Limiting dilution xenotransplantation assays of uncultured HSPCs show comparable frequency of repopulating cells in aged HSPCs compared with young HSPCs. (A) Experimental scheme using three cell doses in xenotransplantation time course experiments (n = 4) where human CD45⁺ engraftment and lineage in the BM of NSG (NOD.Cg PrkdcscidIl2rgtm1Wjl/SzJ) mice were assessed at 4, 12, and 20 wk for the indicated human HSPCs cell sources (number of donors for each source: CB = 4; young BM = 5; middle-aged BM = 3; old BM = 5). (B) The CD34⁺ repopulating cell frequency for 4, 12, and 20 wk for CB and BM samples from indicated age groups. The number reported in brackets in the table display the total number of transplanted mice for each source or age group considering all cell doses at each time point. (C–E) Box plots showing human CD45⁺ cell frequencies in the murine BM in mice transplanted with 60,000 (60K), 10,000 (10K), and 1,000 (1K) HSPCs for CB and BM samples at 4 (C), 12 (D), and 20 (E) wk. Transplanted mice with engraftment below 0.05% in the murine BM were considered not engrafted. Statistical test: Mann–Whitney; 4 wk, 60K: CB vs. Y, P < 0.0001; CB vs. M, P = 0.0001; CB vs. O, P = 0.0002; Y vs. M, P = 0.0001; Y vs. O, P = 0.009. 12 wk, 60K: CB vs. Y, P < 0.0001; CB vs. O, P = 0.028; Y vs. M, P < 0.0001; Y vs. O, P < 0.0001. 10K: Y vs. M, P = 0.0002; Y vs. O, P = 0.0004; 20 wk, 60K: CB vs. Y, P = 0.0002; CB vs. O, P = 0.0005; Y vs. M, P = 0.0016; Y vs. O, P = 0.0055; M vs. O, P = 0.0094. 10K: CB vs. M, P = 0.0357; CB vs. O, P = 0.0167. (F–H) Box plots showing human CD45⁺ cell frequencies in the murine BM in mice transplanted with 60K, 10K, and 1K HSPCs for CB and BM samples at 4 (F), 12 (G), and 20 (H) wk normalized on the HSC+MPP content of the infused cells. Statistical test: Mann–Whitney; 4 wk, 60K: CB vs. Y, P < 0.0001; CB vs. M, P = 0.0295; CB vs. O, P = 0.0005. 10K: CB vs. M, P = 0.0426; 12 wk, 60K: CB vs. Y, P = 0.0206. 10K: Y vs. O, P = 0.0028. 20 wk, 60K: CB vs. Y, P = 0.0008; CB vs. O, P = 0.0007; Y vs. M, P = 0.0109; M vs. O, P = 0.0268. 10K: CB vs. M, P = 0.0357; CB vs. O, P = 0.0167. (I) Experimental scheme for secondary transplant. CD34⁺ cells isolated from three young and three old donors were intrafemorally transplanted in 23 primary NSG (NOD.Cg PrkdcscidIl2rgtm1Wjl/SzJ) recipients (each donor into three to four mice). At 12 wk after transplant, human CD45⁺ cells from the BM of primary mice were collected and transplanted into 23 secondary recipients. The murine BM of the secondary recipients was analyzed at 12 wk. Transplanted mice with engraftment below 0.05% (for primary transplants) and 0.005% (for secondary transplants) in the murine BM were considered not engrafted. The threshold for secondary mice was set on not transplanted control mice. Not engrafted mice were plotted in the graphs but excluded from the statistical analyses. (J) Box plots showing human CD45⁺ cell frequencies in the murine BM in primary and secondary recipients at 12 wk, normalized on the content of HSC+MPP infused. (K and L) Olive Tail Moment average values of neutral (K) or alkaline (L) comet experiments conducted in CD34⁺ isolated at 12 wk after secondary transplantation of CD34⁺ cells from young and old donors (CD34⁺ isolated from the mice transplanted with the same donor were pooled). * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001.