Mesenchymal stem cell adipogenic differentiation medium 2

The hMSCs were obtained from Lonza and maintained in a xenofree hMSC culture medium (ROHTO Pharmaceutical). For RNAi experiments, hMSCs were transfected with Silencer Select siRNA (Ambion) by using Lipofectamine RNAiMAX reagent (Life Technologies) according to the protocol supplied by the manufacturer. Incubation with adiponectin-containing media started 36 h after transfection. The multilineage differentiation of hMSCs to adipocytes, osteoblasts, and chondrocytes was tested by using Mesenchymal Stem Cell-Adipogenic Differentiation Medium 2, -Osteogenic Differentiation Medium, and -Chondrogenic Differentiation Medium, respectively (PromoCell), according to the protocol supplied by the manufacturer. After the incubation with differentiation medium, staining with Oil Red O, Alizarin Red S, and Alcian Blue was performed to detect the adipocytes, osteoblasts, and chondrocytes, respectively.

Human bone marrow-derived mesenchymal stromal cells were procured from PromoCell (hBM-MSC, C-12974). Cells were cultured in flat bottom transparent 6-well plates with 4 × 10⁴ cells/well seeding density and fed Mesenchymal Stem Cell Growth Medium 2 (C-28009, PromoCell) unless otherwise noted. Cultured cells were kept in incubators with 37°C temperature and 5% CO₂ between experiments. Adipogenic differentiation of hBM-MSCs were induced in cells that reached confluency by swapping cell culture medium with Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (C-28016, PromoCell). Induced cells were cultured for 14 days with periodical medium changes (every third day) for cells to reach a mature adipocyte state. For treatment groups, HNG was present in the initial differentiation medium only and subsequent medium changes did not contain HNG. A blank group without differentiation inducement and HNG treatment, and a control group with adipogenic differentiation but not HNG were cultured.

Bone marrow-derived human mesenchymal stem cells (hBM-MSCs) were procured from PromoCell (C-12974, Heidelberg, Germany). Cells were seeded in 6-well plates (1 × 10⁶ cells/well) and cultured using Mesenchymal Stem Cell Growth Medium (C-28009, PromoCell). Incubation of the plates was carried out in an environment with 37°C temperature and 5% CO₂ atmosphere. For adipogenic differentiation of hBM-MSCs, cells were grown to confluence prior to swapping cell culture medium with Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (C-28016, PromoCell). Following the introduction of differentiation, medium cells were incubated for 10 days (unless otherwise noted), and the medium was changed every third day without disturbing the cell monolayer. Luteolin and luteolin-OSO₃Na were supplied along with initial differentiation inducement and were not present in consequent media changes.