Fig. 6: KIT^lo human BM HSCs exhibit enhanced lymphoid potential.

A–E Young and old human BM CITE-seq dataset generated and published by Sommarin et al.⁷⁶ A–C UMAP of CD34+ young and old human BM annotated with HSC subsets (A), and age (B, C) HSC cluster composition by age. D Kit^lo gene signature (top 50 marker genes from 239 mouse genes identified with human orthologues) was overlaid on human HSC UMAP. E Violin plot for Kit^lo gene score by age. Statistical analysis was performed using the Wilcoxon test. F, G FACS analysis of human BM samples. F Violin plot of KIT protein expression on phenotypic HSCs (p-HSC: CD34 + CD38-CD10-CD45RA-CD90 + ) by age (young: 20–40 yrs; MA/old: >40 yrs). G Scatterplot showing distribution of KIT^hi and KIT^lo HSC subsets within p-HSCs in human BM samples with varying age. The two-tailed P-values generated for (G) were based on a single multivariable generalized estimating equation (GEE) logistic model. The model was adjusted for age, KIT^lo and KIT^hi groups, and their interaction, with an exchangeable working correlation structure to account for the matched data structure. H Experimental schema to evaluate in vitro T cell and lympho-myeloid potential of human BM HSC subsets based on differential KIT expression using human artificial thymic organoids (H-ATO) and MS5 assay, respectively. I Following 8–10-weeks of culture, enumeration of CD19+ B cells and T cell subsets within CD34+ T cell precursors (Early Thymic Progenitors, ETP: CD34⁺CD1a⁻CD7⁻; CD1a^neg- ProT: CD34⁺CD1a⁻CD7⁺; CD1a^pos-ProT: CD34⁺CD1a⁺CD7⁺), and mature T cells (DP: CD34⁻CD5⁺CD7⁺CD4⁺CD8⁺; SP4: CD34⁻CD5⁺CD7⁺CD4⁺CD8⁻; SP8: CD34⁻CD5⁺CD7⁺CD4⁻CD8⁺). Refer to Supplementary Fig. 10E for gating strategies to define the above populations. Aggregated data from 8 independent BM donors, each performed in duplicates, across two independent experiments (KIT^lo = 8; KIT^hi = 8). J Following 4 weeks of culture, enumeration of Lineage output (Neutrophil-Granulocytes: hCD45+CD15+; monocyte-macrophages: hCD45+CD14+; B cells: hCD45+CD19+; NK cells: hCD45+CD56+) after culturing 200 KIT^lo and KIT^hi HSCs.⁷⁹Aggregated data from 5 independent BM donors, each performed in triplicate or triplicates, across two independent experiments (KIT^lo = 5; KIT^hi = 5). Refer to Supplementary Fig. 10F for gating strategies to define the above populations. K Our model illustrates that hematopoietic stem cells (HSCs) with distinct lymphoid-primed chromatin states and differential ZBTB1 activity, which determines their lymphoid potential, can be distinguished by their CD117/KIT expression. In young bone marrow, Kit^lo HSCs (blue) with high ZBTB1 activity exhibit enhanced lymphoid potential, thymic recovery, and T-cell reconstitution, in contrast to Kit^hi HSCs (red) with reduced lymphoid potential. The age-related decline in lymphoid potential results from both a proportional shift toward Kit^hi HSCs and a global decrease in ZBTB1 activity across all HSC populations. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P-values calculated either by nonparametric paired two-tailed Mann–Whitney U test (I and J), unpaired Mann–Whitney U test (F). Panels (H) and (K) were created in BioRender. Lab, K. (2025) https://BioRender.com/fl4hwgn. Source data are provided as a Source Data file, Source Data Fig. 6.