Human MSCs inhibited the initial differentiation of DCs from CD14⁺ monocytes. (A) Monocytes cultured in the presence of GM-CSF and IL-4 for 7 days and then LPS for another 48 hours (Ctr-Mo) show clustered and protruding veils resembling mature DCs (top row). In contrast, monocytes with MSC coculture (MSC-Mo) display macrophage-like morphology (bottom row) (for left, middle, and right columns for both rows: original magnifications × 40, × 200, and × 1000, respectively). Cell morphology was evaluated by phase contrast microscopy and Wright-Giemsa staining (Sigma, Saint Louis, MO). Images in panel A were visualized using either an Olympus BH-2 microscope (right column) or a Nikon TE2000-U microscope (left and middle columns) equipped with Plan Fluor 4×/0.13 and 20×/0.45 and Splan oil 100×/1.25 objective lenses (Nikon, Tokyo, Japan). Images were captured with a Nikon Coolpix 995 camera and processed with Adobe Photoshop 7.0 software (Adobe, San Jose, CA). (B) Flow cytometry was employed to analyze the surface molecules expressed by Ctr-Mo (top row) and MSC-Mo (bottom row). Horizontal bars indicate the positive region; the corresponding percentage is shown above each bar. Results shown are representative of 3 independent experiments. (C) CD14 and CD1a expression of monocyte-derived cells in MSC/monocyte cocultures at ratios ranging from 1:10 to 1:200 was assessed by flow cytometry. Results are representative of 2 independent experiments.