Umbilical cord blood-derived endothelial progenitor cells (National Cancer Center Research Institute, Tokyo)

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.