RNA sequencing on cardiac fibroblasts harvested at different time points following induction of osteogenic differentiation

Purpose: To analyze cardiac fibroblasts gene expression at different time following induction of osteogenic differentiation Methods: Freshly isolated cardiac fibroblasts (Passage 0 or passage 1) were plated at a density of 2.5 x 104 cells/cm2 in growth medium. After overnight incubation, osteogenesis was induced using differentiation medium (α-MEM supplemented with 10% FBS, 10 nM dexamethasone (Sigma, D4902), 20 mM β-glycerol phosphate (Sigma, G9422), and 50 μM L-ascorbic acid (Sigma, A4403). Cardiac fibroblasts harvested at Day0 (before differentiation medium treatment), Day7, Day14 and Day21 were used for RNA sequencing. Results: Cardiac fibroblasts harvested at different time points following induction of differentiation revealed clusters of genes whose expression was significantly altered in a temporal specific manner. Genes regulating cell cycle that were highly expressed in undifferentiated cardiac fibroblasts were down-regulated at the onset of differentiation and remained at low expression levels throughout the duration of osteogenic differentiation, consistent with the principle that induction of differentiation is associated with reduced rates of proliferation. In contrast, genes that were minimally expressed in cardiac fibroblasts were induced in a specific temporal manner during the course of osteogenic differentiation and included sets of genes known to regulate inflammation, extracellular matrix proteins and cell metabolism. Conclusions: Cardiac fibroblasts subjected to osteogenic differentiation progressively adopted an osteogenic signature. Cardiac fibroblasts harvested at Day0 (before differentiation medium treatment), Day7, Day14 and Day21 were used for RNA sequencing. 2 samples / each time point.