Mscbm

Human bone marrow MSCs purchased from Lonza (Basel, Switzerland) were cultured in mesenchymal stem cell basal medium (MSCBM, Lonza). Cells were sub-cultured after 15 days following thawing from passage one and then after every seven days for successive passages. EVs were isolated from cells until passage six.

Human bone marrow-derived Mesenchymal Stem Cells (MSCs; PT-2501, Lonza, Walkersville, MD, USA) and Vascular Smooth Muscle Cells (VSMCs; coronary artery smooth muscle cells, CC-2583, Lonza) were cultured as described previously [27 (link)]. Briefly, MSCs and VSMCs were expanded in Mesenchymal Stem Cell Basal Medium (MSCBM, PT-3238, Lonza) supplemented with Mesenchymal Stem Cell Medium SingleQuot Kit (MSCGM, PT-4105, Lonza) and Smooth muscle cell Basal Medium (SmBM, CC-3181, Lonza) supplemented with Smooth muscle Medium-2, SingleQuot Kit (SmGM-2, CC-4149, Lonza) respectively. For induction of MSCs differentiation into osteoblasts (referred also as MSC/osteoblasts) and VSMC development into C-VSMCs (VSMC/C-VSMC), cells were cultured in DMEM medium (GIBCO, Paisley, UK) containing 10% FCS, penicillin/streptomycin, 1.8 mM CaCl₂ (Sigma, St. Louis, MO, USA) and 20 mM HEPES (Sigma), pH 7.5. Additionally, this medium was freshly supplemented with 0.1 mM ascorbic acid (Sigma), 10 mM ß-glycerophosphate (Sigma) and 100 nM dexamethasone (DEX, Sigma). In the present study 2 independent MSC and VSMC donors were used, one for the gene expression array and the other for validation purposes. All analyses were performed on samples collected at the beginning of cell culture (day 0, before induction of differentiation) and during week 1, 2 and 3 of culture.

Human bone marrow-derived MSCs were purchased from Osiris Therapeutics, Inc. (Columbia, MA) and subcultured in MSCGM medium (MSCBM supplemented with 10% serum, 2% L-glutamine and 0.1% gentamicin sulfate amphotericin b, Lonza Group Ltd.). The fibronectin coated micropatterns were put in 6-well plates and a glass ring (inner diameter 1.5 cm) was placed over each micropattern plate. An aliquot of 3 mL serum-free medium (DMEM medium supplemented with 4500 mg/L glucose, 584 mg/L glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.1 mM nonessential amino acids, 0.4 mM proline, 50 mg/L ascorbic acid) was added to each well. And then 200 μL cell suspension solution (2.7 × 10⁴ cells/mL in serum-free DMEM medium) was added within the glass ring (3000 cells/cm²). After 6 h culture for cell attachment, the glass rings were taken out and the medium was changed to serum-containing medium for cytoskeleton development. Before medium change, cell morphology was observed by an optical microscope. After another 18 h culturein serum medium (totally 24 h), the samples were used for immunofluorescence staining and cell mechanical test.

Primary cells from bone tissue samples from donors with osteoporosis or osteoarthrosis and MSCs (Poietics human mesenchymal stem cells, Lonza, Morristown, NJ, USA) were used for differentiation experiments. Primary cells from donors (OP or OA) were seeded in complete-growth Dulbecco’s modified eagle medium (DMEM) (Gibco, Billings, MT, USA), supplemented with 2 mM L-glutamine (Gibco), 10% foetal bovine serum (FBS) (Gibco) and 1% antibiotic/antimycotic solution (Gibco) in 24-well plates at concentration of 1 × 10⁵ cells/well. MSCs (Poietics human mesenchymal stem cells, Lonza, ZDA) were seeded at a concentration of 5 × 10⁴ cells/well in complete-medium MSCBM (Lonza, ZDA). The next day, cells were washed with 1× phosphate buffer solution (PBS) and treated with osteogenic/adipogenic medium for 21 days. For osteogenic differentiation, 5 mM β-glycerophosphate, 50 µg/mL ascorbic acid-2-phosphate, and 100 nM dexamethasone were added to complete growth medium. For adipogenic differentiation, 0.5 µM dexamethasone, 50 µM isobutylmethylxanthine, 10 µM indomethacin and 10 µg/mL human recombinant insulin was added to the complete growth medium. For control samples, complete growth medium was added to wells. Growth medium in each well was replaced every 3 days with a fresh one. At days 0, 7, 10, 14, 17, and 21, cells were collected and stored at −80 °C for RNA isolation.

The cultured cells of both MSC-lung and MSCs-CPAM were seeded in a 6-multiwell plate with a density of 8 × 10⁴ for each well. After seeding cells were maintained for 24 h in the standard medium MSCBM (Lonza) in incubator at 37°C with a humidified atmosphere at 5% CO₂. Then cultured cells of MSC-lung and MSCs-CPAM were subjected to hypoxic treatment by inserting the plates into the ProOx Model P110 (BioSpherix, New York, NY, United States) hypoxia chamber for 24 h at 0.2% of hypoxia. Cells were observed under inverted light microscopy (Leica Microsystem, Milan, Italy) to evaluate the morphological features in normoxic and hypoxic conditions. Cultured cells of MSC-lung and MSCs-CPAM were maintained in incubator with normoxic condition and used as control cells.