Lineage-specific exosomes promote the odontogenic differentiation of human dental pulp stem cells (DPSCs) through TGF1/smads signaling pathway via transfer of microRNAs

BackgroundExosomes 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.MethodsExosomes 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 TGF1/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.ResultsEndocytosis 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 TGF pathway. 16 genes targeted by 15 differentially expressed microRNAs were involved in TGF signaling. Consistently, automated western blot found that OD-Exo activated TGF1 pathway by upregulating TGF1, TGFR1, p-Smad2/3, and Smad4 in DPSCs. Accordingly, once the TGF1 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 TGF1, TGFR1, p-Smad2/3, and Smad4 by downregulating the inhibitory molecule LTBP1.ConclusionsThe 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 TGF1/smads signaling pathway by downregulating LTBP1.