Inhibition of ROS elevation by antioxidant treatment in an in vitro RIBE model lessens the functional deterioration of human HSPCs. (A) Experimental diagram of in vitro antioxidant treatment RIBE model; human BM cells were treated with single antioxidant alone or in combination for 30 minutes before irradiation, and the antioxidants continued to exist in the coculture system for 40 hours. (B) Flow cytometric analysis of fold change of ROS levels by DCF-DA staining of human CD34⁺ cells from each group. (C-D) Human CD34⁺ cells from each group were immunostained for p-p53 and γ-H2AX (p-p53 and γ-H2AX, green; 4′,6-diamidino-2-phenylindole, blue). Scatter plots represent foci per cell from each group (scale bars, 7 μm). (E) Frequency of Annexin V⁺ cells of human CD34⁺ cells from each group. (F) Collected human CD34⁺ cells from panel A were transplanted into NOG mice. Mean human cell engraftment (left) was detected and bars represent the fold difference of engraftment levels from each group (right, n = 6-8 per group). Frequency of human (G) CD34⁺CD38⁻ cells and (H) CD34⁺CD38⁺ cells in the BM of recipient. (I) Mean engraftment level of human cells in the spleen of recipients. (J) Number of hematopoietic clones formed by human CD34⁺ cells of each group (*P < .05; **P < .01; ***P < .001).