Int J Stem Cells.  2023 May;16(2):202-214. 10.15283/ijsc21170.

Exosomes from Tension Force-Applied Periodontal Ligament Cells Promote Mesenchymal Stem Cell Recruitment by Altering microRNA Profiles

• Chang M
• Chen Q
• Wang B
• Zhang Z
• Han G

• KMID: 2542833
• DOI: http://doi.org/10.15283/ijsc21170

Abstract

Background and Objectives
To investigate the role of exosomes from periodontal ligament cells (PDLCs) in bone marrow mesenchymal stem cell (BMSC) migration.
Methods and Results
Human PDLCs were applied cyclic tension stretching. Exosomes were extracted from cultured PDLCs by ultracentrifugation, then characterized for their size, morphology and protein markers by NTA, TEM and western blotting. The process that PKH26-labeled exosomes taken up by BMSCs was assessed by confocal microscope. BMSC migration was examined by Transwell assay. Exosomes derived from PDLCs were identified. Cyclic tension stretch application on PDLCs can enhance the migration ability of BMSCs through exosomes. The exosomal miRNA expression profiles of unstretched and stretched PDLCs were tested by miRNA microarray. Four miRNAs (miR-4633-5p, miR-30c-5p, miR-371a-3p and let-7b-3p) were upregulated and six (miR-4689, miR-8485, miR-4655-3p, miR-4672, miR-3180-5p and miR-4476) were downregulated in the exosomes after stretching. Sixteen hub proteins were found in the miRNA-mRNA network. Gene Ontology and KEGG pathway analyses demonstrated that the target genes of differentially expressed exosomal miRNAs closely related to the PI3K pathway and vesicle transmission.
Conclusions
The exosomes derived from cyclic tension-stretched PDLCs can promote the migration of BMSCs. Alternation of microRNA profiles provides a basis for further research on the regulatory function of the exosomal miRNAs of PDLCs during orthodontic tooth movement.

Keyword

Periodontal ligament; microRNA; Exosome; Stem cell; Tooth movement

Figure

• 

  Fig. 1 Characterization of exosomes derived from human periodontal ligament cells (PDLCs). (A) Transmission electron microscope images confirmed the presence of exosomes, seen as cup-shaped vesicles. Scale bar: 50 nm. The picture in the white square is an enlarged photo. (B, C) The particle size distribution and concentration of exosomes were measured by nanoparticle tracking analysis. (D) Western blot analysis of the exosome-specific markers (CD63, CD9, CD81, ALIX, and TSG101), endoplasmic reticulum protein (calnexin), and fibroblastic marker (vimentin). PDLCs, microvesicles, and supernatant were used as the control.

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  Fig. 2 Internalization of exosomes by bone marrow mesenchymal stem cells. PKH-26-labelled exosomes (red) were internalized in the cytoplasm of BMSCs after 1 h and accumulated after 2 and 6 h of coincubation. The nuclei of the BMSCs were stained with DAPI (blue). The cytoplasm of the BMSCs was stained with CFDA-SE (green). Scale bar: 25 and 5 μm.

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  Fig. 3 Exosomes were collected from CTS-applied PDLCs (Exos_CTS) and unstretched PDLCs (Exos_US). (A) Western blot analysis of CD63, CD81, ALIX, TSG101, calnexin and vimentin in Exos_CTS and Exos_US. PDLCs were used as the control. (B) Internalization of Exos_CTS and Exos_US by bone marrow mesenchymal stem cells. PKH-26-labelled exosomes (red) were internalized in the cytoplasm of BMSCs after 2 h of coincubation. The nuclei of the BMSCs were stained with DAPI (blue). The cytoplasm of the BMSCs was stained with CFDA-SE (green). Scale bar: 25 and 5 μm.

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  Fig. 4 Periodontal ligament cells (PDLCs) were applied 48 h after cyclic tension stretching. (A) Conditioned medium (CM_B) was collected from the cyclic tension stretch (CTS)-applied PDLCs and removed microvesicles (CM_C) and exosomes (CM_UC). BMSCs were stained with Crystal Violet solution. Representative images and the migration rate of BMSCs for different CMs. (B) Exosomes were collected from CTS-applied PDLCs (Exos_CTS) and unstretched PDLCs (Exos_US). Representative images and migration rate of migrated BMSCs treated with different exosomes. *p<0.05.

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  Fig. 5 (A) After siRNA-Drosha transfection for 48 hr, the knockdown efficiency of Drosha protein in PDLCs was evaluated by western blot analysis. Exosomes were collected from CTS-induced PDLCs after siRNA-mediated knockdown of Drosha and then cultured with BMSCs in Transwell plates. (B) Representative images of migrated BMSCs treated with Exos_CTSsiRNA-Drosha and Exos_CTSsiRNA-control.

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  Fig. 6 The exosomal miRNA differential expression profiles in unstretched and cyclic tension-stretched PDLCs. (A) The Volcano plot of differentially expressed exosomal miRNAs. (B) The heatmap of differentially expressed exosomal miRNAs.

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  Fig. 7 (A) Venn graph showing 1,060 target genes of differentially expressed miRNAs, predicted by miRWalk and miRDB databases. (B) Protein-protein network of target genes.

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  Fig. 8 (A) Top four modules from the protein-protein interaction network. (B) Protein-protein network of genes from the top four modules. (C) Protein-protein network of 16 hub genes.

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  Fig. 9 Enrichment map of Gene Ontology and the Kyoto Encyclo-paedia of Genes and Genomes pathway analyses.

Reference

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