Sdc2⁺ HSCs exhibit increased self-renewal capacity. (A) Representative gating strategy to isolate 34^–KSL cells (HSCs), Sdc2^– HSCs, or Sdc2⁺ HSCs. (B) Experimental design for primary competitive repopulation assay of 500 Scd2⁺ HSCs, Sdc2^– HSCs, and 34-KSL HSCs from CD45.1⁺ mice into CD45.2⁺ recipients. (C) Representative flow cytometric analysis of donor CD45.1⁺ engraftment in the PB of recipient CD45.2⁺ mice at 16 weeks posttransplant. (D) Percentages of total donor CD45.1⁺ cells, B cells, T cells, and myeloid cells in the PB over time in recipient CD45.2⁺ mice in the groups shown (n = 7 mice pooled for donor cell isolation; n = 10-17 recipient mice per group). (E-F) Percentages of donor CD45.1⁺ KSL cells and CD45.1⁺CD150⁺CD48^–KSL cells at 16 weeks in the BM of recipient CD45.2⁺ mice (n = 10-17 recipient mice per group). (G) Percentages of total donor CD45.1⁺ cells, B cells, T cells, and myeloid cells in the PB over time after secondary competitive transplant in the treatment groups shown (n = 10-17 recipient mice per group). (H) Percentages of donor CD45.1⁺ CD150⁺CD48^–KSL cells in the BM of recipient mice at 16 weeks postsecondary transplant (n = 10-17 recipient mice per group). (I) Graph of limiting dilution analysis of engraftment of Sdc2⁺ HSCs, Sdc2^– HSCs, or CD150⁺ HSCs (CD45.1⁺) at 16 weeks after competitive transplant into lethally irradiated C57BL/6 (CD45.2⁺) mice. CD45.1⁺ donor engraftment ≥0.1% was considered positive for engraftment (n = 10 mice pooled for donor cell isolation; n = 4-21 recipient mice per dose). (J) HSC frequency estimates in CD150⁺ HSCs, Sdc2⁺ HSCs, and Sdc2^– HSCs are displayed based on Poisson statistical analysis of engraftment data shown. (K) Representative gating strategy used to isolate CD150⁺34^–KSL Sdc2⁺ cells and CD150⁺34^–KSL Sdc2^– cells for competitive transplantation of 300 isolated cells. (L) Percentages of total donor CD45.1⁺ cells, B cells, T cells, and myeloid cells in the PB over time after primary competitive transplant (n = 10 mice pooled for donor cell isolation; n = 8-10 recipient mice per condition). Error bars = standard error of the mean; statistics denote one- or two-way analysis of variance followed by Holm-Šidák’s corrected unpaired Student t test. *P < .05, **P < .01, ***P < .001, ****P < .0001 for comparison of Sdc2⁺ HSCs vs Sdc2^– HSCs in panels D and G or comparison of CD150⁺34^–KSL Sdc2⁺ cells vs CD150⁺34^–KSL Sdc2^– cells in panel L. ^#P < .05, ^##P < .01, ^###P < .001 for comparison of Sdc2⁺ HSCs and 34^–KSL cells (HSCs) in panels D and G. IgG, immunoglobulin G; n.s., not significant; SSC-A, side scatter area.