Dental pulp stem cells (DPSCs), due to the ease of isolation and their capacities of multi-lineage differentiation, are considered as attractive resources for regenerative medicine. In a previous study, we showed that TNF-alpha. promoted the osteogenic differentiation of DPSCs via the NF-kappa B signaling pathway. However, the mechanisms of such differentiation were largely unknown. Here, we examined the gene expression profiles between undifferentiated, partially differentiated and fully differentiated DPSCs induced by TNF-alpha by performing the next-generation sequencing technique (RNA-Seq). Our results revealed a continuous transition of the transcriptome changes during TNF-alpha promoted osteogenic differentiation of DPSC. Bioinformatics analysis revealed a relatively general to specific transformation of the involved signaling pathways from the early to late stages of differentiation. Gene regulatory network analysis highlighted novel, key genes that are essential for osteogenic differentiation at different time points. These results were further validated by quantitative RT-PCR, confirming the high reliability of the RNA-Seq. Our data therefore will not only provide novel insights into the molecular mechanisms that drive the osteogenic differentiation of DPSCs, but also promote the studies of bone tissue engineering that utilizes DPSCs as a crucial resource. (C) 2016 Elsevier Inc. All rights reserved.