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

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)

1. Preparations
   1. 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).
   2. Pre-warm medium A and 100 ml Hanks' Balanced Salt Solution (HBSS) in a 37 °C water bath.
   3. Allow collagen-coated chamber slides to warm to room temperature for 30 min.
   4. Add 100 µl 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.
   5. Aliquot pre-warmed HBSS into two 10 cm² plates containing 50 ml each of HBSS.
2. Isolate Embryonic Hearts from Pregnant Dam at 11.5 Days Post Coitum (dpc).³⁰
   1. Anesthetize mouse with 0.5 ml of 13 mg/ml ketamine/0.88 mg/ml xylazine cocktail in 1x Dulbecco'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.
   2. Lay the mouse facing ventral side up and spray the abdomen with 70% ethanol to reduce contamination by hair.
   3. 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.
   4. Pinch the peritoneum with forceps and cut with scissors to expose the abdominal cavity.
   5. Carefully remove the decidua by cutting along the mesometrium. Transfer the dissected decidua to pre-warmed HBSS in the first 10 cm² plate.
   6. Separate the decidua by cutting between embryos.
   7. 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.
   8. Pinch the thoracic wall with forceps and tear the tissue apart at the midline, exposing the chest cavity.
   9. 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.
3. 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.
4. Once all hearts have been transferred to individual wells, incubate the chamber slide for 30 min at 37 °C, 5% CO₂ to allow sufficient adherence to the collagen matrix.
5. Carefully add 20-50 µl 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.
6. Culture the hearts at 37 °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.

2. Genetic Ablation and Induction of EMT in Epicardial Outgrowths

Caution: Handle adenoviruses with care according to approved biosafety guidelines.

1. Prepare 15 ml medium B (M199 supplemented with 1% FBS and 1% Pen/Strep) and pre-warm to 37 °C in a water bath.
2. 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.
3. Carefully remove epicardium-depleted hearts from outgrowth cultures using forceps. Transfer hearts to 1.5 ml tubes with 800 µl Trizol and store at -80 °C for later RNA isolation and gene expression analysis.
4. Wash each well with DPBS to remove blood cells and excess cellular debris.
5. Add 500 µl explant medium B supplemented with adenovirus(es) to each outgrowth culture and incubate for 24 hr at 37 °C, 5% CO₂.
6. For EMT induction, remove media and replenish with 37 °C explant medium B supplemented with recombinant transforming growth factor (TGF)-β1 [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 °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-β1 ranging from 0.5 ng/ml – 10 ng/ml^26,31-33. Higher concentrations of ligand may result in non-specific binding to all TGF-β receptors. The concentration of TGF-β1 should reflect the specific experimental goals.

3. Gene Expression Analysis of Epicardial Outgrowths

1. Thaw epicardium-depleted hearts previously stored in Trizol (2.2).
2. Aspirate media from EPDC cultures and wash twice with DPBS.
3. Add 800 µl room temperature Trizol to explant cultures.
4. 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.
5. Transfer lysates to 1.5 ml tubes and proceed with total RNA isolation per manufacturer's instructions. Add 10 µg glycogen prior to precipitation to increase RNA yield. A given explant will yield approximately 0.5-1.0 µg RNA.
6. Remove contaminating DNA with DNase I and reverse transcribe 0.5 µg mRNA per manufacturer's instructions.
7. 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^–ΔΔCt method ³⁵. Typical results are shown in Figure 2A.

4. Protein Analysis of Epicardial Outgrowths

1. Aspirate media from EPDC cultures and wash twice with DPBS.
2. Add 10 µl 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 ¼ 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.
3. Sonicate lysed explants on low (160 W) for 5 min in an ice water bath with 30 sec ON and OFF cycles.
4. Centrifuge lysates for 10 min at 10,000 x g, 4 °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 µg total protein. We have found that pooling explants from two hearts and loading 2 µg protein per well is sufficient to detect smooth muscle α-actin (ACTA2) and GAPDH ⁹. The detection of other target proteins may require pooling multiple explant samples as determined by the end user.

