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 collagen-coated chamber slides to warm to room temperature for 30 min.</li>
		<li>Add 100 &#181;l medium A to each well of the chamber slide and gently rotate to evenly coat the collagen surface. Repeat for 8-12 wells. Remove media after coating chambers.</li>
		<li>Aliquot pre-warmed HBSS into two 10 cm2 plates containing 50 ml each of HBSS.</li>
	</ol>
	</li>
	<li>Isolate Embryonic Hearts from Pregnant Dam at 11.5 Days Post Coitum (dpc).30
	<ol>
		<li>Anesthetize mouse with 0.5 ml of 13 mg/ml ketamine/0.88 mg/ml xylazine cocktail in 1x Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) via intraperitoneal injection. Confirm proper anesthetization using a toe pinch test. A gentle pinch is sufficient to determine if the mouse is responsive. Any movement or paw withdraw indicates the animal is not sufficiently anesthetized to do surgery. Euthanize by cervical dislocation.</li>
		<li>Lay the mouse facing ventral side up and spray the abdomen with 70% ethanol to reduce contamination by hair.</li>
		<li>Pinch the skin with forceps and cut a lateral incision at the abdomen with surgical scissors. Hold the skin firmly and then pull apart in opposing directions towards the head and tail.</li>
		<li>Pinch the peritoneum with forceps and cut with scissors to expose the abdominal cavity.</li>
		<li>Carefully remove the decidua by cutting along the mesometrium. Transfer the dissected decidua to pre-warmed HBSS in the first 10 cm2 plate.</li>
		<li>Separate the decidua by cutting between embryos.</li>
		<li>Transfer an embryo to the second 10 cm2 plate with pre-warmed HBSS under a dissecting microscope. Carefully remove the extraembryonic tissue and yolk sac, then decapitate the embryo.</li>
		<li>Pinch the thoracic wall with forceps and tear the tissue apart at the midline, exposing the chest cavity.</li>
		<li>Place the forceps behind the heart. Grasp the outflow tract and pull the heart away from the embryo. Separate the pulmonary tract and lungs from the heart, if still attached.</li>
	</ol>
	</li>
	<li>Transfer the heart to a single well of the collagen-coated slide and place it dorsal side down (Figure 1B). Repeat steps 1.2.7-1.3 for each embryo.</li>
	<li>Once all hearts have been transferred to individual wells, incubate the chamber slide for 30 min at 37 &#176;C, 5% CO2 to allow sufficient adherence to the collagen matrix.</li>
	<li>Carefully add 20-50 &#181;l of pre-warmed explant medium A around each heart. Important: Do not add the medium too quickly or to a level above the heart, otherwise hearts may detach from the collagen substrate.</li>
	<li>Culture the hearts at 37 &#176;C, 5% CO2 for approximately 24 hr to allow epicardial outgrowth. Note: Outgrowths can be observed as early as 3-4 hr of culture (Figure 1C) and primarily consists of mesothelial cells displaying cobblestoned morphology, with minimal mesenchymal cell contamination (Figures 1D, E). Representative results were acquired using a dissection microscope equipped with a camera.</li>
</ol>
2. Genetic Ablation and Induction of EMT in Epicardial Outgrowths
Caution: Handle adenoviruses with care according to approved biosafety guidelines.
<ol>
	<li>Prepare 15 ml medium B (M199 supplemented with 1% FBS and 1% Pen/Strep) and pre-warm to 37 &#176;C in a water bath.</li>
	<li>For excision of floxed alleles, prepare explant medium B supplemented with 2.5 x 104 pfu/ml of adenovirus expressing Cre recombinase (Ad/Cre) or control virus. 
	Note: Adenoviral mediated overexpression of candidate genes can also be carried out to evaluate gain of function phenotypes. Viral titers should be determined by the end user.</li>
	<li>Carefully remove epicardium-depleted hearts from outgrowth cultures using forceps. Transfer hearts to 1.5 ml tubes with 800 &#181;l Trizol and store at -80 &#176;C for later RNA isolation and gene expression analysis.</li>
