Increased HSPCs in TGFβ1ΔMk/ΔMk mice have normal function. (A) Schematic for the competitive repopulation assay is shown. Marrow CD45.2 donor cells from either TGFβ1FL/FL or TGFβ1ΔMk/ΔMk mice were mixed in a 1:1 ratio with marrow donor cells from congenic CD45.1 mice, then transplanted into lethally irradiated mice (n = 8 per group). CD45.2 chimerism in peripheral blood is shown at the indicated times after transplant (left). Chimerism of blood and BM LKS+ signaling lymphocyte activation molecule (SLAM; Lin−, Kit+, Sca1+, CD150+, CD48−) cells is shown 16 weeks after transplant for mice having received TGFβ1FL/FL (gray) or TGFβ1ΔMk/ΔMk (black) donor cell grafts, as assessed by flow cytometry. (B) Schematic for secondary transplantation of marrow harvested from primary recipients is shown. CD45.2 chimerism in peripheral blood is shown at the indicated times after secondary transplant (left). Chimerism in blood and in peripheral blood and marrow LKS+SLAM is shown 16 weeks after secondary transplant as for panel A (16 mice were transplanted in 2 independent experiments; 8 mice were transplanted per group in each experiment). (C) CFC enumeration of functional myeloid HPCs is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk marrow cells (n = 5 per group). The total number of CFU granulocyte, erythroid, monocyte, Mk progenitors (CFU-GEMM), CFU granulocyte macrophage (CFU-GM), and burst-forming unit erythroid progenitors (BFU-E) is shown per leg (femur and tibia). (D) The number of spleen CFUs (CFU-S12) 12 days after transplantation is shown per 100 000 donor cells transplanted (n = 4 per group). (E) Gating and representative flow cytometry data are shown for the indicated HSPC populations. (F) Quantification of the number of marrow cells with the immunophenotype of common myeloid progenitor (CMP), GMP, MEP, erythroid progenitors (EP), pre-GMP, pre-MEP is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice (n = 12 per group). (G) The number of LKS+ (Lin−Kit+Sca1+), MPP and LKS+SLAM cells per leg is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice (n = 12 per group). (H) Gating and representative flow cytometry data are shown for LKS+SLAM cell cycle as reported by Ki67 and Hoechst staining. (I) Cell cycle of BM LKS+SLAM HSCs is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk (mice n = 6 per group). All of the quantified data are shown as mean plus or minus SD (*P < .05, **P < .01, ***P < .001, or if not shown, the comparison was not significant). BMT, BM transplantation; SSA, side scatter area.