Functional characterization of cord blood–derived endothelial cells. (A) Western blotting of cell lysates of HUVECs and 2 samples of cord blood–derived cell monolayers showing expression of eNOS (top left panel). The bottom panel shows the same blot reprobed with an anti–β-actin antibody to demonstrate equal protein loading. (B) Flow cytometric analysis of VCAM-1 expression in cord blood–derived endothelial cells cultured for 8 hours in the presence or absence of IL-1β (25 ng/mL). The dotted vertical line marks the upper limit of the IgG isotype control. Representative of 3 experiments. (C) Representative microscopic fields of cord blood–derived endothelial cells, from 2 independent donors, forming cordlike structures after 16 hours' culture on Matrigel. (D) Confocal microscopy of Matrigel/cord blood–derived endothelial cell plugs transplanted into NOD/SCID mice, showing the development of human blood vessels. Functional human blood vessels are identified by the uptake of FITC-ISB4, coexpression of human CD34 and VWF, and endothelial markers PECAM-1 and VE-cadherin (arrowheads). Lower panels show background for red and blue fluorophores using isotype control IgGs. Vascular structures are still visible by the green fluorescence of FITC-ISB4. Reference bar is 50μm.