Mesenchymal stem cell adipogenesis 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 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.

Adipogenesis was induced by plating hC-MSCs at density of 6 × 10⁴ cells/well in a 24-well plate using the Mesenchymal Stem Cell Adipogenesis Kit (Chemicon International, Temecula, CA, USA) according to the manufacturer’s instructions. Induction medium was replaced every 2 to 3 days for 2 to 3 weeks and alternated with maintenance medium (DMEM 10% fetal bovine serum (FBS) and 0.02 mg/ml insulin). Three complete cycles of induction/maintenance medium stimulated optimal adipogenic differentiation, forming adipocytes. Control cells were culture in basal medium (DMEM plus 10% FBS). To confirm their identity, hC-MSCs were fixed and the cytoplasmic presence of lipid droplets was assessed by Oil Red O staining and transmission electron microscopy (TEM). The cells cultured were also processed for reverse transcriptase (RT)-PCR analysis as specified above to investigate the expression of adipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARγ).

P3 SHEDs were seeded at a density of 2.0 × 10⁵ cells/well into 6-well plates with routine DEME. When the cells become 80% confluence, the culture medium was replaced with adipogenic medium (Mesenchymal Stem Cell Adipogenesis Kit, Chemicon, USA). SHEDs were grown in the adipogenic medium for 21 days. The cells were fixed with 4% formalin for 10 min at room temperature. After washed by PBS and incubated by isopropanol, the cells were stained with working solution of the Oil Red O for 30 min. The Oil Red O-positive cells were analyzed under microscopy by computer as described previously [28 ]. Adipogenesis was determined by lipid accumulation in fat vacuoles.

Cell differentiation was performed using StemPro Adipogenesis Differentiation Kit (Gibco, United States) and StemPro Osteogenesis Differentiation Kit (Gibco, United States) according to the manufacturer’s instructions. SCs were seeded in 12-well plates at a density of 100,000 cells per well and cultured during 2–3 days until reaching 100% confluency, and growth media have been replaced by differentiation induction media. For adipogenic differentiation, the medium was changed every 2–3 days, and cells were fixed with 4% formaldehyde (Panreac, United States) for 30 min on day 14; lipid droplets were stained with Oil Red O solution from Mesenchymal Stem Cell Adipogenesis Kit (Chemicon, United States) according to the manufacturer’s instructions. For osteogenic differentiation, the medium was changed every 3–4 days, and cells were fixed with 4% formaldehyde for 30 min on day 21; mineral deposits were stained by Alizarin red solution from Mesenchymal Stem Cell Osteogenesis Kit (Chemicon, United States) according to the manufacturer’s instructions. Visualization and image acquisition were performed on an inverted Leica DMi8 microscope with a DFC7000T camera, and images were processed in Fiji package (Schindelin et al., 2012 (link)) based on ImageJ (NIH, United States).