In vivo functional characterization of pre-activated human-aged HSPCs. (A) Experimental scheme for xenotransplantation experiment where human CD34⁺ cells from young (n = 4) and old donors (n = 4) were pre-cultured overnight prior transplantation into NSG (NOD.Cg PrkdcscidIl2rgtm1Wjl/SzJ) mice (n = 3 mice for each donor). (B) Percentage of human engrafted cells (CD45⁺ cells) in PB over time as well as in BM and spleen (SP) at euthanasia in mice transplanted with young or aged HSPCs. Statistical test: Mann–Whitney; BM Y vs. O, P = 0.0099. (C–E) Percentage of human myeloid cells (CD13⁺ cells [C]), B cells (CD19⁺ cells [D]), and T cells (CD3⁺ cells [E]) in PB over time as well as BM and SP at 20 wk in mice transplanted with young or old HSPCs. Statistical test: Mann–Whitney; PB myeloid cells 20 wk Y vs. O, P = 0.0008; PB B cells 20 wk Y vs. O, P = 0.0215; Spleen B cells Y vs. O, P = 0.0149. PB T cells 20 wk Y vs. O, P = 0.0018. (F) Number of colonies generated starting from the same amount of CD34⁺ cells isolated from transplanted mice at 20 wk. Statistical test: Mann–Whitney; Y vs. O, P = 0.0022. (G–L) Relative mRNA expression of cell cycle inhibitor p21 and inflammatory genes IL1β, IL6, IL8, MCP1, and TNFα in human CD34⁺ cells isolated from BM of mice transplanted with young and aged HSPCs (20 wk). Statistical test: Mann–Whitney; p21 Y vs. O, P = 0.0087; IL1β Y vs. O, P = 0.0087; IL6 Y vs. O, P = 0.0173; IL8 Y vs. O, P = 0.0043; MCP1 Y vs. O, P = 0.0043; TNFα Y vs. O, P = 0.0043. Gene expression was measured by quantitative real-time PCR and represented as 2^−ΔCt relative to housekeeping. (M) Frequencies of cells showing nuclear γH2AX foci in human CD34⁺ cells isolated from BM of mice transplanted with young and old HSPCs at 20 wk. Statistical test: Mann–Whitney test. * < 0.05 ** < 0.01; *** < 0.001.