22 hydroxycholesterol 22oh c

Human mesenchymal stem cells (hMSCs) were obtained from Cambrex (Walkersville, MD). Cells were positive for CD105, CD166, CD29, and CD44 and negative for CD14, CD34, and CD45. Human MSCs were grown in hMSC growth medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 4 mmol/L L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin). The growth medium was changed every 3 days. The natural LXR agonist 22-hydroxycholesterol (22OH-C) was purchased from Sigma. Recombinant human Wnt1 protein was purchased from Abcam (Cambridge, MA). Cells were subjected to serum depletion for 16 hours. Then, cells were exposed to 22OH-C (0.01 μmol/L) or vehicle for 6 hours, after which Wnt10b mRNA expression was determined using quantitative RT-PCR. For analysis of Wnt ligand, cells were serum depleted for 16 hours, as above, before being exposed to human recombinant Wnt1 protein (final concentration 20 ng/mL) or vehicle for 6 hours. Cells were then harvested and total RNA was analyzed by quantitative RT-PCR to determine LXRα mRNA expression.