Bone marrow-derived endothelial progenitor cells (University of Milan)

Bone marrow samples were collected from healthy donors and placed in  tubes containing lithium heparin and then layered on a Ficoll gradient. The low-density mononuclear cells (LDMNCs) were washed twice and resuspended in Iscove's modified Dulbecco's medium (IMDM), supplemented with fetal bovine serum (10%, FBS) and  EDTA (5 mM).

CD133-enriched cells were isolated, using magnetic bead separation, plated in fibronectin-coated tissue culture flasks, and cultured in M199 medium supplemented with  FBS (10%), vascular endothelial growth factor (VEGF,50 ng/ml ),  basic-fibroblast growth factor (bFGF, 1 ng/ml) and  insulin-like growth factor-1 (IGF-1, 2 ng/ml), for 3-4 weeks. Endothelial cells were further enriched by magnetic selection for the UEA-1 marker. Selected cells were seeded on fibronectin plates and cultured in the same medium, as described above.