Cell-cell contact is required for MSC-DC generation. (A) FACS for the influence of transwell system on phenotype of MSC-DC. The MSC-DCs and trans-DCs (maDCs cultured in the transwell plates to avoid cell-cell contact with MSC monolayers for 7 days) were stained with antibodies to Ia, CD11b, CD11c, CD80, CD86, and CD40, analyzed by flow cytometry. Dotted lines, background staining. Numbers in histograms indicate the geometric mean fluorescence of each DC population. (B) FACS for the influence of transwell system on expression of notch ligands on the surface of MSC-DC. The MSC-DCs and trans-DCs were stained with antibodies to Jagged-1, Jagged-2, and Delta-1, analyzed by flow cytometry. Dotted lines, background staining. Numbers in histograms indicate the geometric mean fluorescence of each DC population. (C) Effects of transwell system on the immune inhibitory function of MSC-DC. CFSE-labeled lymphocytes (H-2K^b) were cultured with MSC-DCs (H-2K^d) or trans–MSC-DCs (H-2K^d) in the absence or presence of ConA (5 μg/mL) stimulation, and 3 days later, cells in each well were collected and measured by FACS. Dotted line, unstimulated lymphocytes.