	<li>Wash each well with DPBS to remove blood cells and excess cellular debris.</li>
	<li>Add 500 &#181;l explant medium B supplemented with adenovirus(es) to each outgrowth culture and incubate for 24 hr at 37 &#176;C, 5% CO2.</li>
	<li>For EMT induction, remove media and replenish with 37 &#176;C explant medium B supplemented with recombinant transforming growth factor (TGF)-&#946;1 [10 ng/ml] or vehicle (4 mM hydrochloric acid (HCl) containing 1 mg/ml bovine serum albumin (BSA)). Culture explants for an additional 24 hr at 37 &#176;C, 5% CO2. Proceed with gene/protein expression analyses or immunocytochemistry as detailed in the subsequent protocols. 
	Note: Induction of epicardial EMT has been stimulated with various concentrations of TGF-&#946;1 ranging from 0.5 ng/ml - 10 ng/ml26,31-33. Higher concentrations of ligand may result in non-specific binding to all TGF-&#946; receptors. The concentration of TGF-&#946;1 should reflect the specific experimental goals.</li>
</ol>
3. Gene Expression Analysis of Epicardial Outgrowths
<ol>
	<li>Thaw epicardium-depleted hearts previously stored in Trizol (2.2).</li>
	<li>Aspirate media from EPDC cultures and wash twice with DPBS.</li>
	<li>Add 800 &#181;l room temperature Trizol to explant cultures.</li>
	<li>Incubate epicardium-depleted hearts and EPDC cultures in Trizol for 5 min at room temperature. Pipette up and down ten times to homogenize the lysed sample. 
	Note: Hearts may require further pipetting to completely homogenize.</li>
	<li>Transfer lysates to 1.5 ml tubes and proceed with total RNA isolation per manufacturer&#39;s instructions. Add 10 &#181;g glycogen prior to precipitation to increase RNA yield. A given explant will yield approximately 0.5-1.0 &#181;g RNA.</li>
	<li>Remove contaminating DNA with DNase I and reverse transcribe 0.5 &#181;g mRNA per manufacturer&#39;s instructions.</li>
	<li>Validate the purity of epicardial explants and/or induction of EMT with quantitative reverse transcription (qRT)-PCR 34 using SYBR green and the markers described in Table 1. Normalize mRNA levels to glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and calculate relative expression using the 2-&#916;&#916;Ct method 35. Typical results are shown in Figure 2A.</li>
</ol>
4. Protein Analysis of Epicardial Outgrowths
<ol>
	<li>Aspirate media from EPDC cultures and wash twice with DPBS.</li>
	<li>Add 10 &#181;l ice-cold protein lysis buffer (50 mM Tris pH 7.5, 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid pH 8.0, 1% Triton X-100, 1x protease inhibitor cocktail) to each chamber well and place the chamber slide on ice. Cut off approximately &#188; of the bottom on a P200 pipette tip and use the remaining piece to scrape the cells off of the slide. Pipette up and down to lyse epicardial cells and transfer to a 1.5 ml tube.</li>
	<li>Sonicate lysed explants on low (160 W) for 5 min in an ice water bath with 30 sec ON and OFF cycles.</li>
	<li>Centrifuge lysates for 10 min at 10,000 x g, 4 &#176;C. Transfer supernatant to a fresh tube and determine the protein concentration using a protein assay36. 
	Note: A given explant will yield up to 1.5 &#181;g total protein. We have found that pooling explants from two hearts and loading 2 &#181;g protein per well is sufficient to detect smooth muscle &#945;-actin (ACTA2) and GAPDH 9. The detection of other target proteins may require pooling multiple explant samples as determined by the end user.</li>
</ol>
5. Immunocytochemistry of Epicardial Outgrowths
<ol>
	<li>Remove media from explant cultures and wash twice with DPBS.</li>
	<li>Gently add 500 &#181;l of 4% (v/v) paraformaldehyde or ice-cold methanol to each well and fix explants for 5 min at room temperature or 10 min at -20&#160;oC, respectively. Fix according to antibody specifications as recommended by the manufacturer.</li>
	<li>Remove fixative and rinse each well with DPBS three times for 5 min.</li>
