This research is supported by The Netherlands Organization for Scientific Research (NWO) (VENI 016.146.079) and a LUMC Research fellowship both to AMS, and LUMC Bontius Stichting (MJG).

All experiments with human tissue specimens were approved by the ethics committee of the Leiden University Medical Center and conforms to the Declaration of Helsinki. All steps are performed with sterile equipment in a cell culture flow cabinet.
1. Preparations
<ol>
	<li>Prepare EPDC medium by mixing Dulbecco&#39;s modified Eagle&#39;s medium (DMEM low- glucose) and Medium 199 (M199) in a 1:1 ratio. Add 10% heat inactivated fetal bovine serum (FBS, heat inactivated for 25 min at 56 &#1...

<ol>
	<li>Smits, A. M., Dronkers, E., Goumans, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epicardium+as+a+source+of+multipotent+adult+cardiac+progenitor+cells:+Their+origin,+role+and+fate.">The epicardium as a source of multipotent adult cardiac progenitor cells: Their origin, role and fate.</a> Pharmacological research. 127, 129-140 (2017).</li><li>Risebro, C. A., Vieira, J. M., Klotz, L., Riley, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+the+human+embryonic+and+foetal+epicardium+during+heart+development.">Characterisation of the human embryonic and foetal epicardium during heart development.</a> Development. 142 (21), 3630-3636 (2015).</li><li>Zhou, B., Ma, Q., 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+progenitors+contribute+to+the+cardiomyocyte+lineage+in+the+developing+heart.">Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart.</a> Nature. 454 (7200), 109-113 (2008).</li><li>Smits, A., Riley, P. <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+Heart+Repair.">Epicardium-Derived Heart Repair.</a> Journal of Developmental Biology. 2 (2), 84-100 (2014).</li><li>Chen, T. H. P., Chang, 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=Epicardial+Induction+of+Fetal+Cardiomyocyte+Proliferation+via+a+Retinoic+Acid-Inducible+Trophic+Factor.">Epicardial Induction of Fetal Cardiomyocyte Proliferation via a Retinoic Acid-Inducible Trophic Factor.</a> Developmental Biology. 250 (1), 198-207 (2002).</li><li>Pennisi, D. J., Ballard, V. L. T., 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> Developmental Dynamics. 228 (2), 161-172 (2003).</li><li>Lavine, K. J., Yu, K., 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> Developmental Cell. 8 (1), 85-95 (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> Developmental biology. 255 (2), 334-349 (2003).</li><li>M&#228;nner, 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> Developmental Dynamics. 233 (4), 1454-1463 (2005).</li><li>Weeke-Klimp, A., Bax, N. A. 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=Epicardium-derived+cells+enhance+proliferation,+cellular+maturation+and+alignment+of+cardiomyocytes.">Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes.</a> Journal of Molecular and Cellular Cardiology. 49 (4), 606-616 (2010).</li><li>Eralp, I., Lie-Venema, 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=Coronary+Artery+and+Orifice+Development+Is+Associated+With+Proper+Timing+of+Epicardial+Outgrowth+and+Correlated+Fas+Ligand+Associated+Apoptosis+Patterns.">Coronary Artery and Orifice Development Is Associated With Proper Timing of Epicardial Outgrowth and Correlated Fas Ligand Associated Apoptosis Patterns.</a> Circulation Research. 96 (5), (2005).</li><li>Kelder, T. P., Duim, S. 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=The+epicardium+as+modulator+of+the+cardiac+autonomic+response+during+early+development.">The epicardium as modulator of the cardiac autonomic response during early development.</a> Journal of Molecular and Cellular Cardiology. 89, 251-259 (2015).</li><li>van Wijk, B., Gunst, Q. D., Moorman, A. F. M., van den Hoff, M. J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration+from+activated+epicardium.">Cardiac regeneration from activated epicardium.</a> PloS one. 7 (9), e44692 (2012).</li><li>Zhou, B., Honor, L. 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> The Journal of clinical investigation. 121 (5), 1894-1904 (2011).</li><li>Smart, N., Bollini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+cardiomyocytes+from+within+the+activated+adult+heart+after+injury.">De novo cardiomyocytes from within the activated adult heart after injury.</a> Nature. 474 (7353), 640-644 (2011).</li><li>Zangi, L., Lui, K. O., 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=Modified+mRNA+directs+the+fate+of+heart+progenitor+cells+and+induces+vascular+regeneration+after+myocardial+infarction.">Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.</a> Nature Biotechnology. 31 (10), 898-907 (2013).</li><li>Moerkamp, A. T., Lodder, K., 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=Human+fetal+and+adult+epicardial-derived+cells:+a+novel+model+to+study+their+activation.">Human fetal and adult epicardial-derived cells: a novel model to study their activation.</a> Stem Cell Research &amp; Therapy. 7 (1), 1-12 (2016).</li><li>Clunie-O'Connor, C., Smits, A. 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=The+Derivation+of+Primary+Human+Epicardium-Derived+Cells.">The Derivation of Primary Human Epicardium-Derived Cells.</a> Current Protocols in Stem Cell Biology. 35, 2C.5.1-2C.5.12 (2015).</li><li>Van Tuyn, J., Atsma, D. 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+Cells+of+Human+Adults+Can+Undergo+an+Epithelial-to-+Mesenchymal+Transition+and+Obtain+Characteristics+of+Smooth+Muscle+Cells+In+Vitro.">Epicardial Cells of Human Adults Can Undergo an Epithelial-to- Mesenchymal Transition and Obtain Characteristics of Smooth Muscle Cells In Vitro.</a> Stem Cells. 25 (2), 271-278 (2007).</li><li>Bax, N. A. M., van Oorschot, A. A. 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Here we describe a detailed protocol to isolate and culture primary epicardial cells derived from human adult and fetal hearts. Extensive characterization of these cells has been previously published17. We have shown that both cell types can be maintained as epithelial cobblestone-like cells when cultured with the ALK5 kinase inhibitor SB431542. EMT is an integral part of epicardial activation in vivo during both development and the post-injury response. EMT can be studied using this meth...

