Cell culture

The immortalized human fetal osteoblast cell line hFOB1.19 (hFOB1.19) was purchased (ATCC® CRL-11372™). It was cultured according to the protocol specified by the repository and regularly tested for mycoplasma contamination. hFOB1.19 cells were maintained in growth medium (GM): DMEM:Ham’s F12 (Sigma, D6421) supplemented with 10% fetal bovine serum (FBS), 2.5 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, 300 µg/ml G418 (Sigma, G8168). All the experiments were carried out at 34 °C and 5% CO₂. Two monoclonal hFOB1.19 cell lines expressing enhanced green fluorescent protein (eGFP) under the control of the i) human full-length osteocalcin promoter (hFOB hOC_eGFP) and ii) the human ubiquitinase (UbC) promoter (hFOB_eGFP) were generated previously by lentivirus transduction¹⁵. Cultivation was conducted at 34 °C in growth media. Osteogenesis was triggered in spheroids by incubating the cells at 34 °C in osteogenic medium (OM): DMEM:Ham’s F12 mixture containing 2% FBS, 500 µM ascorbic acid and 10 nM dexamethasone (Sigma, D4902). For adipogenic differentiation, the cells were incubated in adipogenic medium (AM) supplemented with 0.5 mM isobutyl‐methylxanthine (Sigma, I5879), 1 μM dexamethasone, 10 μM insulin (Sigma, l9278), and 60 μM indomethacin (Sigma, I7378). Spheroids were formed by incubating 10³ cells in ultralow attachment U-bottom 96-well plates (Greiner)⁴⁶. Further cultivation was carried out in 96-well-plates at 34 °C and 5% CO₂, with medium changes performed every second day. eGFP fluorescence was quantified by summing all pixel values in the region of interest (raw integrated density) by means of image analysis (ImageJ, Fiji open source) at 3 and 6 days post-aggregation. eGFP signals related to growth and osteo- and adipogenic media were normalized to the area, and differences were statistically evaluated via unpaired t tests.

THP-1 cells were purchased (ACC16; DMSZ Leibniz-Institut-Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH). They were expanded in RPMI 1640 medium supplemented with 5% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Human mesenchymal stem cells-hMSCs (Cambrex, East Rutherford, NJ) were expanded in α-MEM supplemented with 20% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Both THP1 cells and hMSCs were cultivated as monocultures at 37 °C. For all co-culture experiments, complete DMEM:Ham’s F12 growth medium was used.