CD34⁻KSL HSCs express IL-27R subunits, gp130 and WSX-1, and IL-27 induces their proliferation in synergy with SCF. (A) Semiquantitative RT-PCR for analyzing WSX-1 and gp130 mRNA expression was carried out using cDNAs prepared from cells including mouse BM CD34⁻c-Kit⁺Sca-1⁺ lineage marker⁻ HSCs (CD34⁻KSL), CD34⁺KSL progenitors, lineage marker⁻ cells (Lin-), Gr-1⁺ neutrophils, Mac-1⁺ monocytes/macrophages, TER119⁺ erythroblasts, and B220⁺ B cells; spleen Thy-1.2⁺ T cells, NK1.1⁺ natural killer cells, and B220⁺ B cells; and thymic CD4⁻CD8⁻ T cells (DN), CD4⁺CD8⁺ T cells (DP), CD4⁺CD8⁻ T cells (CD4SP), and CD4⁻CD8⁺ T cells (CD8SP). The intensity of each band was densitometrically measured, and relative intensity was calculated after normalization by the intensity for GAPDH. (B) CD34⁻KSL cells were stained with anti–WSX-1 or control antibody followed by incubation with Alexa-488–conjugated antimouse IgG and DAPI. Three representative cells were shown. At least 50 cells were analyzed with a confocal microscope, and similar results were obtained. (C) CD34⁻KSL cells (single cell/well) were cultured in serum-free medium supplemented with 0.5% BSA and SCF (10 ng/mL) in the presence of IL-27 (10 ng/mL). At several time points, the number of cells per well was counted under an inverted microscope or standard trypan blue exclusion method. Each dot shows number of cells in individual wells. Horizontal lines are median values. (D) Individual day-14 colonies were stained with anti–Gr-1 and anti–Mac-1 and analyzed by flow cytometry. Numbers represent the percentage of cells in each area.