MSCs were isolated from single cell suspensions (SCS) of TN and FL LN and BM tissues. One million cells/cm2/mL were plated in RPMI 1640 with 10% FBS (Life Technologies), penicillin/streptomycin (Cellgro), (R10) supplemented with 12.5 ng/mL basic fibroblast growth factor (R&D Systems). Non-adherent cells were discarded after 2 days of culture. Adherent cells were expanded until confluent, replacing media every 3 days. MSCs were used from passages 2–8.
Adipogenic induction was achieved by culturing confluent MSCs in adipogenic induction/maintenance media according to the manufacturer’s protocol (Lonza). Cells were fixed in 10% formalin and stained with an Oil Red O solution (Sigma) to visualize lipid vacuoles. Osteoblast differentiation was induced by culture for 3 weeks in R10, supplemented with 10 µM dexamethasone, 0.1 mM ascorbic acid and 10 mM β-glycerophosphate (Sigma). Early osteoblast differentiation was assessed by alkaline phosphatase staining after fixation and incubation with a solution containing Naphthol AS-MX-phosphate and Fast Red LB salts (Sigma).
Adipogenic induction was achieved by culturing confluent MSCs in adipogenic induction/maintenance media according to the manufacturer’s protocol (Lonza). Cells were fixed in 10% formalin and stained with an Oil Red O solution (Sigma) to visualize lipid vacuoles. Osteoblast differentiation was induced by culture for 3 weeks in R10, supplemented with 10 µM dexamethasone, 0.1 mM ascorbic acid and 10 mM β-glycerophosphate (Sigma). Early osteoblast differentiation was assessed by alkaline phosphatase staining after fixation and incubation with a solution containing Naphthol AS-MX-phosphate and Fast Red LB salts (Sigma).