Peripheral blood-derived hematopoietic stem cells (Cairo University Children Hospital)

G-CSF-mobilized peripheral blood (200 ml) was obtained from leukapheresis, and CD34+ cells were isolated from the blood by adding 1 volume of blood to 5–10 volumes of lysis buffer (155 mM NH4Cl, 10mM KHCO3, and 0.1 mM EDTA) for 30 min at 4°C, to remove RBCs and to separate mononuclear cells (MNCs) for transfusion. The collected MNCs were washed twice with phosphate-buffered saline (PBS), [pH 7.4], supplemented with bovine serum albumin (0.5%) and EDTA (5 mM), and centrifuged at 1500 rpm for 10 min. The MNCs were counted, adjusted to 2 × 10^9/ml, and centrifuged. CD34+ cells were isolated using the CD34+ positive cell selection kit. The cells were cultured in α-MEM/Ham F12 (1:1) containing BSA (15%), penicillin/streptomycin (1%), GM-CSF (1 ng/ml), and growth factor (multiplication-stimulating factor buffalo rat liver extract 20 mg/ml) and incubated for 5–7 days at 37°C in 5% CO2.