4000b dmi

Cells were grown from cryopreserved stocks to confluence in twelve-well-plates and then subjected to specific differentiating conditions using the osteogenic differentiation medium PT-4120, and chondrogenic medium PT-3003 supplemented with 10 ng/mL TGFβ3, PT-4124 (all from Lonza) under conditions described by the manufacturer.
Multilineage differentiation potential of MSCs was assessed by histochemical staining and phase contrast microscopy (Leica 4000b DMI, Leica Microsystems GmbH. Germany).
The degree of mineralization in osteogenic cultures was assessed staining with 2% Alizarin Red S (Sigma-Aldrich-Aldrich). Briefly, cells were washed three times with phosphate buffered saline (PBS) pH 4.2 and fixed 20 minutes with 4% paraformaldehyde. Fixed cells were washed and stained and washed again to remove excess of stain. Similarly, chondrogenic potential was evaluated measuring production ECM proteoglycan produced by chondrocytes after staining for 20 minutes with 1% Alcian blue (Sigma-Aldrich).

Cells from cryopreserved stocks were grown to confluence in 12-well plates and subjected to osteogenic and chondrogenic differentiation using the appropriate induction media (Lonza osteogenic medium Cat# PT-4120, and chondrogenic Cat# PT-3003) supplemented with 10 ng/mL TGFβ3 Cat# PT-4124 under conditions described by the manufacturer.
The multilineage differentiation potential of MSCs was assessed by histochemical staining and phase-contrast microscopy (Leica 4000b DMI, Leica Microsystems GmbH., Wetzlar, Germany). The degree of mineralization in osteogenic cultures was assessed by staining with 2% Alizarin Red S (Sigma-Aldrich, Saint Louis, MO, USA). Briefly, the cells were washed three times with phosphate buffered saline (PBS) pH 4.2 and fixed 20 min with 4% paraformaldehyde. The fixed cells were washed, stained, and washed again to remove excess stain. Similarly, the chondrogenic potential was evaluated by measuring the production of proteoglycans produced by chondrocytes after staining for 20 min with 1% Alcian blue (Sigma-Aldrich, Saint Louis, MO USA) (Figure S4).