Fig. 9: HFD-induced adipogenic differentiation of mesenchymal stem cells promote subchondral bone marrow osteoclast differentiation in a ferroptosis-dependent manner.

a IF staining of Perilipin-1 (red) on the subchondral bone of joints from OA patients (n = 6). Scale bar, 100 µm. b A schematic diagram of an in vitro cell co-culture model involving murine BMSC and osteoclasts. Created in BioRender. Zhao, C. (2024) https://BioRender.com/s11w531. c TRAP staining. The upper layer of stem cells was subjected to two different culture media: normal medium and adipogenesis medium. Scale bar, 100 µm. d TRAP staining. In the last column, ruxolitinib (10 μM) was included in the adipogenesis medium. Scale bar, 100 µm. e, f Quantification of mature TRAP⁺ osteoclasts in (c, d) (n = 6). g, h TRAP staining (h) and quantification of mature TRAP⁺ osteoclasts (g) (n = 6). Scale bar, 100 µm. In the third column, the upper layer of stem cells was supplemented with PFT β (10 μM), and in the last two columns, the upper layer of stem cells was pre-infected with lentivirus expressing empty vector or FOXO3. i, j FerroOrange staining (red) (i) and lipid peroxidation staining (green) (j). Scale bar, 50 µm. k–m IF staining of Gpx4 (red), Slc7a11 (red) and Fsp1 (red). Scale bar, 50 µm. n, o Exchange of cells in the co-culture system (o). Created in BioRender. Zhao, C. (2024) https://BioRender.com/s11w531. The upper layer of pre-osteoclasts was subjected to normal medium, RANKL-containing medium, and RANKL + Fer-1-containing medium. Oil Red (red) staining was used to assess the extent of adipogenic differentiation in the lower layer of stem cells (n). Scale bar, 100 µm. p Oil Red (red) staining. The upper layer of pre-osteoclasts was treated with normal medium, RANKL-containing medium, RANKL plus PFT β-containing medium, or cells pre-infected with lentivirus. Scale bar, 100 µm. q–s IF staining of Perilipin (red) (q), γH2AX (red) (r), and Lamin-B1 (red) (s). The cell grouping and treatment methods were the same as in (n). Scale bar, 100 µm. t–v IF staining of Perilipin (red) (t), γH2AX (red) (u) and Lamin-B1 (red) (v). The cell grouping and treatment methods were the same as in (p). Scale bar, 100 µm. The above experiments were repeated at least three times. Data are represented as mean ± s.d. One-way ANOVA was performed. All exact p-values have been indicated in the figures. Source data are provided as a Source Data file.