Isolation of Endothelial Cells and Their Progenitor Cells from Human Peripheral Blood

Purpose: We have developed techniques to isolate endothelial cell (EC) progenitors from human peripheral and umbilical cord blood.

Methods: Human adult peripheral and umbilical cord blood monocytes were isolated by centrifugation, and progenitor cells were separated with the use of magnetic polystyrene beads that were coated with a monoclonal antibody specific for the CD34 cell-membrane antigen. Cells were propagated in selective media, and developing cultures were immunostained for CD31, CD34, factor VIII, and vascular endothelial growth factor cell receptors. ECs that developed 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 resembled ECs at approximately 2 weeks, with growth being best supported by EC growth media plus 20% calf serum with iron. Immunostaining of colonies was positive for CD31 and factor VIII. After 18 days in culture, CD34(+) cells from adult peripheral blood were noted, which had the typical cobblestone appearance of ECs and immunostained positively for CD31 and factor VIII-related antigens. Cultures of umbilical cord-derived cells and adult peripheral blood-derived cells developed complex line formations within 1 week in culture that stained positively for vascular endothelial growth factor receptor-2. Urokinase-transfected ECs were shown to overexpress urokinase. Prosthetic grafts lined with transfected cells showed 87.33% +/- 4.97% cell adherence after 2 hours in a pulsatile flow system at clinically relevant shear stress.

Conclusion: We conclude that endothelial progenitor cells can be isolated from human adult peripheral and umbilical cord blood and developed into EC cultures as a source of cells for vascular graft seeding and gene therapy.