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