Vascular endothelial growth factor induces endothelial cells from vascular progenitors. (A-C) Cells after three-dimensional culture of Flk1⁺ cells (Flk-d3). (A-B) Exclusive induction of endothelial cells (ECs) and mural cells (MCs) from Flk1⁺ cells. (A) Expression of EC and MC markers. (Top panels) Double immunostaining of CD31 (green) and αSMA (red) cultured with differentiation medium (DM) alone (control). Note that no CD31⁺ cells appeared. (Other panels) Vascular endothelial growth factor (VEGF) treatment (50 ng/mL). EC sheets appeared. Double staining with pan-EC markers (CD31, VE-cadherin, Claudin5, or eNOS; green) and MC markers (αSMA, SM22α, or Calponin; red). (Bottom panels) Double staining with eNOS (green) and CD31 (red). ECs and MCs were exclusively induced. Nuclei are stained with DAPI (blue). Scale bar represents 50 μm. (B) Flow cytometry. x-axis: VE-cadherin; y-axis: CD31. Percentages of CD31⁺/VE-cadherin⁺ ECs in total Flk1⁺ cell–derived cells are indicated. (C) Flow cytometry. x-axis: CD45; y-axis: CD31. Percentages of CD31⁺/CD45⁻ ECs and CD31⁺/CD45⁺ blood cells in total Flk1⁺ cell–derived cells are indicated. Note that almost no CD45⁺ cells were induced in this culture.