Name: epicardial outgrowth culture assay and ex vivo assessment of epicardial-derived cell migration
Uploaded: 2016-03-18T14:00:00
Description: Watch this Scientific Journal Video about Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration at JoVE.com

E.M.S. was supported by grants from the National Institutes of Health (NIH) [grant number R01HL120919]; the American Heart Association [grant number 10SDG4350046]; University of Rochester CTSA award from the NIH [grant number UL1 TR000042]; and startup funds from the Aab CVRI. M.A.T. was supported in part by a grant to the University of Rochester School of Medicine and Dentistry from the Howard Hughes Medical Institute Med-Into-Grad Initiative; and an Institutional Ruth L. Kirschstein National Research Service Award from the NIH [grant number GM068411].

NOTE: All experimentation with mice were approved by the University Committee on Animal Resources at the University of Rochester.
1. Epicardial Outgrowth Culture Assay (Figure 1A)
<ol>
	<li>Preparations
	<ol>
		<li>Prepare 5 ml medium A for primary epicardial cell isolation by supplementing Medium 199 (M199) with 5% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (Pen/Strep).</li>
		<li>Pre-warm medium A and 100 ml Hanks&#39; Balanced Salt Solution (HBSS) in a 37 &#176;C water bath.</li>
		<li>Allow coll...

Here we outline detailed methods to isolate primary epicardial cell by outgrowth and track EPDC migration in ex vivo heart cultures. For outgrowth cultures, the appropriate time to remove the epicardium-depleted heart varies slightly between experiments. Periodic monitoring of the explants after an overnight incubation is recommended to gauge the extent of epicardial outgrowth and to extract the heart before fibroblasts appear. To ensure purity, hearts should be removed within 24 hr of explanting to prevent accu...

The epicardium is a single layer of mesothelial cells that lines the heart and impacts cardiac development, maturation and repair. Through a highly coordinated exchange of paracrine signals, the intimate dialogue between the myocardium and epicardium is indispensable for cardiac growth and the formation of non-myocyte cardiac lineages1. Epicardial-derived cells (EPDC) emerge from a subset of epicardial cells through an epithelial-to-mesenchymal transition (EMT)2, invade the subepicardial space and underlying myocardium, and differentiate largely into coronary vascular mural cells and cardiac fibroblasts, and to a lesser extent, endothelial cells and cardiomyocytes3-9.
The mechanisms regulating epicardial EMT and EPDC invasion have been attributed to the coordinated action of various molecules including secreted ligands10-13, cell surface receptors and adhesion molecules14,15, regulators of apical-basal polarity16,17, small GTPases18,19, and transcription factors2,20,21. While many of the molecular effectors of EPDC migration have been defined, a better understanding of the physiological signals that stimulate EPDC mobilization in the embryo may accelerate the development of strategies to manipulate this process in the adult for improved cardiac repair.
Studies aimed at identifying novel regulators of EPDC mobilization rely on purification of this cell population or tracing the migration of individual epicardial cells. One or a combination of marker genes, including Wt1, Tcf21, Tbx18, and/or Aldh1a2, is commonly used to identify the fetal epicardium1. However, the use of these markers to track migrating EPDC is not optimal as expression of epicardial markers wanes over the course of heart development and is often lost when epicardial cells undergo transdifferentiation into mesenchymal cells.
The application of the Cre/loxP system, in conjunction with lineage-tracing reporters, has been useful in permanently labeling the epicardium and its descendants during heart development and following ischemic injury in the adult7,22,23. Several epicardial-restricted Cre lines have been generated and are widely used to label EPDC and for conditional gene deletion strategies1. These studies have led to the characterization of various epicardial lineages, and the identification of critical mediators of EPDC motility and differentiation. However, accumulating evidence also suggests the epicardium is a heterogeneous population of progenitor cells4,24,25. Thus, only a subset of epicardial cells will be targeted using a given Cre driver.
In order to label the entire epicardium regardless of Cre specificity, a number of laboratories have utilized outgrowth culture systems or ex vivo organ culture methods to faithfully isolate or label epicardial cells without depending upon the expression of a genetic lineage markers26-28. For ex vivo migration studies, embryonic hearts are isolated prior to epicardial EMT and cultured in medium supplemented with a green fluorescent protein (GFP)-expressing adenovirus (Ad/GFP)9,18. This approach allows for efficient labeling of the entire epicardium rather than a subset of cells by Cre-mediated recombination. Heart cultures are subsequently exposed to known inducers of EMT to stimulate the mobilization of EPDC28,29. Ex vivo and in vivo analyses are complemented by in vitro epicardial explant cultures, which are a particularly useful approach for exploring detailed mechanisms driving EPDC migration.
Here, we describe methods for isolating primary epicardial cells for in vitro studies of epicardial EMT, as well as an organ culture system for ex vivo analyses of EPDC motility. We have recently demonstrated the utility and robustness of this method by genetically modulating the myocardin-related transcription factor (MRTF)/serum response factor (SRF) signaling axis to manipulate actin-based migration of EPDC9. While our findings highlight one signaling pathway necessary for regulating EPDC migration, these methods are suitable for unraveling the mechanisms that collectively orchestrate EPDC migration and differentiation. Furthermore, the explant and ex vivo culture systems could be implemented in functional screens to identify novel regulators of EPDC mobilization for therapeutic applications in cardiac regeneration.

