a) Scanning two-dimensional fluorescent images of the constructs containing cocultured green fluorescence protein (GFP)-expressing ECs were obtained after the constructs were perfused with red fluorescent dextran. Three xy images at different depths of the cell sheet (CS) regions ( b, e and h), border region ( c, f and i) and vascular bed (VB) region ( d, g and j) are presented. ( b– d) Without FGF-2 treatment, the GFP-positive EC networks remain within the cell sheets, but dextran is hardly observed within the cell sheets. ( e– g) In the case of FGF-2-treated constructs, red fluorescent dextran passed through the GFP-positive EC tubular structures. ( h– j) When 4-μm-diameter red fluorescent spheres were perfused, many microspheres were observed in the GFP-positive blood vessels of the cell sheets when treated with FGF-2. ( k) Immunostaining with CD31 (red: all ECs) and GFP (green: ECs originated from the cell sheet) demonstrated that cocultured ECs migrated into the vascular bed and connected to the blood vessels within the bed. An arrow indicates the communication between migrating ECs originated from the cell sheet and vascular bed originated ECs. ( l) Fused blood vessels containing both vascular bed (red) and cell sheet (green)-derived ECs were present in the vascular bed. ( m) By contrast, when GFP-negative cell sheets were overlaid on the GFP-expressing vascular bed, GFP-positive cells never migrated into the cell sheet layers.