HSC CD34+ lineage negative purified from peripheral blood; To obtain PB HSC cells, 12 healthy donors received glycosylated recombinant human (rh) G-CSF (Lenograstim, Rhone-Poulenc Rorer, Milan, Italy) administered subcutaneously at 10 g/kg/d for 5 6 d. Leukaphereses were performed on days 5 and 6 as previously described (Lemoli et al, 1997; Lemoli et al, 2000). Haematopoietic stem cell purification and subsequent studies were always performed using day-5 collections at the peak time of PB CD34+ cells. The protocol was approved by the ethical committee of the University Hospital and each donor gave written informed consent. Mononuclear light density (<1·077 g/ml) cells (MNC) were enriched using Ficoll-Paque (Pharmacia, Uppsala, Sweden) and resuspended in 1% bovine serum albumin (BSA; Sigma Chemical Co, St Louis, MO, USA). MNC were stained for 30 min at 4°C with a mixture of lineage-specific antibodies (CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD38, CD66b, CD41 and Glycophorin A) provided by the manufacturer (Stem Cell Technologies, Vancouver, Canada), followed by addition of secondary antibody conjugated to magnetic dextran-iron particles for a further 30 min at 4°C. Cells were then eluted through a magnetized column (StemSep; Stem Cell Technologies) to remove Lin+ cells. Lin cells were then collected according to the manufacturers instructions, incubated with anti-human CD34-fluorescein isothiocyanate (FITC) monoclonal antibody (MoAb) (HPCA-2; Becton Dickinson, San Jose, CA, USA) and sorted on a FACStar Plus equipment (Becton Dickinson). The sorting gates were set on isotype control antibody-stained samples. Sort windows were adjusted to minimize the chance of weakly positive cells contaminating the CD34 cell population. Aliquots of sorted Lin CD34+ and Lin CD34 cells, were reanalysed by FacScan to assess their purities, which were 98·5 ± 0·7% and 99·8 ± 0·1% respectively.Contamination of CD34+ cells in the CD34 cell population was ruled out by reverse transcription-polymerase chain reaction (RT-PCR) for CD34 mRNA .
