1. Rinse MSC with 2 ml PBS, trypsinize with 0.5 ml trypsin, transfer to 15 ml tube, add 10 ml DMEM with 10% FBS. 2. Pellet cells at 435 x g for 5 min at 4 °C, resuspend in 2 ml DMEM with 10% FBS, seed in 6-well plate at 5,000-10,000 cells per well. 3. Incubate overnight at 37 °C, 5% CO2. 4. Wash cells twice with 2 ml PBS, add osteogenic differentiation medium, change medium every 2 days for 10 days. 5. At day 10, wash cells with PBS, stain with Alizarin red S for 5 min, rinse with distilled water. 6. Incubate wells with 1 μg IL-27 in 500 μl serum-free DMEM for 1 hr on ice. 7. Wash cells 5 times with 1 ml ice-cold serum-free DMEM, scrape in 100 μl urea/phosphate lysis buffer. 8. Transfer lysates to 1.5 ml tubes, dilute 10 times with SDS-PAGE loading buffer, quantify protein concentration, analyze 50 μg protein by SDS-PAGE and Western blot. 9. Detect IL-27 using biotinylated goat anti-mouse p28 Ab and HRP-labeled streptavidin.