Isolation of endothelial cells and their progenitor cells from human peripheral blood

Purpose: We have developed techniques to isolate endothelial cell. (EC) pro genitors from human peripheral and umbilical cord blood. Methods: Human adult peripheral and umbilical cord blood Monocytes were iso lated by centrifugation, and progenitor cells were separated with the use o f magnetic polystyrene beads that were coated with a monoclonal antibody sp ecific for the CD34 cell-membrane antigen. Cells were propagated in selecti ve media, and developing cultures were immunostained for CD31, CD34, factor VIII, and vascular endothelial growth factor cell receptors. ECs that deve loped were transfected with a gene for prourokinase and used to line ePTFE grafts, which were evaluated in vitro in a pulsatile flow system. Results: Umbilical cord monocyte cultures demonstrated colonies that resemb led ECs at approximately 2 weeks, with growth being best supported by EC gr owth media plus 20% calf serum with iron. Immunostaining of colonies was po sitive for CD31 and factor WI. After 18 days in culture, CD34+ cells from a dult peripheral blood were noted, which had the typical cobblestone appeara nce of ECs and immunostained positively for CD31 and factor VIII-related an tigens. Cultures of umbilical cord-derived cells and adult peripheral blood -derived cells developed complex line formations within 1 week in culture t hat stained positively for vascular endothelial growth factor receptor-2. U rokinase-transfected ECs were shown to overexpress urokinase. Prosthetic gr afts lined with transfected cells showed 87.33% +/- 4.97% cell adherence af ter 2 hours in a pulsatile now system at clinically relevant shear stress. Conclusion: We conclude that endothelial progenitor cells can be isolated f rom human adult peripheral and umbilical cord blood and developed into EC c ultures as a source of cells for vascular graft seeding and gene therapy.