	<li>Permeabilize the cells with 500 &#181;l of 0.1% (v/v) Triton X-100 in DPBS for 5 min.</li>
	<li>Remove permeabilization solution and rinse with DPBS three times for 5 min.</li>
	<li>Block the cells with 10% serum in DPBS for 30 min at room temperature.</li>
	<li>Proceed with primary antibody staining per manufacturer&#39;s recommendations and optimal conditions as determined by the end user.</li>
	<li>Remove antibody solution and rinse with DPBS three times for 5 min.</li>
	<li>Apply secondary antibody per manufacturer&#39;s instructions and counterstain with an appropriate nuclear dye.</li>
	<li>Remove antibody solution and rinse with DPBS three times for 5 min.</li>
	<li>Remove chamber slide walls per manufacturer&#8217;s instructions and add aqueous mounting medium directly to the cells.&#160; Place a coverslip over the cells and seal with nail polish. Typical results are shown in Figure 2B-2D using inverted confocal scanning microscopy.</li>
</ol>
6. Ex Vivo Culture Assay (Figure 3A)
<ol>
	<li>Preparations
	<ol>
		<li>Prepare 12 ml ex vivo culture medium using a (1:1) mixture of Dulbecco&#39;s Modified Eagle Medium (DMEM):M199 supplemented with 10% FBS and 1% Pen/Strep.</li>
		<li>In a 37 &#176;C water bath, pre-warm ex vivo culture medium and 100 ml HBSS.</li>
		<li>Aliquot 1 ml pre-warmed medium into 8-12 wells of a 24 well plate.</li>
		<li>Transfer pre-warmed HBSS to two 10 cm2 plates, containing 50 ml each.</li>
	</ol>
	</li>
	<li>Isolate mouse embryonic hearts in HBSS (as described in 1.2) from pregnant dams at 12.5 dpc.</li>
	<li>Transfer each heart to a single well of the 24 well plate containing ex vivo culture medium. Incubate the plate at 37 &#176;C, 5% CO2 with gentle rocking manually or using a rocking platform to prevent adherence. Immediately proceed to step 7.1.</li>
</ol>
7. Epicardial Labeling and EMT Induction in Ex Vivo Cultures
<ol>
	<li>To label the epicardium, prepare 12 ml of 37 &#176;C ex vivo culture medium supplemented with 3.0 x 106 pfu/ml of Ad/GFP. For targeted ablation of floxed alleles, transfer 6 ml of Ad/GFP supplemented culture medium to a new 15 ml conical tube. Add Ad/Cre or control adenovirus to a final concentration of 1.5 x 106 pfu/ml. 
	Note: Gain of function analyses can also be carried out with appropriate adenoviral mediated gene delivery.</li>
	<li>Carefully remove the culture medium from each well of the 24 well plate using a P1,000 pipette tip.</li>
	<li>Gently add culture medium supplemented with adenovirus(es) to each well.</li>
	<li>Incubate the plate at 37 &#176;C, 5% CO2 for 24 hr with gentle rocking.</li>
	<li>For EMT induction, prepare 12 ml of 37 &#176;C ex vivo culture medium containing 10 ng/ml TGF-&#946;1 and 20 ng/ml platelet-derived growth factor (PDGF)-BB or vehicle (4 mM HCl containing 1 mg/ml BSA).</li>
	<li>Carefully remove culture medium from each well of the 24 well plate using a P1,000 pipette tip. Gently rinse the hearts with 1 ml DPBS.</li>
	<li>Remove the DPBS and replenish hearts with ex vivo culture medium containing TGF-&#946;1 and PDGF-BB or vehicle.</li>
	<li>Incubate for 24 hr at 37 &#176;C, 5% CO2 with gentle rocking.</li>
</ol>
8. Cryopreservation and Immunohistochemistry of Ex Vivo Cultures
<ol>
	<li>Prepare the Hearts for Cryopreservation.
	<ol>
		<li>Remove the culture medium and wash hearts twice with ice-cold DPBS.</li>
		<li>Add 1 ml of 4% (v/v) paraformaldehyde to each heart and fix for 2 hr at 4 &#176;C with gentle rocking.</li>
		<li>Remove the fixative and wash hearts twice with DPBS. Transfer the hearts to fresh wells with 1 ml 10% (w/v) sucrose prepared in DPBS. Incubate the plate overnight at 4 &#176;C with gentle rocking.</li>
		<li>Carefully transfer hearts to new wells with 18% (w/v) sucrose prepared in DPBS. Incubate the plate overnight at 4 &#176;C with gentle rocking.</li>
