*4.10. MSC Metabolic Activity and Proliferation Assays*

MSC were seeded in a 96-well plate at 2000 cells/well and allowed to reach 40% to 60% confluence. Then, metabolic activity and proliferation assays were performed. Metabolic activity was evaluated by resazurin assay, incubating cells with 10% Alamar blue (ThermoFisher Scientific, Waltham, MA, USA) in MSC culture conditions for 4 h. Fluorescence was measured (530 nm excitation and 590 nm emission) in a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA). This procedure was repeated at 1, 3 and 7 days of culture in five replicates. Samples without cells were used as negative controls.

To determine the MSC proliferation index, cells were fixed after 48 h in culture in absence/presence of TNF-α or IL-4 (Immunotools, Friesoythe, Germany) at 10 ng/mL and 100 ng/mL. After permeabilization step with Triton X-100 0.1% and block with 5% BSA, cells were labelled for Ki-67 (1:150; clone SP6, ThermoFisher Scientific, Waltham, MA, USA) followed by AlexaFluor647-conjugated secondary antibody, and nuclei stained with DAPI 1 μg/mL. Cells incubated with secondary antibody only were used as a negative control. Immunofluorescence images were acquired in the IN Cell Analyzer 2000 (GE Healthcare, Chicago, IL, USA) using a Nikon 20x/0.45 NA Plan Fluor objective, capturing 30 sequential and non-overlapping images of the center of each well. Images were analyzed by Fiji Software and proliferation index determined by counting number of cells with Ki-67<sup>+</sup> labelling inside the nuclei relative to the total number of nuclei counted.

#### *4.11. MSC Di*ff*erentiation Assay*

For MSC characterization, cells were seeded in 6 well-plate at a density of 6000 cells/well and grow until reached 40–60% of confluence. Then, cells were incubated in basal or differentiation MSC media without/with osteogenic (α-MEM 10−<sup>7</sup> M dexamethasone (Sigma-Aldrich Inc., St Louis, MO, USA), 10−<sup>2</sup> M <sup>β</sup>-glycerophosphate and 5 <sup>×</sup> 10−<sup>5</sup> M ascorbic acid) and adipogenic (10−<sup>4</sup> M dexamethasone, <sup>5</sup> <sup>×</sup> <sup>10</sup>−<sup>4</sup> M IBMX, 10 <sup>μ</sup>g/mL insulin, 10−<sup>4</sup> M indomethacin) supplements for 21 and 28 days, respectively. Media was changed twice a week and at the end of the osteogenic or adipogenic differentiation, and cells were fixed and stained with Alizarin red or Oil red O, respectively.

For chondrogenic differentiation, a cell pellet of 2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells was incubated in 15 mL falcon conical tubes in differentiation media (α-MEM supplemented with 4.5 g/L glucose, 2.5 <sup>×</sup> 10-4 M ascorbic acid, 40 μg/mL l-proline, 100 μg/mL sodium pyruvate, 100 μg/mL ITS, 10−<sup>7</sup> M dexamethasone, and 10 ng/mL TGF-β3) for 28 days. Then, cell pellet was fixed with paraformaldehyde 4%, processed for histologic analysis, and stained by H&E and Toluidine blue stain.

For MSC gene expression, cells were cultured in basal conditions for 3 and 7 days, in absence/presence of TNF-α or IL-4 (10 ng/mL and 100 ng/mL). Cells stimulated with osteogenic and chondrogenic media as above were used as positive controls. Cells were collected for RNA isolation and expression of gene markers for osteogenic and chondrogenic differentiation was analyzed.
