Figure 1.
ESSCs induce BM HSC differentiation into CD11bhiIalo DCs. (A) HSCs were purified by FACSDiva according to the phenotype of HSCs. Flt3–Lin– bone marrow cells were first gated and further gated by using the marker of CD117 and Sca-1, and, finally, Lin–Flt3–CD117+Sca-1+cells were sorted as HSCs. (B) The morphology of HSCs were differentiated in clonal formation assay from small round to large DC-like cells. Photos were taken by a digital imaging system on the indicated days. Top panel (×400), HSC from normal mice. Bottom panel (×100), HSCs from GFP transgenic mice. Photos were taken with a Leica DMZRB (Leica, Wetzlar, Germany) using Meta Imaging Series 5.0 (Molecular Devices, Downingtown, PA). Images in the top panel were taken with a 40×/0.55 NA objective; those in the lower panel, with a 10×/0.25 objective. (C) The phagocytic ability of imDCs, mDCs, DC-like cells (CD11bhiIalo), and LPS-treated DC-like cells (LPS-CD11bhiIalo) as tested by measuring OVA-FITC phagocytosis using FACS. Filled histograms represent imDCs, mDCs, DC-like cells (CD11bhiIalo), or LPS-treated DC-like cells (LPS-CD11bhiIalo) cultured with 100 μg/mL OVA-FITC at 37°C for 2 hours and were marked with geometric mean fluorescence. Open histograms represent cells cultured with 100 μg/mL OVA-FITC at 4°C for 2 hours as a control. One of at least 3 independent experiments with similar results is shown. (D) The expression of lineage markers on the surface of DC-like cells. After HSCs grown on ESSC monolayers for 10 days, nonadherent DC-like cells were collected and stained with the indicated fluorescein-conjugated antibodies. Dotted lines represent cells stained with isotype-matched control mAbs. (E) The expression of functional molecules on DC-like cells. DC-like cells purified with microbead-conjugated anti-CD11b mAbs, further stimulated with LPS (10 ng/mL for 4 days) or not, were labeled with fluorescein-conjugated mAbs, comparing with imDCs and mDCs. Open histogram lines represent cells stained with isotype-matched control mAbs. Filled histograms are labeled with the geometric mean fluorescence of each DC population. (F) The cytokine profiles and NO secretion of CD11bhiIaloDCs, imDCs, and mDCs (stimulated with or without 0.5 μg/mL LPS for 24 hours) as measured by ELISA and Griess assay, respectively. Data are mean + SD of triplicate wells.

ESSCs induce BM HSC differentiation into CD11bhiIalo DCs. (A) HSCs were purified by FACSDiva according to the phenotype of HSCs. Flt3–Lin– bone marrow cells were first gated and further gated by using the marker of CD117 and Sca-1, and, finally, Lin–Flt3–CD117+Sca-1+cells were sorted as HSCs. (B) The morphology of HSCs were differentiated in clonal formation assay from small round to large DC-like cells. Photos were taken by a digital imaging system on the indicated days. Top panel (×400), HSC from normal mice. Bottom panel (×100), HSCs from GFP transgenic mice. Photos were taken with a Leica DMZRB (Leica, Wetzlar, Germany) using Meta Imaging Series 5.0 (Molecular Devices, Downingtown, PA). Images in the top panel were taken with a 40×/0.55 NA objective; those in the lower panel, with a 10×/0.25 objective. (C) The phagocytic ability of imDCs, mDCs, DC-like cells (CD11bhiIalo), and LPS-treated DC-like cells (LPS-CD11bhiIalo) as tested by measuring OVA-FITC phagocytosis using FACS. Filled histograms represent imDCs, mDCs, DC-like cells (CD11bhiIalo), or LPS-treated DC-like cells (LPS-CD11bhiIalo) cultured with 100 μg/mL OVA-FITC at 37°C for 2 hours and were marked with geometric mean fluorescence. Open histograms represent cells cultured with 100 μg/mL OVA-FITC at 4°C for 2 hours as a control. One of at least 3 independent experiments with similar results is shown. (D) The expression of lineage markers on the surface of DC-like cells. After HSCs grown on ESSC monolayers for 10 days, nonadherent DC-like cells were collected and stained with the indicated fluorescein-conjugated antibodies. Dotted lines represent cells stained with isotype-matched control mAbs. (E) The expression of functional molecules on DC-like cells. DC-like cells purified with microbead-conjugated anti-CD11b mAbs, further stimulated with LPS (10 ng/mL for 4 days) or not, were labeled with fluorescein-conjugated mAbs, comparing with imDCs and mDCs. Open histogram lines represent cells stained with isotype-matched control mAbs. Filled histograms are labeled with the geometric mean fluorescence of each DC population. (F) The cytokine profiles and NO secretion of CD11bhiIaloDCs, imDCs, and mDCs (stimulated with or without 0.5 μg/mL LPS for 24 hours) as measured by ELISA and Griess assay, respectively. Data are mean + SD of triplicate wells.

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