Functional characterization of MAPC-derived endothelial cells. 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 for 14 days. (a) Histamine-mediated release of vWF from MAPC-derived endothelium. MAPC-derived endothelial cells were plated at 10⁴ cells/cm² in fibronectin-coated chamber slides. After 24 hours, cells were treated with 10 μM histamine in serum-free medium with VEGF for 25 minutes. Cells were then stained with Ab’s against vWF (FITC) and myosin (Cy3). vWF staining is found throughout the cytoplasm in untreated cells, but decreased and was detectable only in the perinuclear region following treatment with histamine. Staining with Ab’s against myosin shows cytoskeletal changes with increased numbers of myosin stress fibers, and widening of gap junctions. Representative example of three experiments from three donors. Scale bar = 60 μm. (b) MAPC-derived endothelium takes up a-LDL. MAPCs induced to differentiate with VEGF for 3, 7, and 9 days with VEGF were incubated with DiI-Ac-LDL. Cells were colabeled with Ab’s against Tek, Tie1, or vWF, and analyzed by confocal microscopy. After 3 days, we detected expression of Tek, but no uptake of a-LDL. After 7 days, cells expressed Tie1, but again did not take up a-LDL. However, acquisition of expression of vWF on day 9 was associated with uptake of a-LDL. Representative example of ten experiments from six donors. Scale bar = 100 μm. (c) Vascular tube formation by MAPC-derived endothelium. MAPC-derived endothelial cells were replated in Matrigel with VEGF. After 6 hours, typical vascular tubes could be seen. Representative example of six experiments from six donors. Scale bar = 200 μm.