The epicardium, a single-cell epithelial layer that envelopes the heart, is of vital importance for cardiac development and repair (reviewed in Smits et al.1). Developmentally, the epicardium arises from the proepicardial organ, a small structure located at the base of the developing heart. Around developmental day E9.5 in mouse, and 4 weeks post-conception in human, cells start to migrate from this cauliflower structure and cover the developing myocardium2. Once a single epithelial cell layer is formed, a portion of the epicardial cells undergoes&#160;epithelial to mesenchymal transition (EMT). During EMT, cells lose their epithelial characteristics, such as cell-cell adhesions, and obtain a mesenchymal phenotype which gives them the capacity to migrate into the developing myocardium. The formed epicardial derived cells (EPDCs) can differentiate into several cardiac cell types including fibroblasts, smooth muscle cells, and potentially cardiomyocytes and endothelial cells3, although differentiation of the latter two cell populations remains subject to debate (reviewed in Smits et al.4). Furthermore, the epicardium provides instructive paracrine signals to the myocardium to regulate its growth and vascularization5,6,7,8. Multiple studies have demonstrated that impaired epicardial formation leads to developmental defects in cardiac muscle9,10, vasculature11, and conduction system12, emphasizing the essential contribution of the epicardium to the formation of the heart.
Although in the adult heart the epicardium is present as a dormant layer, it becomes reactivated upon ischemia13. Epicardial reactivation post-injury recapitulates several of the processes described for cardiac development, including proliferation and EMT14, albeit less efficiently. Interestingly, although the exact mechanism is not fully understood, the epicardial contribution to repair can be improved by treatment with, e.g., Thymosin &#946;415 or modified VEGF-A mRNA16, resulting in ameliorated cardiac function after myocardial infarction. The epicardium is therefore considered an interesting cell source to enhance endogenous repair of the injured heart.
Mechanisms of cardiac development are often recapitulated during injury, although in a less efficient manner. In search of epicardial activators, it is paramount that we can determine and compare the full capacity of the fetal and adult epicardium. Moreover, from a therapeutic point of view, it is important that, in addition to animal experiments, we extend knowledge regarding the response of the human epicardium. Here, we describe a method to isolate and culture human adult and fetal epicardial derived cells (EPDCs) in an epithelial-cell-like morphology and to induce EMT. With this model, we aim to explore and compare adult and fetal epicardial cell behavior.
The main advantage of this protocol is the use of human epicardial material, which has not been thoroughly studied. Importantly, the described isolation and cell culture protocol provides a single uniform method to derive both fetal and adult cobble EPDCs, enabling a direct comparison between these two cell sources. Additionally, since the epicardium is isolated based on its location, it is ensured that the cells are actually epicardially derived17.
While human EPDC isolation methods have been established previously, these mostly rely on outgrowth protocols where pieces of cardiac or epicardial tissue are plated onto a cell culture dish18,19. This approach thereby selects specifically for cells that partially lose their epithelial phenotype in order to migrate, and that are more prone to undergo spontaneous EMT. In the current protocol, the epicardium is first processed into a single cell solution which allows the isolated EPDCs to maintain their epithelial state. This method therefore provides a solid in vitro model to study epicardial EMT.

