Igf2bp2 deletion impairs repopulation and colony-forming capacity of young but not old HSCs. Freshly isolated HSCs from Igf2bp2^+/+ and Igf2bp2^−/− mice were analyzed. (A) Myeloid-biased HSCs (CD150^highCD34⁻LSK; n = 500) from male mice at young age (range: 3-6 months) and old age (range: 22-26 months) were plated for 3 rounds to determine the colony-forming capacity (8-12 mice per group). Statistical analysis by 3-way analysis of variance (ANOVA; using "program package R v3.6.3, function aov()") revealed that age (P < .0001), genotype (P < .0001), and round of plating (P < .0001) significantly affected the colony-forming capacity. Moreover, age and genotype had a significant combinatorial effect (P = .0119). Post hoc testing with 2-way ANOVA for separate age groups indicated that Igf2bp2 deletion had a stronger effect on impairing the colony-forming capacity of myeloid-biased HSCs from young mice (effect size: −31.84 units; P < .0001) compared with aged mice (effect size: −15.42 units; P = .0031). (B,D-E) Myeloid-biased HSCs (CD150^highCD34⁻LSK; n = 100) from young donors (3-6 months) or 1000 total CD150⁺ (high and low) HSCs (CD150⁺CD34⁻LSK) from old donors (27 months) were transplanted along with 1 × 10⁶ competitor BM cells (CD45.1). Young HSCs, 9-10 donors and recipients (1:1 transplantation) per group; old HSCs, 4 to 5 recipient per group. (B) Analysis of the total chimerism of donor-derived cells in PB at the indicated time points after transplantation. (D-E) Donors were analyzed 16 or 20 weeks after transplantation. Cell cycle status of donor-derived HSCs: quantification (D) and representative fluorescence-activated cell sorting plots (E). (C) Percentage of homed myeloid-biased HSCs (CD150^highCD34⁻LSK) from 3-month-old Igf2bp2^−/− vs Igf2bp2^+/+ donor mice (n = 5 mice per group). (B-D) Statistical significance of genotype-dependent difference in young or old donors was determined by Welch’s t test. All data are expressed as the mean ± SD; ns, nonsignificant.