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CD34+/CD133+ cells were isolated from human bone marrow cells.
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.