Kinetix camera

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 assess the adipogenic potential, iMSCs, pMSCs and ASC52telo were cultured in 12-well plates in complete growth medium for 15 days until the monolayer was achieved. Adipogenic differentiation was induced by replacing the growth medium with DMEM low glucose (Gibco, #11885084) supplemented with 10% of FBS, insulin (10 μg/mL), IBMX (0.5 mM) and dexamethasone (1 μM), according to the previously described protocol [8 (link)]. Control cells were cultured in DMEM low glucose + 10% FBS medium. The medium was replaced every two days. On day 21, cells were washed with HBSS (Hanks’ Balanced Salt Solution) (PanEko, Moscow, Russia, #P020n) supplemented with 20 nM of HEPES (Dia-M, Moscow, Russia, #3350.0100) and then incubated in HBSS with 20 nM HEPES and 1 mM Nile red for 1 h in a cell culture incubator at 37 °C. The images of the stained cultures were captured with a Nikon Eclipse Ti2 microscope equipped with a Kinetix camera (Teledyne Photometrics) and analyzed using NIS Elements AR 5.40.02 software. The effectiveness of MSC adipogenic differentiation was evaluated by the staining intensity of cells with lipophilic dye Nile red (Sigma-Aldrich, #19123).

Alizarin Red was used to estimate the efficiency of osteogenic differentiation. Cells were fixed with 10% paraformaldehyde for 30 min. Then, they were stained with the Osteogenesis Quantitation Kit (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. Cell cultures were stained with Nile Red (Sigma-Aldrich, St. Louis, MO, USA) to determine the efficiency of adipogenic differentiation. Cells were washed with HBSS with 20 nM HEPES and then stained for 1 h in a cell culture incubator in HBSS with 20 nM HEPES containing 1 mM of Nile Red. Obtained samples were analyzed via Nikon Eclipse Ti2 microscope with a Kinetix camera (Teledyne Photometrics, Tucson, AZ, USA). Image capture and analysis was performed using NIS Elements AR 5.40.02 software.