Embryoid body vessel development in response to different VEGF ligands. (A) Coexpression of HS and VEGFR-2 in 2D embryoid body (EB) cultures (top panels) and coexpression of NRP1 and VEGFR-2 in angiogenic sprouts in 3D EB cultures (bottom panels). Schematic representation of 2D (top) and 3D (bottom) cultures is shown to the far left. Scale bar represents 100 μm. (B) Formation of peripheral CD31-positive capillary plexus in 2D embryoid body cultures (↙ in top panels, see bottom panels for higher magnification) was induced in response to treatment with VEGF-A165 and VEGF-E-NZ2, but not VEGF-A121 or vehicle. Scale bar represents 300 μm. (C) Quantification of the CD31-positive peripheral capillary plexus area in panel B. The outer rim (defined as the outer 12.5% segment of the EB diameter) was quantified using the Image analysis 2000 software.