Serial transplantation of aged and young BM results in a loss of clonal complexity. (A-D) CD45.2⁺Conf-VE^Cre BM was serially transplanted. (A) Schematic of serial transplantation of CD45.2⁺Conf-VE^Cre BM. For primary transplant, 5 × 10⁶ BM cells were transplanted from young (age 2 months) or old (age 24 months) donors into distinct cohorts of primary CD45.2⁺/CD45.1⁺ recipients. For serial transplants, 5 × 10⁶ BM cells were transplanted. For each age group (young and old), at least 3 independent donor mice were transplanted into distinct recipient cohorts. Each cohort was composed of at least 5 mice and was transplanted with an independent donor. (B) Recipient PB was analyzed for the distribution of Confetti colors in their PB at 4, 10, and 16 weeks posttransplant. MtMV was used to estimate the number of repopulating units (see “Methods”). Primary and secondary transplants are shown (see supplemental Figure 4 for additional cell doses transplanted in primary transplants and PB lineages; see also Figure 5). (C) Recipient BM HSCs were examined at 4 months posttransplant for the MtMV in the Confetti colors (see supplemental Figure 6D for additional BM HSPC compartments). (B-C) Averages are shown; error bars denote standard deviation (*P < .05; #P < .1) (D) The median frequency of the most prevalent color is shown for the PB at 4 weeks (i) and 16 weeks (ii) and for MPPs (iii) and HSCs (iv) at 16 weeks for each transplantation stage. Whisker plots show interquartile range. Analysis of variance was run to test for the statistical significance of the transplantation stage and age for each cell type. Age did not result in statistical differences for any cell type. Transplantation stage had a significant effect in all cell types (P < .05) except HSCs (*P < .05). (B-D) Each bar or point represents the average or median obtained from at least 3 independent cohorts of mice (each cohort n ≥ 5) from 3 independent initial young or old donors. Source data are provided in supplemental Table 1. Trx, transplant; WBM, whole bone marrow.