Ascorbic 2 phosphate

Osteogenic differentiation was induced using osteogenic differentiation medium (regular MSC medium supplemented with 50 mg/ml ascorbic 2-phosphate (Sigma-Aldrich), 10 nM dexamethasone (Sigma-Aldrich) and 10 mM ß-glycerol phosphate (Sigma-Aldrich) at P1-P3. Next, osteogenic differentiation was determined using Real-time PCR (see below) and Alizarin Red on day 21 of differentiation. Images were captured using a digital camera system (Olympus DP72) connected to an inverted optical microscope (Olympus CKX41-Olympus Optical CO). For quantitation the Alizarin Red staining, differentiated osteocytes were gently washed by PBS and fixed with 4 % paraformaldehyde in PBS (Sigma-Aldrich) for 15 min. Subsequently, the cells were washed with distilled water, exposed to Alizarin Red S (Sigma-Aldrich) for 20 min at room temperature, and washed again with distilled water for 5 min for 4 times. For quantification of Alizarin Red S staining, the dye was desorbed using 10 % (wt/vol) cetylpyridinium chloride (Sigma, C9002) for 1 hour and absorbance was determined at 540 nm using a Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific™, USA). To analyze the ALP activity in osteocytes, Alkaline Phosphatase Assay Kit (abcam; ab83369) was used. ALP activity was determined based on the manufacturer's protocol and the results were measured at 405 nm (Thermo Scientific™, USA).

PDCs were pelleted and resuspended to a concentration of 20 × 10⁶ cells/mL, plated in 10 μL micromasses, cultured for 7 days (50 μg/mL ascorbic‐2‐phosphate [Sigma A8960‐5G], 10 ng/mL recombinant mouse transforming growth factor [TGF]‐ß1 [PeproTech, Rocky Hill, NJ, USA, 100–21]) and stained with Alcian Blue as previously described.⁽⁵⁾

For both smooth muscle [12 (link)] and osteogenic [13 ] differentiation, 30,000 cells/cm² were seeded single-cell on collagen-coated plates. To induce smooth muscle differentiation, cells were put in DMEM (1X, 4.5 g/L D-glucose) supplemented with 1% pen–step, 1% L-Glutamine, 1% sodium pyruvate, 2% HS (all from ThermoFisher Scientific), and transforming growth factor beta 1 (TGFβ1) (50 ng/ml; Peprotech, New Jersey, USA). For osteogenic differentiation, cells were exposed to αMEM supplemented with 10% FBS, 1% L-Glutamine, 1% pen–strep (all from ThermoFisher Scientific), 50 µM Ascorbic-2-phosphate, 10 mM β-glycerolphosphate, and 10 mM dexamethasone (all from Sigma). Full myogenic differentiation of the cdMiPs was performed according to the protocol published by Shelton et al. [10 ].