Characterization of murine MSCs. Flow cytometry analysis was conducted on in vitro-cultured Epo⁺ MSCs to determine cell surface antigen expression of CD31, CD34, CD44, CD45, CD73, CD90, CD105, and Mac1, as described in “Materials and methods” (A). The dashed line represents the isotype control, and the solid line represents the specific antibody. Epo⁺ MSCs, undifferentiated (Bi, × 200 magnification; objective, ×20/0.4 NA), were cultured in conditions inductive of osteogenic or adipogenic differentiation. Staining with Alizarin Red S following osteogenic differentiation revealed the mineralization of the extracellular matrix (Bii, × 50; objective, ×5/0.12 NA). Adipogenic differentiation was visualized with light microscopy (Biii, × 400; objective, ×40/0.55 NA) as well as by Oil Red O staining of lipid droplets (Biii inset, × 400; objective, ×40/0.55 NA). The immunosuppressive effects of C57Bl/6-derived MSCs against allogeneic mixed lymphocyte cultures were determined as described in “Materials and methods” (C). C57Bl/6 and Balb/c splenocytes were cocultured with or without C57Bl/6-derived MSCs, and IFNγ production was assessed by ELISA after 3 days. MSCs were pretreated with recombinant mouse IFNγ and extensively washed in PBS prior to addition to the mixed lymphocytes cultures (n = 4 per group, mean ± SEM).