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Abstract: Bone marrow-derived human mesenchymal stem cells (hMSCs) have the in vitro capacity to differentiate into osteoblasts, chondrocytes or adipocytes, depending on the applied stimulus. In order to identify novel regulators of osteogenesis in hMSCs, osteo-transcriptomics was performed whereby differentiation induced by dexamethasone (DEX), DEX+ bone morphogenetic protein 2 (BMP2), and DEX+ Vitamin D3 (1,25(OH)2D3) was studied over a course of 12 days. Microarray analysis revealed that 2095 genes were significantly regulated by DEX+ 1,25(OH)2D3, of which 961 showed accelerated expression kinetics compared to treatment by DEX alone. The majority of these genes were accelerated 24–48 h after onset of osteogenic treatment. Gene ontology (GO) analysis of these 1,25(OH)2D3-accelerated genes indicated their involvement in biological processes related to cellular differentiation and cell cycle regulation. When compared to cells treated with DEX or DEX+BMP2, treatment with DEX+ 1,25(OH)2D3 clearly accelerated osteoprogenitor commitment and osteoblast maturation, as measured by alkaline phosphatase (ALP) activity and calcification of the matrix. Cell cycle progression, as observed after initial growth arrest, was not significantly accelerated by 1,25(OH)2D3 and was not required for onset and progression of osteogenesis. However, expression of c-Myc was accelerated by 1,25(OH)2D3, and binding sites for c-MYC were enriched in promoters of genes accelerated by 1,25(OH)2D3. Lentiviral overexpression of c-MYC strongly promoted DEX+ BMP2-induced osteoblast differentiation and matrix maturation. In conclusion, our studies show for the first time that 1,25(OH)2D3 strongly accelerates expression of genes involved in differentiation of hMSCs and, moreover, identify c-MYC as a novel regulator of osteogenesis. [Copyright &y& Elsevier]