Loss of miR-146a promotes HSC aging in young, naïve mice. (A) Strategy for examining the impact of miR-146a deletion on frequency of immunophenotypic and serially transplantable HSCs. (B) Frequency of ESLAMs in BM of WT vs miR-146a^−/− mice (n = 16; 21 mice; P by Student t test). (C) Extreme limiting dilution analysis (ELDA) plot for secondary transplants with WT_Ly5.2 vs miR-146a^−/−_Ly5.2 BM (n = 15 mice per genotype, with 4 dilutions). (D) Number of serially transplantable HSCs estimated from ELDA analysis in panel C. SEM, standard error of the mean (P by ELDA). (E) Proportion of single ESLAMs with myeloid, balanced, or lymphoid output profile in single-cell transplants from WT vs miR-146a^−/− (n = 49; 14 transplants). Data for WT mice are from a previously published study.³⁴ P by Fisher’s exact test comparing myeloid outputs. (F) GSEA hallmark gene sets upregulated in miR-146a^−/− vs WT LSK HSPCs (NES >1.5). (G) GSEA plots showing enrichment of cytokine signaling hallmark gene sets in genes upregulated in miR-146a^−/− vs WT LSK HSPCs. (H) miR-146a expression in young (10-12 weeks), middle-aged (12 months), and old (18 months) WT ESLAMs (n = 10 young, 6 middle-aged, 4 old mice; P by Student t test). (I) Overlap between differentially methylated genes in aged vs young WT HSCs from Sun et al⁵⁴  (labeled Aged) and in miR-146a^−/− (n = 6) vs WT (n = 12) HSPCs (labeled: miR-146a^−/−). P by Fisher’s exact test. (J) GSEA plots showing enrichment of genes up- or downregulated in aged HSCs⁵⁴  in miR-146a^−/− vs WT LSK HSPCs.