Fig. 5: Old Kit^lo HSCs exhibit enhanced T cell potential.

A, B Evaluation of in vitro lymphoid progenitor and T cell differentiation potential of Kit^hi (red) and Kit^lo HSCs (blue) from or 24-mo (old) C57BL/6 mice using the S17 lymphoid assay and murine artificial thymic organoid (M-ATO), respectively, as shown in Fig. 2A. Enumeration of absolute number of lymphoid progenitor cells following 14 days in S17 lymphoid assay (A) and absolute number of T cell subsets following 8 weeks of culture in M-ATOs (B). Refer to Supplementary Fig. 13A, B for gating strategies to define the above populations. Aggregated data across 3 independent experiments, each performed in triplicate (Kit^lo = 3; Kit^hi = 3). C Experimental schema for competitive allogeneic HCT (allo-HCT) using Kit^hi (red) and Kit^lo (blue) HSCs from 22–24-mo (old) C57BL/6 mice with competitor bone marrow (BM) cells from B6.SJL-PtprcaPepcb/BoyJ mice transplanted into lethally irradiated young BALB/cJ recipients. D–H Eight weeks after competitive HCT, enumeration of absolute number of donor-derived T cells in the PB (Kit^hi = 9; Kit^lo = 9) (D), LSK cell subsets and CLP cells (Kit^hi = 10; Kit^lo = 10) (E), total thymic cellularity (Kit^hi = 9; Kit^lo = 9). F Donor-derived T cell precursor thymocyte subsets (Kit^hi = 9; Kit^lo = 9) (G), stromal subsets within thymic CD45-compartment (Kit^hi = 9; Kit^lo = 9) (H). Aggregated data across two independent experiments. I Experimental schema to evaluate thymic function following competitive allo-HCT using old HSCs. J Frequency of donor-derived Recent Thymic Emigrants (RTE: CD3⁺GFP⁺) in the PB at 8 weeks post-HCT. Refer to Supplementary Fig. 13C for the gating strategy to define the above population. All data are from n = 5 mice/group (Kit^lo = 5; Kit^hi = 5). K Experimental schema for competitive allogeneic HCT (allo-HCT) using Kit^hi (red) and Kit^lo HSCs (blue) from 22–24-mo (old) C57BL/6 mice with competitor bone marrow (BM) cells from B6.SJL-PtprcaPepcb/BoyJ mice transplanted into lethally irradiated middle-aged BALB/cJ recipients. L–N Eight weeks after competitive HCT, enumeration of the absolute number of donor-derived T cells in PB (L), donor-derived CLPs in the BM as defined in Fig. 3D (M), and thymic cellularity (N). All data are from n = 5 mice/group (Kit^lo = 5; Kit^hi = 5). Refer to Supplementary Fig. 12 for gating strategies to define the above populations. O Frequency of T cell subsets following 6 weeks of culture in M-ATO of old Kit^hi HSCs following Zbtb1 OE. Refer to Supplementary Fig. 13B for gating strategies to define the above populations. Aggregated data across three independent experiments, each performed in duplicates (Control = 3; Zbtb1-OE = 3). Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P-values calculated by a nonparametric unpaired two-tailed Mann–Whitney U test. Panels (C), (I), and (K) were created in BioRender. Lab, K. (2025) https://BioRender.com/oeh4i7x. Source data are provided as a Source Data file, Source Data Fig. 5.