Ang1-induced CD31⁺/CD144⁺ ECs have a neovasculogenic potential in vitro and in vivo. Purified Flk-1⁺ MPCs differentiated from mouse ESCs were cultured for 3 days on OP9 cells. Ang1 was administered on days 0 and 2. CD31⁺/CD144⁺ cells were sorted by FACS on day 3 and used as Ang1-EC-ESCs for further experiments. (A) Phase contrast images showing Ang1-EC-ESCs. Scale bar represents 100 μm. (B) Immunostaining images showing Ang1-EC-ESCs. Nuclei were stained with Hoechst. Scale bar represents 100 μm. (C) Images showing network formations of Ang1-EC-ESCs and HUVECs. Ang1-EC-ESCs and HUVECs were plated on Matrigel-covered plates and incubated in EGM-2. The tube structures were observed after 24 hours. Scale bar represents 200 μm. (D-E) Ang1-mESC-ECs were injected subcutaneously into the dorsal skin of cyclosporine A–treated CD31-deficient mice in Matrigel. The implanted Matrigel was harvested 14 days after injection of FITC-lectin 20 minutes before the mice were killed; The presence of CD31⁺ vessel-like structures containing FITC-lectin can be identified in regions 1 and 2, indicating that effective perfusion had taken place in the implanted Ang1-EC-ESCs. The bottom 2 panels are the magnified views of indicated regions. Scale bars represent 50 μm.