Mesenchymal stem cell osteogenesis kit

hTSCs were seeded at a concentration of 3 × 10⁴ cells/cm² in a growth medium, and after 24 hours, cells were switched to an osteogenic or adipogenic medium for 17 days or 21 days, respectively. Osteogenic differentiation was obtained by culturing cells in the presence of DMEM-low glucose (Merck) supplemented with 4 mM L-glutamine (Euroclone), 1% antibiotic-antimycotic mixture (Euroclone), 10% FBS (HyClone, Thermo Fisher Scientific), 10 nM cholecalciferol (Merck Millipore), and the mesenchymal stem cell osteogenesis kit (Merck Millipore) according to the manufacturer's instructions. Adipogenic differentiation was induced by culturing cells in the presence of DMEM-low glucose supplemented with 4 mM L-glutamine, 1% antibiotic-antimycotic mixture, 10% FBS, and the mesenchymal stem cell adipogenesis kit (Merck Millipore), according to the manufacturer's instructions. To evaluate the effects of ganglioside GM1 treatment (Santa Cruz Biotechnology) on differentiation, hTSCs were cultured for 17 days in an osteogenic medium or 21 days in adipogenic medium supplemented with 1, 10, 50, and 100 μM GM1. To evaluate the effects of the platelet-derived growth factor-BB (PDGF-BB, Thermo Fisher Scientific) on osteogenic differentiation, cells were cultured in an osteogenic medium containing PDGF-BB at the final concentration of 10 ng/ml. The differentiation medium was changed every 2-3 days.

To assess the osteogenic potential, iMSCs, pMSCs and ASC52telo were cultured in 12-well plates in complete growth medium for 15 days until the dense monolayer was achieved. Osteogenic differentiation was induced using a Mesenchymal Stem Cell Osteogenesis Kit (Merck, Darmstadt, Germany, #SCR028) according to the manufacturer’s instructions. The medium for osteogenic differentiation was based on DMEM low-glucose medium (Gibco, #11885084) supplemented with 10% of FBS, CaCl2 (100 mg/L), dexamethasone (0.1 μM), ascorbic acid (0.2 mM), glycerol-2-phosphate (10 mM), and L-glutamine (2 mM). Control cells were cultured in DMEM low glucose + 10% FBS medium. The medium was replaced every two days. On day 15, the medium was removed, and cells were fixed in 70% ethanol for 1 h. Cells were washed with water and stained with Alizarin red (Sigma, #A5533-25G). The images of the stained cultures were captured with a Nikon Eclipse Ti2 microscope equipped with a Kinetix camera (Teledyne Photometrics, Tucson, AZ, USA) and analyzed using NIS Elements AR 5.40.02 software. The effectiveness of MSC osteogenic potential was assessed by the staining intensity of mineral deposits, primarily, calcium carbonate in the extracellular matrix.

To induce adipocyte or osteocyte differentiation, we incubated culture-expanded BM-MSCs at passage 3 with either adipogenic medium or osteogenic medium using a Mesenchymal Stem Cell Adipogenesis Kit or Mesenchymal Stem Cell Osteogenesis Kit (Chemicon International, Temecula, CA). Three or four days later, intracellular lipid droplets were stained with a Lipid Assay kit (Cosmo Bio, Tokyo) according to the manufacturer’s instructions. Alkaline phosphatase activity was visualised using an Alkaline Phosphatase (ALP) staining kit (Primary Cell Co., Hokkaido, Japan) according to the manufacturer’s instructions.