Single-cell functional analyses of aged LT-HSCs. (A) Experimental scheme for in vitro multi-lineage differentiation assay on bulk CD34⁺ and HSCs (500 cells/well) from young (n = 3) and old (n = 3) donors. (B) Differentiation output of bulk CD34⁺ and HSCs from young and old donors at different time points. (C) Experimental scheme for in vitro multi-lineage differentiation assay on single CD49f^+/− HSCs (100 cells/donor) from young (n = 3) and old (n = 3) donors. (D and E) Differentiation efficiency of single CD49f⁺ HSCs (D) and CD49f- HSCs (E) from young and old donors. Pearson’s Chi-squared test with Yates’ continuity correction was used to measure statistical significance of the observed differences. CD49f⁺: Y vs. O, P < 0.0001; CD49f⁻: Y vs. O, P < 0.0001. (F–H) Pie charts representing the lineage scores of young and old total HSCs (F), CD49f⁺ HSCs (G), and CD49f⁻ HSCs (H), based on the most abundant differentiation outputs toward lymphoid, myeloid, erythroid, and megakaryocyte lineages detected at the end of the single-cell in vitro differentiation assay. Cells showing differentiation toward all the four lineages were scored as “multi-lineage”. (I) Graph showing the absolute cell count of single HSCs clones output scored according to their lineage production. **** < 0.0001.