<table border="0" cellpadding="0" cellspacing="0" width="798">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Dulbecco&#8217;s modified Eagle&#8217;s medium + GlutaMAX</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:119px;">21885-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Medium 199&#160;</td>
			<td>Gibco</td>
			<td>31150-022</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Fetal Bovine Serum&#160;</td>
			<td>Gibco</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Trypsin 0.25%</td>
			<td>Invitrogen</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Penicillin G sodium salt</td>
			<td style="width:133px;">Roth</td>
			<td style="width:119px;">HP48</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Streptomycin sulphate</td>
			<td style="width:133px;">Roth</td>
			<td>HP66</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Trypsin 1:250 from bovine pancreas</td>
			<td style="width:133px;">Serva</td>
			<td>37289</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">EDTA</td>
			<td style="width:133px;">Sigma</td>
			<td>E4884</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Gelatin from porcine skin</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Culture plates 6 well</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Culture plates 12 well</td>
			<td style="width:133px;">Corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Culture plates 24 well</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">SB 431542</td>
			<td style="width:133px;">Tocris</td>
			<td style="width:119px;">1614</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:133px;">Merck</td>
			<td style="width:119px;">102931</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">100-1000&#181;L Filtered Pipet Tips</td>
			<td style="width:133px;">Corning</td>
			<td style="width:119px;">4809</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">10-ml pipet</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td style="width:119px;">607180</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">5-ml pipet</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td style="width:119px;">606180</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Cell culture dish 100/20 mm</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td style="width:119px;">664160</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">PBS</td>
			<td>Gibco</td>
			<td style="width:119px;">10010056</td>
			<td>Or home-made and sterilized</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Eppendorf tubes 1.5 mL</td>
			<td style="width:133px;">Eppendorf</td>
			<td style="width:119px;">0030120086</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">15-ml centrifuge tubes</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">50-ml centrifuge tubes</td>
			<td style="width:133px;">Greiner bio-one</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">10 mL Syringe</td>
			<td>Becton Dickinson</td>
			<td>305959</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Needles 19 Gauge</td>
			<td>Becton Dickinson</td>
			<td>301700</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Needles 21 Gauge</td>
			<td>Becton Dickinson</td>
			<td>304432</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">EASYstrainer Cell Sieves, 100 &#181;m</td>
			<td>Greiner bio-one</td>
			<td>542000</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">TGF&#946;3&#160;</td>
			<td style="width:133px;">R&#38;D systems</td>
			<td style="width:119px;">243-B3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Monoclonal Anti-Actin, &#945;-Smooth Muscle</td>
			<td style="width:133px;">Sigma</td>
			<td>A2547&#160;</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Anti-Mouse Alexa Fluor 555</td>
			<td style="width:133px;">Invitrogen</td>
			<td>A31570</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:119px;">A12379</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Equipment</td>
			<td style="width:133px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Name</td>
			<td style="width:133px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Pipet P1,000</td>
			<td style="width:133px;">Gilson</td>
			<td style="width:119px;">F123602</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Pipet controller</td>
			<td style="width:133px;">Integra</td>
			<td style="width:119px;">155 015</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Stereomicroscope</td>
			<td style="width:133px;">Leica</td>
			<td style="width:119px;">M80</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:379px;">Inverted Light Microscope</td>
			<td style="width:133px;">Olympus</td>
			<td style="width:119px;">CK2</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Centrifuge</td>
			<td style="width:133px;">Eppendorf</td>
			<td style="width:119px;">5702</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Waterbath</td>
			<td style="width:133px;">GFL</td>
			<td style="width:119px;">1083</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Here, we outline a straightforward protocol to isolate EPDCs from human adult and fetal cardiac tissue (Figure 1). This protocol takes advantage of the easily accessible location of the epicardium on the outside of the heart (Figure 1A). Staining of the heart auricle after dissection demonstrates that the WT1+ epicardium is removed while the underlying subepicardial extracellular matrix and myocardial tissue remain intact (<stron...

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

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.

