Multilineage Differentiation Potential of Cultured MSCs

The in vitro differentiation potential of cultured MSC was evaluated as previously described5 (link)20 (link). Briefly, cells were cultured in osteogenic induction medium (Dulbecco’s modified Eagle’s medium high glucose/L-glutamine (PPA) containing β-glycerophosphate (Sigma Aldrich), L-ascorbic acid-2-phosphate (Wako Chemicals, Neuss, Germany) and dexamethasone (Sigma Aldrich) followed by a Alizarin Red (Sigma Aldrich) staining. For adipocyte differentiation, cells were seeded in AdipoDiff medium (Miltenyi Biotec) and further stained with Oil Red O (Sigma Aldrich). Pictures of adipocyte and osteoblast differentiation were taken with a Nikon Eclipse TE2000-E microscope equipped with a Nikon DS-U2/L2 USB camera using NIS Elements software. Chondrogenic differentiation was performed by culturing cell pellets in chondrocyte induction medium (Miltenyi Biotec) supplemented with TGF-β. Chondrocyte pellets were fixed and frozen in O.C.T (Tissue-Tek, Sakura, Zoeterwoude, The Netherlands). The pellets were sectioned and stained against Aggrecan (cat no. AF1220, R&D Systems). Pictures of chondrocyte pellets were taken with a Nikon TE200E microscope equipped with Olympus DP80 camera using CellSense software.