Background Exosomes derived from dental pulp stem cells (DPSCs) can be used as biomimetic tools to induce odontogenic differentiation of stem cells, but the regulatory mechanisms and functions of exosome-encapsulated microRNAs are still unknown. The present study aimed to clarify the role of microRNAs contained in the exosomes derived from human DPSCs and their potential signaling cascade in odontogenic differentiation. Methods Exosomes were isolated from human DPSCs cultured undergrowth and odontogenic differentiation conditions, named UN-Exo and OD-Exo, respectively. The microRNA sequencing was performed to explore the microRNA profile contained in UN-Exo and OD-Exo. Pathway analysis was taken to detect enriched pathways associated with the predicted target genes of microRNAs. The regulatory roles of a highly expressed microRNA in OD-Exo were investigated through its inhibition or overexpression (miRNA inhibitors and miRNA mimics). Automated western blot was used to identify the function of exosomal microRNA and the roles of TGFβ1/smads pathway in odontogenic differentiation of DPSCs. A luciferase reporter gene assay was used to verify the direct target gene of exosomal miR-27a-5p. Results Endocytosis of OD-Exo triggered odontogenic differentiation of DPSCs by upregulating DSP, DMP-1, ALP, and RUNX2 proteins. MicroRNA sequencing showed that 28 microRNAs significantly changed in OD-Exo, of which 7 increased and 21 decreased. Pathway analysis showed genes targeted by differentially expressed microRNAs were involved in multiple signal transductions, including TGFβ pathway. 16 genes targeted by 15 differentially expressed microRNAs were involved in TGFβ signaling. Consistently, automated western blot found that OD-Exo activated TGFβ1 pathway by upregulating TGFβ1, TGFR1, p-Smad2/3, and Smad4 in DPSCs. Accordingly, once the TGFβ1 signaling pathway was inhibited by SB525334, protein levels of p-Smad2/3, DSP, and DMP-1 were significantly decreased in DPSCs treated with OD-Exo. MiR-27a-5p was expressed 11 times higher in OD-Exo, while miR-27a-5p promoted odontogenic differentiation of DPSCs and significantly upregulated TGFβ1, TGFR1, p-Smad2/3, and Smad4 by downregulating the inhibitory molecule LTBP1. Conclusions The microRNA expression profiles of exosomes derived from DPSCs were identified. OD-Exo isolated under odontogenic conditions were better inducers of DPSC differentiation. Exosomal microRNAs promoted odontogenic differentiation via TGFβ1/smads signaling pathway by downregulating LTBP1. Electronic supplementary material The online version of this article (10.1186/s13287-019-1278-x) contains supplementary material, which is available to authorized users.
Background To investigate the odonto-immunomodulatory properties of dental pulp stem cell-derived small extracellular vesicles (DPSCs-sEV), which promote odontogenesis by switching macrophages toward the pro-healing M2 phenotype. Methods MicroRNA sequencing was carried out for microRNA profiling of DPSCs-sEV. Automated Western blot, qPCR, ELISA, and flow cytometry were performed to identify the functions of microRNA-enriched DPSCs-sEV in macrophages. A luciferase reporter gene assay was carried out to confirm exosomal miR-125a-3p’s direct target gene. DPSCs-sEV-stimulated macrophage-conditioned media were used to promote odontogenesis in DPSCs and explore the mechanism of immune response in DPSCs-SEV-stimulated odontogenesis. DPSCs-sEV were injected into the exposed pulp tissue of rat incisor to investigate the odonto-immunomodulatory properties of DPSCs-sEV in vivo. Results DPSCs-sEV switched macrophages to the pro-healing M2 phenotype by inhibiting TLR and NFκΒ signaling. MicroRNA sequencing found 81 microRNAs significantly altered in DPSCS-sEV, with miR-125a-3p showing a 12-fold upregulation. Exosomal miR-125a-3p switched macrophages toward the M2 phenotype via inhibiting NFκΒ and TLR signaling via direct IKBKB targeting. Interestingly, DPSCs-sEV and the encapsulated miR-125a-3p enhanced BMP2 release in macrophages, promoting odontogenesis in DPSCs through BMP2 pathway activation. The rat study confirmed that DPSCs-sEV could be used as ideal biomimetic tools to enhance odontogenesis by switching macrophages toward pro-healing M2 cells. Conclusions We firstly defined the odonto-immunomodulatory properties of microRNA-enriched DPSCs-sEV, which could be used as ideal biomimetic tools to enhance odontogenesis by switching macrophages toward the pro-healing M2 phenotype.
Background: Magnesium (Mg 2+)-enriched microenvironment promotes odontogenic differentiation in human dental pulp stem cells (DPSCs), but the regulatory mechanisms remain undefined. The aim of this work was to assess magnesium's function in the above process and to explore the associated signaling pathway. Methods: DPSCs underwent culture in odontogenic medium with the addition of 0, 1, 5, or 10 mM MgCl 2. Intracellular Mg 2+ levels in DPSCs were evaluated flow cytometrically using Mag-Fluo-4-AM. Mg 2+-entry was inhibited by TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB). RNA-Sequencing was carried out for assessing transcriptome alterations in DPSCs during odontogenic differentiation associated with high extracellular Mg 2+. KEGG pathway analysis was performed to determine pathways related to the retrieved differentially expressed genes (DEGs). Immunoblot was performed for assessing magnesium's role and exploring ERK/BMP2/Smads signaling. Results: Mg 2+-enriched microenvironment promoted odontogenic differentiation in DPSCs via intracellular Mg 2+ increase. Consistently, the positive effect of high extracellular Mg 2+ on odontogenic differentiation in DPSCs was blocked by 2-APB, which reduced Mg 2+ entry. RNA-sequencing identified 734 DEGs related to odontogenic differentiation in DPSCs in the presence of high extracellular Mg 2+. These DEGs participated in many cascades such as MAPK and TGF-β pathways. Consistently, ERK and BMP2/Smads pathways were activated in DPSCs treated with high extracellular Mg 2+. In agreement, ERK signaling inhibition by U0126 blunted the effect of high extracellular Mg 2+ on mineralization and odontogenic differentiation in DPSCs. Interestingly, BMP2, BMPR1, and phosphorylated Smad1/5/9 were significantly decreased by U0126, indicating that BMP2/Smads acted as downstream of ERK. Conclusions: Mg 2+-enriched microenvironment promotes odontogenic differentiation in DPSCs by activating ERK/ BMP2/Smads signaling via intracellular Mg 2+ increase. This study revealed that Mg 2+-enriched microenvironment could be used as a new strategy for dental pulp regeneration.
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