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.