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			<td height="40" style="height:40px;width:244px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:163px;">Hyclone&#160;</td>
			<td style="width:141px;">SH3002201</td>
			<td style="width:203px;">DMEM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Hanks&#39; Balanced Salt Solution</td>
			<td style="width:163px;">Hyclone</td>
			<td style="width:141px;">SH3003102</td>
			<td style="width:203px;">HBSS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Medium 199</td>
			<td style="width:163px;">Hyclone</td>
			<td style="width:141px;">SH3025301</td>
			<td style="width:203px;">M199</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Dulbecco&#8217;s Phosphate-Buffered Saline&#160;</td>
			<td style="width:163px;">Hyclone</td>
			<td style="width:141px;">SH3002802</td>
			<td style="width:203px;">DPBS</td>
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			<td height="20" style="height:20px;width:244px;">Fetal Bovine Serum&#160;</td>
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			<td style="width:141px;">354557</td>
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			<td height="20" style="height:20px;width:244px;">Multiwell 24-well plates</td>
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			<td style="width:141px;">08-772-1</td>
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			<td style="width:203px;">Equipped with Leica KL300 LED might source</td>
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			<td style="width:203px;">Equipped with muli-argon laser 405/488/515, FV10-MCPSU laser 405/440/635, 559 laser, and mercury lamp</td>
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			<td height="20" style="height:20px;width:244px;">Protease inhibtor cocktail tablets</td>
			<td style="width:163px;">Roche</td>
			<td style="width:141px;">11836170001</td>
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			<td style="width:163px;">Life Technologies</td>
			<td style="width:141px;">A-11012</td>
			<td style="width:203px;">Secondary antibody</td>
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			<td style="width:203px;">monoclonal antibody, clone 1A4</td>
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			<td>MA1-46028</td>
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			<td style="width:163px;">Millipore</td>
			<td style="width:141px;">AB756P</td>
			<td style="width:203px;">polyclonal antibody</td>
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			<td style="width:141px;">D1306</td>
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			<td height="20" style="height:20px;width:244px;">Fluorescent mounting medium&#160;</td>
			<td style="width:163px;">DAKO</td>
			<td style="width:141px;">S3023</td>
			<td style="width:203px;"></td>
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			<td height="60" style="height:60px;width:244px;">16% Paraformaldehyde solution</td>
			<td style="width:163px;">Electron Microscopy Sciences</td>
			<td style="width:141px;">15710</td>
			<td style="width:203px;">PFA diluted to 4% in DPBS. Caution, hazardous material</td>
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			<td height="20" style="height:20px;width:244px;">Sucrose</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:141px;">S0389</td>
			<td style="width:203px;"></td>
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			<td height="40" style="height:40px;width:244px;">Tissue Freezing Medium</td>
			<td style="width:163px;">Triangle Biomedical Sciences</td>
			<td style="width:141px;">TFM-B</td>
			<td style="width:203px;"></td>
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			<td height="20" style="height:20px;width:244px;">Pap pen</td>
			<td style="width:163px;">DAKO</td>
			<td style="width:141px;">S2002</td>
			<td style="width:203px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Disposable mictrotome blades&#160;</td>
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			<td style="width:141px;">4689</td>
			<td style="width:203px;"></td>
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			<td height="40" style="height:40px;width:244px;">Microscope slides</td>
			<td style="width:163px;">Globe Scientific</td>
			<td style="width:141px;">1358W</td>
			<td style="width:203px;">positively charged, 25 x 75 x 1mm</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Cryostat&#160;</td>
			<td style="width:163px;">Leica</td>
			<td style="width:141px;">CM1950</td>
			<td style="width:203px;"></td>
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			<td height="20" style="height:20px;width:244px;">Forceps</td>
			<td style="width:163px;">Fine Science Tools</td>
			<td style="width:141px;">11295-00</td>
			<td style="width:203px;"></td>
		</tr>
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			<td height="20" style="height:20px;width:244px;">Surgical Scissors</td>
			<td style="width:163px;">Fine Science Tools</td>
			<td style="width:141px;">91460-11</td>
			<td style="width:203px;"></td>
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			<td height="20" style="height:20px;width:244px;">Disposable base molds</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:141px;">22-363-553</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Ketamine</td>
			<td style="width:163px;">Hospira</td>
			<td>NDC 0409-2051-05</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Xylazine</td>
			<td style="width:163px;">Akorn</td>
			<td>NADA 139-236</td>
			<td style="width:203px;"></td>
		</tr>
	</tbody>
</table>

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.

The epicardium can be efficiently isolated using an outgrowth culture assay by taking advantage of its exterior location and exploiting the developmental plasticity and intrinsic migratory behavior of EPDC. Murine embryonic hearts are isolated at E11.5 prior to epicardial EMT and cultured dorsal side down on collagen-coated chamber slides26 (Figure 1A, B). Explanted hearts will continue to contract; however, the epicardium will adhere to the collagen matrix and...

