Candidates for therapeutic drug repurposing in order to treat patients sufferingCandidates for therapeutic drug repurposing

Candidates for therapeutic drug repurposing in order to treat patients suffering
Candidates for therapeutic drug repurposing in order to treat patients suffering from such diseases as obesity and osteoporosis.MethodsCulture and differentiation of hMSCshMSCs harvested from normal human bone marrow were purchased from Lonza (Walkersville, MD, USA) at passage 2. Cells were expanded for no more than five passages in “mesenchymal stem cell growth medium” (MSCGM; Lonza) at 37 in a humidified atmosphere containing 7.5 CO2. Studies were performed with hMSCs from three different donors, encoded 5F0138, 6F4085, and 7F3458. For differentiation experiments, 4.0 ?104 cells per cm2 were seeded in high-glucose-containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 fetal bovine serum (a selected lot from Lonza), 100 U/ml penicillin, and 100 g/ml streptomycin. This medium will be further referred to as “proliferation medium” (PM). The next day, cells were switched to either osteogenic or adipogenic differentiation medium. Cells treated with PM were used as negative controls. Osteogenic differentiation medium (ODM) was composed of PM supplemented with 10-7 M DEX, 0.2 mM ascorbate, and 10 mM -glycerophosphate. Adipogenic differentiation medium (ADM) was composed of PMvan Zoelen et al. Stem Cell Research Therapy (2016) 7:Page 3 ofsupplemented with 10-6 M DEX, 10 g/ml insulin (R D Systems, Minneapolis, MN, USA), 10-7 M rosiglitazone (Sigma-Aldrich, St. Louis, MO, USA), and 500 M IBMX (Sigma-Aldrich). Unless indicated order Biotin-VAD-FMK otherwise, recombinant human TGF1 and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25768400 BMP2 (both from R D Systems) were used at concentrations of 2 ng/ml and 125 ng/ml, respectively. Prostaglandin E2 (PGE2), Batimastat, and Zopolrestat (Sigma-Aldrich) were used at the indicated concentrations. Media were refreshed every 3? days.Alkaline phosphatase assaysTo quantify alkaline phosphatase (ALP) enzymatic activity as a measure of osteogenic differentiation, hMSCs were seeded in 96-well tissue culture plates as already described, after which cells were differentiated for 7 days in osteogenic differentiation medium. ALP enzymatic activity was quantified by measuring the formation of p-nitrophenol from p-nitrophenyl phosphate (PNPP; SigmaAldrich), as described previously [13]. ALP enzymatic activity was corrected for differences in cell number, as determined by a Neutral Red assay. Cells were incubated with Neutral Red dye diluted in PBS for 1 hour at 37 . After washing with PBS, the dye was extracted from the cells using 0.05 M NaH2PO4 in 50 EtOH, after which the absorbance was measured at 540 nm. For histochemical analysis of ALP activity, cells were seeded in 48-well tissue culture plates and differentiated for 7 days in osteogenic differentiation medium. Subsequently, cells were fixed in 3.7 formaldehyde/PBS for 10 min at 22 . After washing with PBS, cells were incubated for 1 hour at 37 in a mixture of 0.1 mg/ml naphtol ASMX phosphate (Sigma-Aldrich), 0.5 N,N-dimethylformamide, 2 mM MgCl2, and 0.6 mg/ml Fast Blue BB salt (Sigma-Aldrich) in 0.1 M Tris Cl, pH 8.5.Mineralization assayfor 30 min with 1 formaldehyde in PBS, and then washed once with water and twice with 60 isopropanol. Cells were then stained for 1 hour with 0.3 w/v Oil Red O (Sigma-Aldrich) in 60 isopropanol. Subsequently, cells were washed once with 60 isopropanol and twice with distilled water. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27797473 For quantification of Oil Red O staining, samples were treated with 100 isopropanol and absorbance was measured at 530 nm. The amount of triglycerides stored in lipid.