Adipocyte Differentiation of OP9 Cells

Cells were incubated at 37°C in a humidified atmosphere with 95% air and 5% CO₂. OP9 stromal cells (CRL-2749) were cultured in α-MEM (HyClone) supplemented with 20% fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 1× penicillin/streptomycin. Primary B-ALL cells harvested from xenografts were enriched via magnetic cell sorting using anti-human CD45 MicroBeads (Miltenyi Biotec) and cultured at a density of 1 × 10⁶/ml in RPMI1640 (HyClone, Thermo Scientific), 10% FBS, 2 mM L-glutamine, 1× P/S. For cytokine supplementation, the culture medium was supplemented with 10 ng/ml IGF-1 (Peprotech, 100–11), 10 ng/ml IL-7 (Peprotech, 200–07), 10 ng/ml IL-6 (Peprotech, 200–07). The culture medium was changed every two days. Adipocyte differentiation was induced following a previous study.^{21 (link)} Briefly, OP9 cells were cultured as a monolayer. Upon reaching confluence, the cells were maintained for an additional 2 days and then incubated in DMEM supplemented with 10% FBS, 1 µM Dexamethasone, .5 mM isobutyl-methylxanthine, 5 µg/mL insulin, and 1 µM Rosiglitazone (Sigma-Aldrich) for 2 days. Subsequently, the medium was replaced with DMEM plus 10% FBS and 1 µM Dexamethasone for 7–14 days, with fresh medium provided every 2 days.