Fig. 5.

Fetal epicardium-derived VSMCs promote axon outgrowth from fetal sympathetic ganglia in vitro. (A) Schematic illustrating the preparation of coronary VSMCs. E12.5 or E13.5 cardiac ventricles were cultured in collagen gel for 2 days. The ventricles were removed from the gel, and migrated epicardial cells were harvested and further expanded on a type IV collagen-coated dish. VSMC aggregations were obtained using a hanging drop culture method. (B,C) Primary culture of epicardial-derived VSMCs stained with antibodies to VSMC markers SM22α (B,C, green) and αSMA (B, red), and SM-MHC (C, green), together with the nuclear dye To-pro-3 (blue). (D-H) Co-culture of coronary VSMCs and sympathetic ganglia (SG). E13.5 fetal SG were cultured with VSMC aggregations obtained as in A and stained with anti-TUJ1 antibody (green) and To-pro-3 (blue). Magnified images (E,F) show the boxed regions in D. Note that directional axon outgrowth towards VSMCs was observed (E). The total lengths of axons in the forward and reverse quadrants (G) were calculated using Volocity (H). n=19. (I-N) Co-culture of myocardium explants and SG. SG were cultured with fetal myocardial tissue explants and labeled with anti-TUJ1 antibody (green) and To-pro-3 (blue). E13.5 myocardial tissue explants failed to stimulate axon outgrowth from E13.5 and E16.5 SG explants (I,K), whereas E16.5 myocardial tissue explants successfully induced directional axon outgrowth from both E13.5 and E16.5 SG explants (J,L). The total number of axons in the forward region (M) for each sample was quantified using Volocity (N). E13.5 SG with E13.5 myo, n=7; E13.5 SG with E16.5 myo, n=8; E16.5 SG with E13.5 myo, n=5, E16.5 SG with E16.5 myo, n=6. ^*P<0.01 (Student’s t-test); error bars indicate s.e.m. Myo, myocardium.