Endothelial precursor cells (EPCs) were isolated from umbilical cord blood samples that were donated at normal full-term deliveries. After 7 to 14 days in culture, cells exhibit spindle-like morphology, express endothelial markers, incorporate DiI-acetylated LDL, and form tubular structures when cultured in matrigel.
Cord blood was suspended in PBS. Mononuclear cells (MNCs) from the cord blood were isolated using Ficoll-based density gradient centrifugation. Cells were then incubated with magnetic beads coated with a phycoerythrin (PE)-conjugated anti-CD34 antibody, and put through a MACS immunomagnetic separation system. The cells were seeded in a cell culture insert over mouse feeder cells, HESS-5, which were physically separated from the CD34-positive cord blood cells, and cultured in the presence of thrombopoietin (TPO), stem cell factor (SCF) and Flk-2/Flt-3 ligand (FL-2) in Stempro-34 SFM medium, for 5 days. The cells were then seeded on vitronectin-coated plates and cultured for an additional 7 days.