Sdc2⁺ HSCs generate HSCs in culture with increased self-renewal ability. (A) Experimental design for ex vivo culture of 34^–KSL HSCs, Sdc2^– HSCs, or Sdc2⁺ HSCs in TSF media (n = 4-5 replicates per group). (B) Mean numbers of total cells after 7-day culture (n = 4-5 replicates per group). (C) Representative flow cytometric analysis of KSL cells and 34^–KSL cells at day 7 in each group (n = 4-5 replicates per group). Percentages (D) and numbers (E) of 34^–KSL cells in each group at day 7 of culture (n = 4-5 replicates per group). Percentages of Syndecan-2⁺ cells of 34^–KSL in each condition at day 3 (F) or day 7 (G) after culture (n = 4-5 replicates per group). Number of syndecan-2^– (H) and syndecan-2⁺ (I) cells of 34^–KSL in each condition at day 7 after culture (n = 4-5 replicates per group). (J) Percentages of syndecan-2⁺ cells of donor-derived 34^–KSL cells at 8 weeks after transplantation of 500 Sdc2⁺ or Sdc2^– HSCs (n = 4-5 replicates per group). (K) At left, representative flow cytometric analysis showing percentages of 34^–KSL cells (HSCs) derived from Sdc2^– HSCs or Sdc2⁺ HSCs in G₀, G₁, and G₂/S/M phases at day 7; at right, percentages of cells in G₀, G₁, and G₂/S/M phases (n = 3 replicates per group). (L) Percentages of 34^–KSL cells derived from Sdc2^– HSCs or Sdc2⁺ HSCs in G₀ relative to total cell output (n = 3 replicates per group). (M) Percentages of total donor CD45.1⁺ cells, B cells, T cells, and myeloid cells in the PB of recipient CD45.2⁺ mice over time after transplantation of day 7 progeny of 34^–KSL HSCs, Sdc2^– HSCs, or Sdc2⁺ HSCs cultured with TSF media (n = 5-6 recipients per group). (N) Percentages of donor CD45.1⁺ CD150⁺CD48^–KSL cells in the BM of recipient mice at 16 weeks posttransplant. Error bars = standard error of the mean; statistics denote Holm-Šidák’s post hoc unpaired Student t test after 1- or 2-way analysis of variance. *P < .05, **P < .01, ***P < .001, ****P < .0001 for Sdc2⁺ HSCs vs Sdc2^– HSCs. ^#P < .01, ^##P < .01, ^###P < .001 for comparison between Sdc2⁺ HSCs and 34^–KSL HSCs. Scf, stem cell factor; SSC-A, side scatter area; TPO, thrombopoietin.