Osteogenic Differentiation of Mesenchymal Stem Cells

For osteogenic differentiation assays, 3x10⁴ culture-expanded MSCs (passage 3) from donor-matched IC-BM and VB-BM samples were cultured in osteogenic media. The osteogenic media was formed of low glucose DMEM (ThermoFisher Scientific Waltham, MA, USA) supplemented with 10% FCS (ThermoFisher Scientific), penicillin and streptomycin (Thermo Fisher Scientific), 100nM dexamethasone, 10mM β-glycerophosphate and 0.05mM ascorbic acid (all from Sigma-Aldrich). After 14 days of culture, the quantification of the calcium level was performed using colorimetric calcium kit (Calcium Liquid, Sentinel Diagnostics, Milan, Italy) as previously described [37 (link)]. To extract calcium, cultured MSCs were treated with 1M of HCl solution. The spectrophotometric reading was taken on MULTISCAN EX reader and analysed using Ascent software (Thermo Fisher Scientific). Additionally, the staining for calcium deposition and alkaline phosphatase (ALP) expression was performed using Alizarin Red dye and fast blue RR salt dye respectively (both from Sigma-Aldrich) as used previously [32 (link), 38 (link)]. The culture plates were scanned using an Epson 3590 flatbed scanner (Epson Ltd, Hertfordshire, UK).