		<li>Transfer each heart to a cryomold containing tissue freezing medium. Immediately freeze blocks using liquid nitrogen and store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Section hearts at 5 &#181;m using a cryostat and place tissue sections on positively charged microscope slides. Store slides at -80 &#176;C until staining.</li>
	<li>Perform immunohistochemistry on sections for detection of the sub-epicardial basement membrane.
	<ol>
		<li>Thaw slides for at least 30 min at room temperature.</li>
		<li>Rehydrate the sections by washing twice in DPBS for 5 min with gentle rocking.</li>
		<li>Permeabilize section with 0.1% (v/v) Triton X-100 in PBS for 5 min at room temperature.</li>
		<li>Wash the slides twice in DPBS for 5 min with gentle rocking.</li>
		<li>Blot off excess DPBS and define the staining region with a hydrophobic labeling pen. Block sections with 10% serum in DPBS for 30 min at room temperature.</li>
		<li>Remove block and apply collagen type IV antibody (ColIV, 1:200) diluted in block. Incubate overnight at 4&#160;&#176;C in a humidity-controlled chamber.</li>
		<li>Wash the slides three times in DPBS for 5 min with gentle rocking.</li>
		<li>Dilute fluorescently conjugated secondary antibody (1:500) and DAPI (1:5,000) in DPBS and apply to the slides. Incubate for 1 hr at room temperature. Choose an appropriate secondary antibody with an emission and excitation wavelength distinct from GFP.</li>
		<li>Wash the slides three times in PBS for 5 min with gentle rocking. Mount slides with mounting medium.</li>
	</ol>
	</li>
</ol>
9. Imaging and Quantification of Ex Vivo Cultures
<ol>
	<li>Have a blinded user image and quantify stained sections. Image at least five non-consecutive tissue sections from a given heart in 4-5 arbitrary locations along the epicardium (edge of the heart) using a fluorescent or confocal microscope at 40X magnification. Typical results are shown in Figure 3B using inverted confocal scanning microscopy.</li>
	<li>Manually count the total number of GFP positive cells in a given image. Identify migrating EPDC as GFP positive cells located within or beyond the ColIV subepicardial basement membrane. Determine the percent of GFP migrating cells as the number of migrating GFP positive cells over the total number of GFP positive cells in a given image. Typical results are shown in Figure 3C-D using inverted confocal scanning microscopy.</li>
</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Hyclone&amp;nbsp;</td><td>SH3002201</td><td>DMEM</td></tr><tr><td>Hanks&#039; Balanced Salt Solution</td><td>Hyclone</td><td>SH3003102</td><td>HBSS</td></tr><tr><td>Medium 199</td><td>Hyclone</td><td>SH3025301</td><td>M199</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline&amp;nbsp;</td><td>Hyclone</td><td>SH3002802</td><td>DPBS</td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gemini&amp;nbsp;</td><td>100-106</td><td>FBS</td></tr><tr><td>Penicillin/Streptomycin Solution</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>Corning Biocoat Collagen I, 4-Well Culture Slides</td><td>Corning</td><td>354557</td><td></td></tr><tr><td>Multiwell 24-well plates</td><td>Falcon</td><td>08-772-1</td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>M50</td><td>Equipped with Leica KL300 LED might source</td></tr><tr><td>Confocal miscroscope</td><td>Olympus</td><td>1X81</td><td>Equipped with muli-argon laser 405/488/515, FV10-MCPSU laser 405/440/635, 559 laser, and mercury lamp</td></tr><tr><td>Bioruptor</td><td>Diagenode&amp;nbsp;</td><td>UCD-200</td><td></td></tr><tr><td>Transforming growth factor beta 1</td><td>R&amp;amp;D Systems</td><td>100-B-001</td><td>TGF-&amp;beta;1</td></tr><tr><td>Platelet-derived growth factor BB</td><td>R&amp;amp;D Systems</td><td>220-BB-010</td><td>PDGF-BB</td></tr><tr><td>Trizol Reagent</td><td>Applied Biosystems</td><td>15596-026</td><td>Caution, hazardous material</td></tr><tr><td>TURBO DNA-free Kit</td><td>Life Technologies</td><td>AM1907</td><td>DNaseI</td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>BioRad</td><td>1708891</td><td></td></tr><tr><td>UltraPure