Abstract
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 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.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 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.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 TGFβ1/smads signaling pathway by downregulating LTBP1.
Highlights
Relevant dental pulp tissue regeneration can serve as a treatment to replace existing root canal therapy used to treat necrotic permanent teeth [1]
The microRNA expression profiles of exosomes derived from dental pulp stem cells (DPSCs) were identified
Endocytosis of UN-Exo and OD-Exo by DPSCs To characterize the presence of exosomes in the isolates, the bilayer membrane and “saucerlike” appearance of representative exosomes were examined by Transmission electron microscopy (TEM), which verified the presence of UN-Exo and OD-Exo ranging from 30 to 150 nm in diameter (Fig. 2a)
Summary
Relevant dental pulp tissue regeneration can serve as a treatment to replace existing root canal therapy used to treat necrotic permanent teeth [1]. Regenerative endodontic treatment is attempted by using a variety of mesenchymal stem cells (MSCs), growth factors, and biomaterials [2, 3]. Exosomes are reported as an ideal biomaterial in regenerative endodontic treatment, with the properties of immunomodulation [4, 5], promoting angiogenesis [6] and inducing differentiation of stem cells [7, 8]. The cargo of exosomes is shown to contain both ubiquitous and cell type-specific biological molecules such as protein, mRNA, and microRNA [9]. It has been revealed that exosomal microRNAs are transferred between cells and can regulate the post-transcriptional gene expression in recipient cells [10]. 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
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