Understanding the mechanism of osteo-/dentinogenic differentiation is beneficial for jaw bone and dental tissue regeneration. DLX5 is highly expressed in dental tissue-derived mesenchymal stem cells (MSCs) and is upregulated by lysine-specific demethylase 4B (KDM4B), enabling it to regulate osteo-/dentinogenic differentiation, while the function of DLX5 in osteo-/dentinogenesis has not been thoroughly elucidated to date. Therefore, we investigated DLX5 function using stem cells from apical papilla (SCAPs). SCAPs were obtained from the human wisdom tooth. Alkaline phosphatase (ALP) assay, Alizarin red staining (ARS), quantitative analysis of calcium, osteo-/dentinogenesis-related gene expression and in vivo transplantation were used to determine the osteo-/dentinogenic differentiation potential. Luciferase and ChIP assays were used to investigate the physical relationship between DLX5 and KDM4B. DLX5 and KDM4B were upregulated during osteogenic induction and were induced by BMP4 in SCAPs. Next, we found that DLX5 enhanced ALP activity, mineralization in vitro, and the expression of dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP1), osteopontin (OPN), and the key transcription factor osterix (OSX). Moreover, transplant experiments showed that DLX5 promoted osteo-/dentinogenesis in vivo. Interestingly, DLX5 enhanced KDM4B transcription by directly binding with its promoter. In addition, KDM4B upregulated DLX5 in SCAPs. These results indicate that DLX5 and KDM4B are positive effectors of BMP signaling and regulate each other via a positive feedback mechanism. DLX5 enhanced osteo-/dentinogenic differentiation via upregulated KDM4B in SCAPs, suggesting that activation of the DLX5/KDM4B signaling pathway might serve as an intrinsic mechanism that promotes tissue regeneration mediated by dental-derived MSCs.

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy.

Stromal-derived factor-1α (SDF-1α) is a chemokine signaling molecule that binds to the transmembrane receptor CXC chemokine receptor-4 (CXCR4) and carries out important functions in development tissue homeostasis. SDF-1α signaling via CXCR4 regulates the recruitment of stem and precursor cells to support tissue-specific repair or regeneration. In this study, we examined the contribution of SDF-1α signaling to the odontogenic differentiation of stem cells from the apical papilla (SCAP) induced by bone morphogenic protein 2 (BMP-2). CXCR4 expression was detected in cultured SCAP and SDF-1α promoted the migration of SCAP in Transwell assays. Blocking SDF-1α signaling by treatment with siRNA significantly affected BMP-2-induced mineralized nodule formation and alkaline phosphatase (ALP) activity. Moreover, blocking SDF-1α signaling inhibited the BMP-2-induced early expression of runt-related factor-2 (Runx-2) and strongly suppressed the induction of dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP) expression by BMP-2. Furthermore, the interaction between SDF-1α and BMP-2 signaling was mediated via intracellular Smads and Erk activation. In conclusion, our results demonstrated that SDF-1α can significantly promote the migration of SCAP. Moreover, we revealed corequirement of the SDF-1α/CXCR4 signaling pathways in the BMP-2-induced odontogenic differentiation of SCAP, and these findings may be applied in new strategies for dental pulp regeneration.

Trio, the Rho guanine nucleotide exchange factor (Rho-GEF), plays diverse roles in cell migration, cell axon guidance and cytoskeleton reorganization. Conserved during evolution, Trio encodes two guanine nucleotide exchange factor domains (GEFs) and activates small GTPases. The Rho-family small GTPases RhoA and Rac1, which are target molecules of Trio, have been described to engage in craniofacial development and tooth formation. However, the exact role of Trio in tooth development remains elusive. In this study, we generated Wnt1-cre;Triofl/fl mice to address the potential function of Trio in tooth development. Wnt1-cre;Triofl/fl mice showed short root deformity as well as decreased expression of odontogenic makers such as RUNX2, OSX, OCN, and OPN. In vitro, Trio was silenced in human stem cells of dental papilla (SCAPs). Compared with the control group, the proliferation and migration ability in the experimental group was disrupted. After knocking down Trio in SCAPs, the cells showed phenotypes of poor odontogenic differentiation and weak mineralized nodules. To study the underlying mechanism, we investigated the p38 MAPK pathway and found that loss of Trio blocked the cascade transduction of p38 MAPK signaling. In conclusion, we identified Trio as a novel coordinator in regulating root development and clarified its relevant molecular events.