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 border="0" cellpadding="0" cellspacing="0" width="751">
	<tbody>
		<tr height="40">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Fetal Bovine Serum&#160;</td>
			<td style="width:163px;">Gemini&#160;</td>
			<td style="width:141px;">100-106</td>
			<td style="width:203px;">FBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Penicillin/Streptomycin Solution</td>
			<td style="width:163px;">Hyclone</td>
			<td style="width:141px;">SV30010</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Corning Biocoat Collagen I, 4-Well Culture Slides</td>
			<td style="width:163px;">Corning</td>
			<td style="width:141px;">354557</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Multiwell 24-well plates</td>
			<td style="width:163px;">Falcon</td>
			<td style="width:141px;">08-772-1</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Dissecting microscope</td>
			<td style="width:163px;">Leica</td>
			<td style="width:141px;">M50</td>
			<td style="width:203px;">Equipped with Leica KL300 LED might source</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:244px;">Confocal miscroscope</td>
			<td style="width:163px;">Olympus</td>
			<td style="width:141px;">1X81</td>
			<td style="width:203px;">Equipped with muli-argon laser 405/488/515, FV10-MCPSU laser 405/440/635, 559 laser, and mercury lamp</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Bioruptor</td>
			<td style="width:163px;">Diagenode&#160;</td>
			<td style="width:141px;">UCD-200</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Transforming growth factor beta 1</td>
			<td style="width:163px;">R&#38;D Systems</td>
			<td style="width:141px;">100-B-001</td>
			<td style="width:203px;">TGF-&#946;1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Platelet-derived growth factor BB</td>
			<td style="width:163px;">R&#38;D Systems</td>
			<td style="width:141px;">220-BB-010</td>
			<td style="width:203px;">PDGF-BB</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Trizol Reagent</td>
			<td style="width:163px;">Applied Biosystems</td>
			<td style="width:141px;">15596-026</td>
			<td style="width:203px;">Caution, hazardous material</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">TURBO DNA-free Kit</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:141px;">AM1907</td>
			<td style="width:203px;">DNaseI</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">iScript cDNA Synthesis Kit</td>
			<td style="width:163px;">BioRad</td>
			<td style="width:141px;">1708891</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">UltraPure Glycogen</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:141px;">10814-010</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">SYBR Green</td>
			<td style="width:163px;">BioRad</td>
			<td style="width:141px;">170-8880</td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<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>
			<td style="width:203px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Alexa Goat-anti-rabbit 488</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:141px;">A11008</td>
			<td style="width:203px;">Secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Alexa Goat anti-rabbit 594</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:141px;">A-11012</td>
			<td style="width:203px;">Secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Alexa Goat anti-mouse 594</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:141px;">A-11005</td>
			<td style="width:203px;">Secondary antibody</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Mouse anti-smooth muscle alpha actin, Cy3-conjugated&#160;</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:141px;">C6198</td>
			<td style="width:203px;">monoclonal antibody, clone 1A4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">Mouse anti-Wilms tumor 1 (WT1)</td>
			<td style="width:163px;">Life Technologies</td>
			<td>MA1-46028</td>
			<td style="width:203px;">monoclonal antibody, clone 6F-H2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Rabbit anti-ZO1</td>
			<td style="width:163px;">Life Technologies</td>
			<td>40-2200</td>
			<td style="width:203px;">polyclonal antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Rabbit anti-Collagen Type 4</td>
			<td style="width:163px;">Millipore</td>
			<td style="width:141px;">AB756P</td>
			<td style="width:203px;">polyclonal antibody</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride&#160;</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:141px;">D1306</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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		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sucrose</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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		<tr height="20">
			<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>
			<td style="width:163px;">Sakura Finetek</td>
			<td style="width:141px;">4689</td>
			<td style="width:203px;"></td>
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		<tr height="40">
			<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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		<tr height="20">
			<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>
		<tr height="20">
			<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>
		</tr>
		<tr height="20">
			<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>
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		<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>
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		<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>
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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

Research

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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

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 ...

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

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

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

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

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

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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 ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...