ES cells differentiate into mature ECs and SMCs by VEGF-A treatment. Indirect immunofluorescence analysis by double staining of endothelial and mural precursors. (A-C) At 5.5 days of ES cell differentiation, ECs stained with PECAM1 (green) and α-SMA-positive cells (red) form a network on different focal planes. Nuclei were stained with DAPI as indicated. Scale bar, 40 μm. (D-E) From day 5.5, EC precursors stained with PECAM1 (green) form primitive capillary tubes, which at 8.5 days are surrounded by SMCs stained with α-SMA (red). Scale bar, 16 μm. (F-G) At 8.5 days of differentiation, ECs are positive for VE-cadherin, which is a late marker of differentiation, and are able to uptake the acetylated low-density lipoprotein (Ac-LDL) particles. Green fluorescent Ac-LDL particles were added directly to the medium of differentiated ECs and analyzed at the microscope. Scale bar, 200 μm. (H) Differentiated ECs at 8.5 days plated on matrigel form capillary-like structure in vitro. Differentiated ECs from VEGF-A-induced EBs were disaggregated and plated on Matrigel. (I) SMCs showed fully differentiated α-SMA fibers. The field was at the periphery of the EBs to observe SMCs. Scale bar, 24 μm.