Watch this Scientific Journal Video about Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration at JoVE.com

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.

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 ...

The overall goals of the outgrowth culture method and the ex vivo migration assay are to isolate primary cultures of epicardial cells for studying epithelial to mesenchymal transitions and to assess the functional migration of epicardial derived cells, respectively. The main advantage of these techniques is that they facilitate the isolation of primary cultures from the epicardium and the testing of the intrinsic behaviors of epicardial derived progenitor cells. The implications of this technique extend toward the development of strategies for manipulating epicardial derived cell biology in cardiac regenerative medicine.In preparation of epicardial cell isolations, pre-warm collagen coated chamber slides to room temperature and add 100 microliters of medium A per well. Remove media after coating chambers ensuring no pooled media remains. To isolate primary epicardial cells first harvest mouse embryos at 11.5 days post coitum.Use forceps to pinch the thoracic wall of embryos and tear the tissue apart at the midline, exposing the chest cavity. Next place the forceps behind the the heart and grasp the outflow tract. Pull the heart away from the embryo and place the cardiac tissue, dorsal side down, in a single well of a collagen coated slide.When all of the hearts have been placed in individual wells, incubate the chamber slide for 30 minutes at 37 degrees Celsius and 5%CO2 to allow the tissue to adhere to the collagen matrix. At the end of the incubation, carefully add 20 to 50 microliters of pre-warmed explant medium A around each heart and return the slide to the cell culture incubator for approximately 24 hours to facilitate the epicardial outgrowth. It's important to add the appropriate amount of medium to the explants, as too much may result in tissue detachment and too little medium may result in dehydration, diminishing the quality of the outgrowths.Outgrowths can be observed as early as three to four hours of culture and by 24 hours, consist primarily of mesothelial cells that display a cobblestoned morphology with a minimal mesenchymal cell contamination. Care should be taken to remove hearts before invasion of mesenchymal cells contaminate the epicardial cell outgrowth. The next day, use forceps to carefully transfer the epicardium-depleted hearts to a one point five milliliter tube.Then add 800 microliters of Trizol and store the samples at negative 80 degrees Celcius for later downstream analysis. Then wash each well with DPBS to remove the blood cells and excess cellular debris. Incubate the outgrowth cultures with 500 microliters of explant medium B supplemented with adenovirus expressing Cre recombinase for 24 hours in a cell culture incubator.For epicardial labeling to track subsequent cell migration isolate the embryonic hearts from day 12.5 post coitum pregnant dams, as just demonstrated, and place them into individual wells of a 24-well plate containing pre-warmed ex vivo culture medium. Incubate the hearts at 37 degrees Celcius and 5%CO2 with gentle rocking. While the hearts are incubating, resuspend three point oh times 10 to the sixth pfu per milliliter of adenovirus GFP and 12 milliliters of 37 degrees Celcius ex vivo culture medium.Next transfer six milliliters of the virus-supplimented culture medium into a new 15 milliliter tube and add adenovirus Cre to a final concentration of one point five times 10 to the sixth pfu per milliliter. Using a P1000 pipette carefully remove the culture medium from each heart and add the adenovirus GFP adenovirus Cre solution to each well. Then return the plate to the cell culture incubator for 24 hours with gentle rocking.For the epithelial to mesenchymal induction the next day use a P1000 pipette to remove the medium from each well. Carefully rinse the hearts with one milliliter of DPBS. Then discard the wash and replenish the hearts with freshly prepared ex vivo culture medium, containing TGF beta one and platelet derived growth factor BB, and incubate for 24 hours in the cell culture incubator with gentle rocking.Epicardial cell explants display a robust expression of epicardial and mesothelial markers and a low expression of cardiomyocyte genes compared to the epicardium-depleted hearts. To further verify their identity, epicardial cell cultures are fixed 48 hours after the removal of the heart and co-stained with antibodies against Wilms'tumor one and ZO-1 to label the mesothelial cells and intercellular tight junctions, respectively. After 24 hours of stimulation the epicardial cells adopt a mesenchymal phenotype with a reduced ZO-1 staining and a robust de novo formation of smooth muscle alpha-actin positive stress fibers, particularly at the periphery of the culture.In contrast, confluent epicardial cells with more defined inter-cellular contacts at the center of a monolayer display smooth muscle alpha-actin associated with cortical regions. Deletion of the miocardin related transcription factor signaling axis genes by Cre-mediated excision of fluxed alleles attenuates the migration of epicardial derived cells into the sub-epicardial space. Conversely, the forced expression of myocardin related transcription factor A results in an increased migration and disruption of the collagen IV basement membrane by the epicardial derived cells.Once mastered, each technique can be completed in 48 to 72 hours if performed properly. While attempting this procedure it's important to isolate embryos at the appropriate developmental time stages to optimize epicardial cell outgrowth from tissue explants and to label the epicardium prior to epicardial derived cell migration. Following this procedure other methods, like genetic manipulation or chemical modifications can be performed to answer questions about identifying novel regulators of epicardial derived cell mobilization and differentiation.

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