Distinctive response of human HSCs and progenitors to oxidative DNA damage. (A) CB Lin⁻CD34⁺CD38⁻ cells and Lin⁻CD34⁺CD38⁺ cells were cultured with or without BSO for 2 days. Intracellular ROS concentrations were determined by the intensity of DCF-DA staining using a flow cytometor. The relative ROS level is presented as a fold induction compared with the MFI value detected in cells cultured without BSO. Data from 2 independent experiments are shown (**P < .01). (B) Two days after BSO treatment, cells were pulsed with BrdU and analyzed by flow cytometry. Data from 2 independent experiments are shown (**P < .01). (C) Relative expressions of p16^INK4a, p14^ARF, and p21^CIP1 in Lin⁻CD34⁺CD38⁻ cells and Lin⁻CD34⁺CD38⁺ cells cultured with or without BSO were analyzed at the end of 2-day culture by quantitative real-time PCR. Each value was normalized to 18S rRNA expression and is presented as a fold increase compared with the levels detected in cells cultured without BSO (**P < .01). (D) CB Lin⁻CD34⁺CD38⁻ cells and Lin⁻CD34⁺CD38⁺ cells cultured with BSO for 2 days were stained with γ-H2AX and p21^CIP1 mAbs (γ-H2AX: green; p21^CIP1: red; DAPI: blue). Bars represent 5 μm. (E) At 2 days after BSO-treatment, the frequency of apoptotic cells was determined by annexin V/PI staining. Data from 3 independent experiments are shown (**P < .01). (F) The number of γ-H2AX foci per cell in Lin⁻CD34⁺CD38⁻ cells and Lin⁻CD34⁺CD38⁺ cells at the indicated time points. Cells cultured with BSO for 2 days were washed, replaced with fresh BSO-free media, and cultured for additional 1, 2, or 3 days (n = 4 in each time point; *P < .05, **P < .01, Lin⁻CD34⁺CD38⁻ cells vs Lin⁻CD34⁺CD38⁺ cells).