β glycerophosphate

MC3T3E1, a murine pre-osteoblast cell line (CRL-2593, from ATCC, Manassas, VA, USA) was used to evaluate the effects of ES on proliferation, viability, and differentiation process. Cells were cultured in Minimum Essential Medium (MEM), containing 10% fetal bovine serum plus antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) (Invitrogen) and antifungal (amphotericin B 100 mg/mL (BioWittaker Lonza). Osteoblasts cells were seeded at a cellular density of 175,000 cells per condition. Plates were kept at 37 °C in a humidified 5% CO2 atmosphere.
At 72 h of osteoblast culture, the cell medium was changed to osteogenic media (α-MEM medium) supplemented with 10 mM ascorbic acid (Merck, Germany), 10 mM of β-glycerophosphate (StemCell Technologies, Canada) and 10 nM of dexamethasone (Sigma). In-vitro experiments were carried out at 4, 7 and 10 days of cell incubation, in which the supernatants were transferred to vials to be stored at − 80 °C until the last day of the experiment, to evaluate cell differentiation.

The potential of ASC to differentiate into osteoblasts and adipocytes was confirmed in monolayer culture. To induce adipogenic differentiation, cells were cultured in MesenCult™ adipogenic stimulatory supplements (human) (STEMCELL Technologies, Canada) supplemented with MesenCult® MSC basal medium (STEMCELL Technologies, Canada). To induce osteogenic differentiation, we used MesenCult™ osteogenic stimulatory supplements (human) (STEMCELL Technologies, Canada) in the osteogenic medium containing 10⁻⁴ M dexamethasone (STEMCELL Technologies, Canada), 1 M β-glycerophosphate (STEMCELL Technologies, Canada), and 10 mg/ml ascorbic acid (STEMCELL Technologies, Canada). Media from both cultures were replaced every 3 d for 21 d in total. The differentiation potential for adipogenesis and the formation of intracellular lipid droplets were assessed by Oil Red O staining after fixation in 10% formalin. The differentiation potential for osteogenesis was assessed by Alizarin Red S (ARS) staining after fixation in 10% formalin. To induce chondrogenic differentiation, we used a chondrogenic medium (LONZA, Switzerland). For histological analysis, pellets were embedded in paraffin and sectioned. Chondrogenic differentiation was assessed by Masson trichrome staining. Morphology and differentiation potential of ASCs are represented for one individual donor from three independent donors.

To qualify isolated cells from human BM as hMSCs, cells were induced to differentiation into adipogenic, osteogenic and chondrogenic lineages. Briefly, for adipogenic differentiation, 5x10³ hMSCs/cm² were exposed to Complete MesenCult Adipogenic Medium containing MesenCult MSC Basal Medium (Stemcell) and 10% Adipogenic Stimulatory Supplement (Stemcell) for 3 weeks. For osteogenic differentiation, 2x10⁵ cells/cm² incubated with Complete MesenCult Osteogenic Medium including MesenCult MSC Basal Medium, Osteogenic Stimulatory Supplement, β-Glycerophosphate, Dexamethasone, Ascorbic acid (all from Stemcell) for 5 weeks. For chondrogenic differentiation, 7.5x10⁶ cells/cm² were cultured for 3 weeks with Stempro Chondrocyte Differentiation Basal Medium (Gibco) containing 10% Stempro Chondrogenesis Supplement (Gibco). Standard histochemical staining methods were applied. Osteogenic, adipogenic and chondrogenic differentiation were confirmed by Toluidine Blue, Oil Red O, Alcian Blue staining, respectively.