Adipogenic differentiation medium

Conventional condition-cultured DPSCs were induced into osteogenic, glial and adipogenic lineages. The cells (5,000 cells/well) were cultured on plain 8-well chambers [except for glial differentiation induction where the wells were coated with 4 µg/ml of laminin (Gibco, CA, USA)] for 1 week before differentiation induction. Osteogenic differentiation was induced by incubating the cells with osteogenic differentiation medium (Gibco, NY, USA) for 3 weeks. Thereafter, immunofluorescence of osteocalcin was performed. For glial differentiation, the cells were supplemented with αMEM containing 1 mM mercaptoethanol (Sigma, Germany) without FCS for 24 h and then for 3 days with 20% FCS αMEM containing 35 ng/ml of retinoic acid (Sigma, China). Thereafter, the cells were incubated in 20% FCS-αMEM supplemented with 5 µM forskolin (Sigma, USA), 10 ng/ml of bFGF, 5 ng/ml of platelet-derived growth factor (PDGF) (Peprotech, London, UK), and 200 ng/ml of recombinant human neuregulin-ß1 (Sigma, USA) for the following 3 weeks. Immunofluorescence detection of the Schwann cell marker S100ß was then performed. Finally, the induction of adipogenic lineage differentiation was achieved by culturing cells with adipogenic differentiation medium (Gibco, NY, USA) for three weeks before performing lipidtox staining.

These procedures were described in detail in our previous study (9 (link)). Briefly, for adipogenic differentiation, BM-MSCs and AT-MSCs were seeded into 24-well plates (8x10⁴ cells per well), cultured for 12 h and treated with adipogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 7 days; the medium was refreshed every 3 days. Adherent cells were stained red with 60% Oil Red O (Sigma-Aldrich; Merck KGaA) for 1 min at room temperature. For osteogenic differentiation, BM-MSCs and AT-MSCs were seeded into 24-well plates (4x10⁴ cells per well), cultured for 12 h and treated with osteogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 21 days; the medium was refreshed every 3 days. Osteogenic differentiation was confirmed by 0.2% Alizarin Red staining for 5 min at room temperature. For chondrogenic differentiation, 2x10⁵ MSCs were collected in 15-ml centrifuge tubes and cultured with chondrogenic differentiation medium (Gibco; Thermo Fisher Scientific, Inc.) for 21 days; the medium was refreshed every 3 days. The chondroid pellets were sectioned (8 µm) with a freezing microtome. The slices were stained with 1% toluidine blue for 3 min at room temperature and were captured using a light microscope (Olympus Corporation) at a x50 magnification.