Differentiation into endothelial cells requires absence of serum and high density of MAPCs. (a) MAPCs (after 65 population doublings; donor age, 22 years) were replated at 2 × 10⁴ cells/cm² in fibronectin-coated wells in serum-free medium with 10 ng/ml VEGF in the presence or absence of serum (2% FCS). After 9 days, cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and stained with Ab’s against vWF and CD34. Representative example of three experiments, one from each of three different donors. (b) MAPCs (after 65 population doublings; donor age, 22 years) were replated at 2 × 10⁴ cells/cm² or ≤ 1 × 10⁴ cells/cm² in fibronectin-coated wells in serum-free defined medium with 10 ng/ml VEGF. After 9 days, cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and stained with Ab’s against vWF and CD34. Representative example of three experiments from three donors. (c) MAPCs (after 45 population doublings; donor age, 28 years) were replated at 2 × 10⁴ cells/cm² in fibronectin-coated wells in serum-free defined medium with 10 ng/ml VEGF. After 9 days, endothelial cells were further expanded in 10% FCS with 10 ng/ml VEGF for more than 20 population doublings. Representative example of six experiments from six donors.