Osteogenic programming of adipose-derived mesenchymal stem cells using a fungal metabolite that suppresses the Polycomb protein EZH2

We report genome-wide expression changes that occur in adipose-derived mesenchymal stem cells upon treatment with CytoD cytoskeletal drug. mRNA-Seq analysis shows that CytoD-treated samples cluster together. In addition, we also see that cells treated with CytoD show upregulation of osteogenic markers, epiregulators, and a number of key molecular function pathways including extracellular matrix, cell membrane gene expression. Overall design: Adipose MSCs were cultured in Advanced-MEM base (Life Technologies), 5% platelet lysate, and 1% non-essential amino acids (Life Technologies), and 2U/ml heparin. Cells used for experiments were of passage 6. Adipose MSCs were seeded at 3,000 cells per cm2 in maintenance medium in 6-well plates and incubated under standard culture conditions for 24 hours before being changed to osteogenic medium containing vehicle (DMSO) or 0.1 µg/ml cytochalasin D (Sigma). Osteogenic medium maintenance media supplemented with 10 nM dexamethasone, 25 µg/ml ascorbic acid, and 10 mM ß-glycerophosphate. Cells in culture were prepared for RNA isolation by lysing with Qiazol. Purified RNA was then submitted for RNA-sequencing.

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Samsonraj RM, Dudakovic A, Manzar B, Sen B, Dietz AB, Cool SM, Rubin J, van Wijnen AJ