Dual-lineage differentiation of BMSCs

Before preparing the mixed differentiation medium, gWAT-CM was diluted 10-fold with fresh medium to obtain 10% gWAT-CM. The mixed induction medium was composed of 50% adipogenic medium and 50% osteogenic medium, with an adipogenic to osteogenic induction ratio of 1:1 as reported^{42 (link)}. Wild-type BMSCs were differentiated in the mixed induction medium supplemented with 10% WAT-CM or BMAds-CM as indicated, in the presence of 0.5 μg/ml recombinant SPP1 protein (rSPP1, Abcam #ab281820), 1 μg/ml SPP1 neutralizing antibody (SPP1 Nab, Novus Biologicals #AF808), 0.5 μg/ml recombinant leptin protein (rLeptin, R&D Systems #490-OB-01M), 250 nM leptin receptor antagonist Allo-aca (MCE #HY-P3212) or an equal volume of vehicle IgG (Cell Signaling Technology #2729). The formation of mineralized nodules was evaluated by alizarin red S staining, and adipocytes were distinguished by oil red O staining.

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Huang T., Lu Z., Wang Z., Cheng L., Gao L., Gao J., Zhang N., Geng C.A., Zhao X., Wang H., Wong C.W., Yeung K.W., Pan H., Lu W.W, & Guan M. (2024). Targeting adipocyte ESRRA promotes osteogenesis and vascular formation in adipocyte-rich bone marrow. Nature Communications, 15, 3769.

Publication 2024

AF808

Corresponding Organization :

Other organizations : Chinese Academy of Sciences, Shenzhen Institutes of Advanced Technology, ShenZhen People’s Hospital, Shantou University, Shantou University Medical College, Kunming Institute of Botany, University of Hong Kong, University of Chinese Academy of Sciences

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Variable analysis

independent variables

10% gWAT-CM
10% BMAds-CM
0.5 μg/ml recombinant SPP1 protein (rSPP1)
1 μg/ml SPP1 neutralizing antibody (SPP1 Nab)
0.5 μg/ml recombinant leptin protein (rLeptin)
250 nM leptin receptor antagonist Allo-aca

dependent variables

Formation of mineralized nodules (evaluated by alizarin red S staining)
Adipocyte formation (distinguished by oil red O staining)

control variables

Wild-type BMSCs
Mixed induction medium (50% adipogenic medium and 50% osteogenic medium)
Vehicle IgG

controls

Positive control: Mixed induction medium supplemented with 10% WAT-CM or BMAds-CM
Negative control: Mixed induction medium supplemented with vehicle IgG

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