DDR in physiologic HSC aging. (A) Bone marrow Lin⁻CD34⁺CD38⁻ cells isolated from middle-aged (n = 6) and elderly individuals (n = 10) as well as HSCT patients (n = 8) were immunostained for γ-H2AX. The number of γ-H2AX foci per cell for each subject is shown as the box and whisker plot. More than 50 cells in random fields on a slide were counted. (B) Lin⁻CD34⁺CD38⁻ cells were isolated from HSCT patients and elderly individuals, and intracellular ROS concentrations were determined by the intensity of DCF-DA staining using a flow cytometer. The relative ROS level is presented as a fold induction compared with the MFI value detected in Lin⁻CD34⁺CD38⁻ cells isolated from CB. Data were collected from 3 independent experiments (*P < .05). (C) Bone marrow CD34⁺ cells (1 × 10⁴ cells) isolated from middle-aged and elderly individuals and HSCT patients were transplanted into NOG mice. Eight weeks after transplantation, engraftment levels of human hematopoietic cells were assessed by flow cytometry (4 recipients in each group).