160;</td>
			<td style="width:131px;">A12379</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3D Collagen Culture kit&#160;</td>
			<td>Millipore&#160;</td>
			<td>ECM 675</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-well chamber slide</td>
			<td>Fisher Scientific</td>
			<td>NNU 154534-PK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Trizol reagent</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:131px;">15596-018</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ViiA 7 Real-Time PCR System</td>
			<td style="width:131px;">Life Technologies</td>
			<td>4453536</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superscript First Strand Synthesis kit</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:131px;">11904-018</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.

Using this culture protocol, primary epicardial cells can be isolated with high purity for downstream applications. The cultured cells are able to undergo EMT, migrate and differentiate just as epicardial cells do in vivo.
To determine the purity of our primary epicardial cell culture, we analyzed the epicardial explants generated from Sema3dGFPCre/+;R26Tom/+ emb...

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

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.

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 ...

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