5. Immunocytochemistry of Epicardial Outgrowths

1. Remove media from explant cultures and wash twice with DPBS.
2. Gently add 500 µl 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 ^oC, respectively. Fix according to antibody specifications as recommended by the manufacturer.
3. Remove fixative and rinse each well with DPBS three times for 5 min.
4. Permeabilize the cells with 500 µl of 0.1% (v/v) Triton X-100 in DPBS for 5 min.
5. Remove permeabilization solution and rinse with DPBS three times for 5 min.
6. Block the cells with 10% serum in DPBS for 30 min at room temperature.
7. Proceed with primary antibody staining per manufacturer's recommendations and optimal conditions as determined by the end user.
8. Remove antibody solution and rinse with DPBS three times for 5 min.
9. Apply secondary antibody per manufacturer's instructions and counterstain with an appropriate nuclear dye.
10. Remove antibody solution and rinse with DPBS three times for 5 min.
11. Remove chamber slide walls per manufacturer’s instructions and add aqueous mounting medium directly to the cells.  Place a coverslip over the cells and seal with nail polish. Typical results are shown in Figure 2B–2D using inverted confocal scanning microscopy.

6. Ex Vivo Culture Assay (Figure 3A)

1. Preparations
   1. Prepare 12 ml ex vivo culture medium using a (1:1) mixture of Dulbecco's Modified Eagle Medium (DMEM):M199 supplemented with 10% FBS and 1% Pen/Strep.
   2. In a 37 °C water bath, pre-warm ex vivo culture medium and 100 ml HBSS.
   3. Aliquot 1 ml pre-warmed medium into 8-12 wells of a 24 well plate.
   4. Transfer pre-warmed HBSS to two 10 cm² plates, containing 50 ml each.
2. Isolate mouse embryonic hearts in HBSS (as described in 1.2) from pregnant dams at 12.5 dpc.
3. Transfer each heart to a single well of the 24 well plate containing ex vivo culture medium. Incubate the plate at 37 °C, 5% CO₂ with gentle rocking manually or using a rocking platform to prevent adherence. Immediately proceed to step 7.1.

7. Epicardial Labeling and EMT Induction in Ex Vivo Cultures

1. To label the epicardium, prepare 12 ml of 37 °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.
2. Carefully remove the culture medium from each well of the 24 well plate using a P1,000 pipette tip.
3. Gently add culture medium supplemented with adenovirus(es) to each well.
4. Incubate the plate at 37 °C, 5% CO₂ for 24 hr with gentle rocking.
5. For EMT induction, prepare 12 ml of 37 °C ex vivo culture medium containing 10 ng/ml TGF-β1 and 20 ng/ml platelet-derived growth factor (PDGF)-BB or vehicle (4 mM HCl containing 1 mg/ml BSA).
6. 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.
7. Remove the DPBS and replenish hearts with ex vivo culture medium containing TGF-β1 and PDGF-BB or vehicle.
8. Incubate for 24 hr at 37 °C, 5% CO₂ with gentle rocking.

8. Cryopreservation and Immunohistochemistry of Ex Vivo Cultures

1. Prepare the Hearts for Cryopreservation.
   1. Remove the culture medium and wash hearts twice with ice-cold DPBS.
   2. Add 1 ml of 4% (v/v) paraformaldehyde to each heart and fix for 2 hr at 4 °C with gentle rocking.
   3. 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 °C with gentle rocking.
   4. Carefully transfer hearts to new wells with 18% (w/v) sucrose prepared in DPBS. Incubate the plate overnight at 4 °C with gentle rocking.
   5. Transfer each heart to a cryomold containing tissue freezing medium. Immediately freeze blocks using liquid nitrogen and store at -80 °C.
2. Section hearts at 5 µm using a cryostat and place tissue sections on positively charged microscope slides. Store slides at -80 °C until staining.
3. Perform immunohistochemistry on sections for detection of the sub-epicardial basement membrane.
   1. Thaw slides for at least 30 min at room temperature.
   2. Rehydrate the sections by washing twice in DPBS for 5 min with gentle rocking.
   3. Permeabilize section with 0.1% (v/v) Triton X-100 in PBS for 5 min at room temperature.
   4. Wash the slides twice in DPBS for 5 min with gentle rocking.
   5. 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.
   6. Remove block and apply collagen type IV antibody (ColIV, 1:200) diluted in block. Incubate overnight at 4 °C in a humidity-controlled chamber.
   7. Wash the slides three times in DPBS for 5 min with gentle rocking.
   8. 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.
   9. Wash the slides three times in PBS for 5 min with gentle rocking. Mount slides with mounting medium.

9. Imaging and Quantification of Ex Vivo Cultures

1. 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.
2. 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.