Glycogen</td><td>Life Technologies</td><td>10814-010</td><td></td></tr><tr><td>SYBR Green</td><td>BioRad</td><td>170-8880</td><td></td></tr><tr><td>Protease inhibtor cocktail tablets</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>Alexa Goat-anti-rabbit 488</td><td>Life Technologies</td><td>A11008</td><td>Secondary antibody</td></tr><tr><td>Alexa Goat anti-rabbit 594</td><td>Life Technologies</td><td>A-11012</td><td>Secondary antibody</td></tr><tr><td>Alexa Goat anti-mouse 594</td><td>Life Technologies</td><td>A-11005</td><td>Secondary antibody</td></tr><tr><td>Mouse anti-smooth muscle alpha actin, Cy3-conjugated&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C6198</td><td>monoclonal antibody, clone 1A4</td></tr><tr><td>Mouse anti-Wilms tumor 1 (WT1)</td><td>Life Technologies</td><td>MA1-46028</td><td>monoclonal antibody, clone 6F-H2</td></tr><tr><td>Rabbit anti-ZO1</td><td>Life Technologies</td><td>40-2200</td><td>polyclonal antibody</td></tr><tr><td>Rabbit anti-Collagen Type 4</td><td>Millipore</td><td>AB756P</td><td>polyclonal antibody</td></tr><tr><td>4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride&amp;nbsp;</td><td>Life Technologies</td><td>D1306</td><td>DAPI</td></tr><tr><td>Fluorescent mounting medium&amp;nbsp;</td><td>DAKO</td><td>S3023</td><td></td></tr><tr><td>16% Paraformaldehyde solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td>PFA diluted to 4% in DPBS. Caution, hazardous material</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Tissue Freezing Medium</td><td>Triangle Biomedical Sciences</td><td>TFM-B</td><td></td></tr><tr><td>Pap pen</td><td>DAKO</td><td>S2002</td><td></td></tr><tr><td>Disposable mictrotome blades&amp;nbsp;</td><td>Sakura Finetek</td><td>4689</td><td></td></tr><tr><td>Microscope slides</td><td>Globe Scientific</td><td>1358W</td><td>positively charged, 25 mm x 75 mm x 1 mm</td></tr><tr><td>Cryostat&amp;nbsp;</td><td>Leica</td><td>CM1950</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11295-00</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>91460-11</td><td></td></tr><tr><td>Disposable base molds</td><td>Fisher Scientific</td><td>22-363-553</td><td></td></tr><tr><td>Ketamine</td><td>Hospira</td><td>NDC 0409-2051-05</td><td></td></tr><tr><td>Xylazine</td><td>Akorn</td><td>NADA 139-236</td><td></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 ...

epicardial-outgrowth-culture-assay-ex-vivo-assessment-epicardial

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JoVE Journal

Developmental Biology

8.2K Views.  University of Rochester School of Medicine and Dentistry. 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. 

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

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

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.

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

Engineering

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

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

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

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

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

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Biology

Electroporation-Based Transgenesis of Cardiac Cells in Mouse Hearts

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

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

Author Spotlight: Cardiac Cell Transgenesis for Rapid Gene Screening

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

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

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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

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

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

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

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