Stempro osteogenic differentiation kit

MSC(AT) were seeded at a density of 100,000 cells/cm² on a 24-well culture plate. Differentiation was also induced 24 h later by using the StemPro™ Osteogenic Differentiation Kit (Thermo Fisher Scientific). Complete differentiation medium supplemented with 1% (v/v) A/A was changed twice a week for 21 days, after which cells were fixed as stated for the adipogenic differentiation protocol. Cells were subjected to both an alkaline phosphatase and a Von Kossa stain to verify their osteogenic differentiation. Firstly, fixed cells were incubated for 40 min at RT in a solution of Fast Violet (Sigma Aldrich) and Naphthol (Sigma Aldrich), and then they were washed with distilled water afterward. With the alkaline phosphatase stain completed, cells were then incubated for 30 min with silver nitrate (Sigma Aldrich), washed three times with distilled water, and kept in PBS. Differentiation phenotype was observed under the microscope and images were taken.

In order to test the effects of miRNA overexpression on the osteogenic differentiation potential, HUVECs were seeded at a density of 4 × 10⁴ cells/well on 24-well plates and left to adhere. After miRNA transfection, Optimem medium was changed with the Stem Pro Osteogenic Differentiation Kit (Thermo Fisher Scientific, Waltham, MA, USA). The miRNA transfection was repeated every three days to keep miRNA levels in cell culture. HUVECs cultured in DMEM with 10% FBS were used as controls. After 14 days, HUVECs were processed for Alizarin Red staining to detect the mineralization process, immunofluorescence, RNA and protein extraction to investigate osteogenic marker expression. For mineralization activity, cells were fixed with formalin, washed twice with PBS and stained with Alizarin Red for 30 min at rt. Images were taken under a phase-contrast light microscope and pictures were taken with a digital camera (Nikon, Tokyo, Japan). Then, Alizarin Red was dissolved by adding 10% cetylpiridinium chloride (in Na₂HPO₄ 10 mM, pH 7) to wells for dissolving Alizarin Red, for 15 min in the dark at rt. Calcium-bound Alizarin Red was measured by reading absorbance O.D. 570 nm by spectrophotometer.

Osteogenic differentiation was performed utilizing a StemPro osteogenic differentiation kit (ThermoFisher, UK) as per the manufacturer’s instructions. OMLP-PC cell lines and cell strains were seeded at a density of 5 × 10³ cells/well. Cells were cultured under normal conditions for 24 h before the medium was removed and replaced with an osteogenic induction medium. The medium was changed every 48 h for 14 days. After 14 days cells were fixed with 4% (v/v) PFA for 20 minutes before staining with 40 mM alizarin red staining solution for 5 minutes. Cell monolayers were extensively washed with ddH₂O. Images were obtained using a brightfield inverted microscope. Data is presented from one of 3 biological repeats.
Adipogenic differentiation was performed utilizing a StemPro adipogenic differentiation kit (ThermoFisher, UK) as per the manufacturer’s instructions. OMLP-PC cell lines and strains were seeded at a density of 3 × 10⁴ cells/well. cultures were re-fed every 48 h for 14 days. After 14 days of culture, cells were washed with PBS and fixed with 4% (w/v) PFA for 20 minutes. Cells were stained with both LipidTox green lipid stain (1:100; Fisher Scientific, UK) and 10 μM Hoechst 33342 in PBS for 30 minutes. Cells were imaged using a Leica SP5 confocal microscope. Data is presented from one of 3 biological repeats.

For osteogenic and adipogenic differentiation, CBti MSCs at the end of P4 were seeded at a density of 4,000 cells/cm2 on cell culture coverslips (Thermo Fisher Scientific) and arranged in 24-well plates (Falcon®, Corning, Corning, NY, USA) in the presence of standard growth medium. At 70–80% cell confluence, the medium was replaced with specific differentiation media, then renewed every 3–4 days for 21 days. To induce adipogenic differentiation, cells were evaluated using the StemPro® Adipogenic Differentiation Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. The presence of intracellular lipid droplets was detected by standard staining with Oil Red O (Diapath, Bergamo, Italy). In parallel, cells were also grown using the StemPro® Osteogenic Differentiation Kit (Thermo Fisher Scientific). The presence of calcium deposits representing osteogenic differentiation were evaluated by von Kossa staining (Sigma-Aldrich). Cells were fixed with 10% formalin for 5 min at room temperature, incubated with 1% silver nitrate solution for 15 min and exposed to ultraviolet light for 2 h. Coverslips were rinsed with distilled water and 5% sodium thiosulfate to remove unreacted silver.