Granulocyte colony-stimulating factor (GCSF)-mobilized peripheral blood cells were obtained from excess leukapheresis samples processed by the Stem Cell Laboratory, Hammersmith Hospital. Informed consent and local research ethics committee approval were granted in all cases.
CD34 cells were diluted 1:4 in Hanks’ buffered saline solution (HBSS) before mononuclear cells (MNCs) were separated, by centrifugation, over a Lymphoprep density gradient at 1,800 rpm for 30 minutes. The MNC fraction was collected and first washed in HBSS, then in MACS (magnetic cell sorting) buffer (phosphate-buffered saline buffer supplemented with bovine serum albumin (0.5%) and EDTA (5 mM, pH 7.2)). CD34 cells were isolated from MNCs, using the CD34-positive cell selection kit. Isolated CD34 cells were plated on 35-mm2 Petri dishes in α -minimal essential medium (α-MEM) supplemented with fetal bovine serum (FBS; 15%) and incubated for 2 hours at 37°C in 5% CO2. After 2 hours, the nonadherent cell fraction was removed by washing the plates three times. Adherent CD34 cells were cultured in α-MEM supplemented with FBS (30%) and cytokines (20 ng/ml stem cell factor [SCF], GM-SCF (1ng/ml), IL-3 (5 ng/ml), and G-CSF (100 ng/ml)) at 37°C in 5% CO2 in air.