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 overall goal of this procedure is to isolate and culture human epicardial-derived cells in order to study epicardial cell behavior. This method can help answer key questions in the field of cardiac research, such as how is the epicardium activated during cardiac development and disease. The main advantage of this technique is that it can be applied to human cardiac tissue samples, which may help translation of results to a clinical setting.Our protocol describes the investigation of epithelial to mesenchymal transition of epicardial-derived cells. However, it can also be used to study proliferation or secretion of factors. To begin, fill a 100-millimeter cell culture dish with PBS inside the laminar flow cabinet.Place approximately 200 microliters of PBS into the lid, then add 5 milliliters of pre-warmed EPDC medium to a 15-milliliter tube. Place the cardiac tissue, isolated from an adult human heart, in the dish. Under a stereo microscope, use forceps to carefully remove the epicardial layer.Next, collect the pieces of epicardial tissue in the lid and use a scalpel to cut them into 0.5 cubed millimeter pieces. Add one milliliter of trypsin to the tissue pieces. Using a P1000 pipette tip, transfer them into a 1.5-milliliter conical centrifuge tube.Then, incubate the tube for 10 minutes at 37 degrees Celsius inside a water bath with constant shaking at 60 RPM. After incubation, clean the tube with 70%ethanol and allow the tissue to settle at the bottom. Next, carefully transfer the supernatant which contains epicardial cells into a 15-milliliter tube with 5 milliliters of EPDC medium to inactivate the trypsin.Add one milliliter of trypsin to the remaining tissue in the centrifuge tube and mix by gentle pipetting. Then, repeat the trypsin incubation step two more times. After the final collection, transfer the remaining epicardial tissue into the tube containing the cell suspension.Thoroughly homogenize the cell suspension by gentle pipetting, then use a 10-milliliter syringe with a 19 gauge needle to mechanically dissociate the cells in the suspension and transfer the suspension into a fresh 15-milliliter tube. To further dissociate the cells, pass the cell suspension through a 10-milliliter syringe with a 21 gauge needle. Next, use a 10-milliliter pipette to transfer the cell suspension onto a 100-micrometer cell strainer on top of a 50-milliliter tube.Allow the suspension to pass through the strainer to remove all remaining clumps. Add 5 milliliters of EPDC medium onto the strainer to collect residual cells. After a brief gentle mixing, transfer the cell suspension into a 15-milliliter tube and centrifuge at 200 g for five minutes at room temperature.Next, remove the supernatant and resuspend the cell pellet in EPDC medium. Remove the coating media from the gelatin-coated plate and add an appropriate amount of cell suspension. Incubate the cells for at least 48 hours at 37 degrees Celsius in 5%CO2.To culture epicardial cells, replenish the medium every three days. Use EPDC medium supplemented with 10 micromolar ALK 5-kinase inhibitor SB431542. Use a microscope to inspect the cells during culture period.Upon reaching confluency, passage the cells in a 1:2 surface ratio. To passage the cells, use a pipette or aspirator to remove the medium and carefully add PBS to the cells. Gently rotate the plate and aspirate PBS.Add trypsin and gently rotate the plate to spread it over the cells. Incubate the plate for one minute at 37 degrees Celsius. Next, tap the plate to mechanically detach the cells and use a microscope to confirm cell detachment.Add the required volume of EPDC medium supplemented with 10-micromolar SB431542 to the plate. Mix by gentle pipetting and transfer the suspension to a new gelatin-coated plate. To induce EMT, passage the cells using the previously described protocol and incubate for at least 24 hours at 37 degrees Celsius in 5%CO2.After reaching 50-70%confluency, aspirate the medium and wash the cells with PBS. Next, stimulate the cells with one nanogram per milliliter of TGF beta-3 supplemented EPDC medium and incubate for five days at 37 degrees Celsius in 5%CO2 and monitor cells daily. After five days, cells that underwent EMT should acquire a spindle-shaped morphology.Culture these cells using the previously described protocol using EPDC medium without any supplementation. To validate proper dissection of the epicardial layer, human adult heart tissue is immunostained with WT1 and cTnI antibodies before and after the dissection. Absence of epicardial marker WT1 after dissection indicates complete removal of the epicardial layer.Presence of myocardial tissue marker cTnI indicates that subepicardial extracellular matrix and myocardial tissue are intact after the dissection. Adult EPDCs cultured in the presence of ALK 5-kinase inhibitor should maintain cobblestone morphology. Transition from cobblestone morphology to a spindle-shaped morphology of EPDCs after TGF beta-3 treatment indicates epithelial to mesenchymal transition.To further confirm EMT, EPDCs are immunostained with a mesenchymal marker alpha Smooth Muscle Actin before and after TGF beta-3 treatment. Presence of mesenchymal marker in TGF beta-3 treated cells insures the induction of EMT. The mRNA levels of EMT-related genes in adult EPDCs are represented here.TGF beta-3-induced downregulation of epicardial marker WT1 and upregulation of mesenchymal markers MMP3, N-Cadherin, periostin, alpha-SMA, and collagen 1A1 confirm the induction of EMT. While attempting this technique, it is important to remember to perform it as efficiently as possible. A shorter isolation time will improve cell survival.Once mastered, this technique can be done in about 60 minutes. Because this method relies on primary cells, each isolation can behave differently. Therefore, it is important to monitor cell morphology and density before starting an experiment.After watching this video, you should have a good understanding of how to handle human primary epicardial-derived cells.

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Developmental Biology

7.6K vistas.  Leiden University Medical Center. The overall goal of this procedure is to isolate and culture human epicardial-derived cells in order to study epicardial cell behavior. This method can help answer key questions in the field of cardiac research, such as how is the epicardium activated during cardiac development and disease. The main advantage of this technique is that it can be applied to human cardiac tissue samples, which may help translation of results to a clinical setting.Our protocol describes the investigation o...