Transcriptional changes, faster exit from quiescence and reduced repopulation capacity of aged pre-activated HSPCs. (A) Volcano plot showing significant (FDR < 0.05) downregulated and upregulated genes between old and young dataset after an overnight activation in culture. (B) Bar plot displaying significant (FDR < 0.05) GSEA categories from the Reactome Database used for the generation of the heatmap shown in Fig. 5 C. Bar length represents the normalized enrichment score (NES) for each category, and the bar color shows the adjusted P-value. (C) Left: Representative overlaid histogram in of the dilution of CellTracker dye during proliferation of young and aged sorted HSCs+MPP populations at different time points. Right: Automatic cell proliferation analyses performed by FlowJo software. (D) Stacked bar graphs showing the frequencies of cells that completed one or more cell division over time in culture in young (Y) and old (O) total CD34⁺ cells. Data are shown as mean ± SEM. Statistical test: Mann–Whitney; 24 h Y vs. O, P = 0.0378. (E) Absolute quantification of human CD45⁺ cells in PB (plotted on the left axis), Spleen, and BM (plotted on the right axis) at 20 wk after transplantation in mice transplanted with young and aged HSPCs. Statistical test: Mann–Whitney; BM Y vs. O, P = 0.051). (F and G) Absolute quantification of human subpopulations in PB and BM of mice transplanted with young or aged HSPCs at 20 wk after transplantation. Statistical test: Mann–Whitney; B cells PB Y vs. O, P = 0.0195; HSPCs BM Y vs. O, P = 0.0357. (H) Composition of human BM HSPCs compartment in mice transplanted with young or aged HSPCs at 20 wk after transplantation. (I) Percentage of human LIN⁻CD3⁺CD38⁻ subpopulations within HSPCs compartment in the BM of mice transplanted with young or aged HSPCs at 20 wk after transplantation. (J) Absolute quantification of human HSPCs subpopulations in the BM of mice transplanted with young or aged HSPCs at euthanasia. Statistical test: Mann–Whitney; HSCs, P = 0.0348; MPP, P = 0.0295; MLP, P = 0.0046; PreBNK, P = 0.017. In all the graphs, data are shown as mean ± SEM. * < 0.05; ** < 0.01.