H insulin

Human multipotent abdominal subcutaneous adipose-derived mesenchymal stem cells (hMADS) were obtained from overweight/obese men with impaired glucose metabolisms and pooled and differentiated into the adipogenic lineage. Therefore, cells were seeded at a density of 2000 cells∙cm⁻² and cultured, as described previously [27 ,28 (link)]. Human multipotent adipose-derived mesenchymal stem cells (hMADS) were kept in a proliferation medium containing DMEM and Ham's F-12 (DMEM-Ham's F-12) Nutrient Mixture (no. 31330-095, Gibco), 10 % FBS (Bodinco BV), and 1x Antibiotic-Antimycotic (Gibco). At ~80 % confluence, a differentiation medium was added to the cells containing DMEM-Ham's F-12, 3 % FBS (Bodinco BV), 1x Antibiotic-Antimycotic (Gibco), 33 μM D-Biotin (no. B4693, Sigma), 17 μM d-pantothenate (no. P5155, Sigma), 0.1 μM h-insulin (no. 91077C, Sigma), 1 μM dexamethasone (no. D4902, Sigma), 250 μM 3-isobutyl-1-methylxanthine (IBMX, no. I5879, Sigma), and 5 μM rosiglitazone (no. ALX-350-125-M025, Enzo Life Sciences). After 7 days, IBMX and rosiglitazone were removed from the medium. All cells were proliferated and differentiated under 21 % O₂, and thereafter, exposed to either 10 % O₂ continuously (resembling physiological normoxia), or to MIH consisting of 3 × 2 h cycles per day, alternating between 5 and 10 % O₂, during the final 7 days of cell culture.

MMTV-PyMT mice were sacrificed at ~12–18 weeks of age for tumor cell isolation. Tumors were minced then digested with 37.5 µg/ml Liberase TH (Roche #5401151001), 37.5 µg/ml Liberase TM (Roche #5401127001), and 25 µg/ml DNAse (Roche #11284932001) in DMEM/F12 (Gibco #31331028) supplemented with HEPES and pen/strep for 1 h at 37 °C. Cells were washed once in 4 °C 1x PBS supplemented with 0.5% BSA and 2 mM EDTA, then were passed through a 70 µm strainer, then washed an additional two times in DMEM/F12 supplemented with HEPES and pen/strep. At this point, cells were stained for flow cytometry or lysed for RNA isolation. Otherwise, cells were seeded onto collagen-coated plates with DMEM/F12 supplemented with 2% FBS, HEPES, pen/strep, 20 ng/ml hEGF (Gibco #PHG0313), and 10 µg/ml hInsulin (Sigma-Aldrich #I9278-5ML).

The cells were incubated under specific conditions to induce differentiation into osteocytes, chondrocytes, and adipocytes, and the multilineage potential was evaluated as previously described [34 (link)]. In brief, osteoblast or osteocyte formation was assessed by measuring the level of alkaline phosphatase (ALP; Sigma-Aldrich) activity. To confirm chondrogenic differentiation, cryosections were analyzed by safranin O staining (Sigma-Aldrich). In particular, to induce adipogenic differentiation, the cells were treated with an adipogenic medium consisting of high-glucose Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 1 mM dexamethasone (Sigma), 0.2 mm indomethacin (Sigma), and 10 mM h-insulin (Sigma) for 2 weeks. Assessment of adipocyte formation was based on the staining of accumulated lipid vacuoles with Oil red O (Sigma-Aldrich). Moreover, we used the fluorescent neutral lipid dye 4.4-difluoro-1, 3, 5, 7, 8-pentametyl-4-bora-3a, 4a-diaza-s-indacene (BODIPY 493/503, Molecular Probes, Carlsbad, California, USA) to confirm the lipid drop formation by confocal microscopy (Zeiss, Germany) [35 (link)]. Lipid vacuole accumulation was quantified by calculating the percentage of stained cells in the total population. All quantitation of stained cells was performed using SABIA software (Meetoo, Seongnam, Korea).