Activation of Functional Somatic Stem Cells Promotes Endogenous Tissue Regeneration

Abstract

Periodontal ligament derived stem cells (PDLSCs) are capable of differentiating into multiple cell types and inducing a promising immunomodulation for tissue regeneration and disease treatment. However, it is still challenging to develop a practical approach to activate endogenous stem cells for tissue self-healing and regeneration. In this study, transcriptome analysis reveals that resveratrol promotes PDLSC stemness through activation of stem cell, osteoprogenitor, and chondroprogenitor markers. Self-renewal and multipotent differentiation abilities are also improved in resveratrol-treated PDLSCs. In addition, immunomodulation of PDLSCs is dramatically increased after resveratrol treatment. Mechanistically, we show that resveratrol activates ERK/WNT crosstalk through elevation of olfactory and growth factor signaling pathways to upregulate the expression levels of RUNX2 and FASL for osteogenesis and immunomodulation, respectively. By using a periodontitis animal model, administration of resveratrol partially rescues bone loss through activation of endogenous somatic stem cells and inhibition of inflammatory T-cell infiltration. Taken together, our findings identify a novel pharmacological approach to achieve autotherapies for endogenous tissue regeneration.

Keywords: autotherapy; immunomodulation; periodontal ligament derived stem cells (PDLSCs); periodontitis; regenerative medicine; resveratrol.

Figure 1.

Transcriptome analysis revealed resveratrol promoted periodontal ligament stem cell (PDLSC) stemness. (A) Volcano plot with the log₂ fold changes in gene expression after resveratrol treatment on the x-axis and the statistical significance (P value) on the y-axis. (B) Heatmap of RNA sequencing (RNA-seq) expression data showing the genes that were differentially regulated following treatment with 5 μM resveratrol. Gene expression is shown in normalized log₂ counts per million. Differentially expressed genes were selected based on a 4-fold change. (C) Venn diagram of differentially expressed genes in resveratrol-treated PDLSCs with P < 0.05. (D) Gene Ontology (GO) enrichment analysis for differentially regulated genes between control and resveratrol-treated group. Only top 19 false discovery rate (FDR) enrichments of GO terms from “biological process” category were listed. (E) Heatmap of RNA-seq expression data showing the PDLSC stemness genes that were differentially regulated following resveratrol treatment. (F–H) Quantitative polymerase chain reaction assay showed the significant increased levels of mesenchymal stem cell (MSC) markers, osteoprogenitor markers, and chondroprogenitor markers in resveratrol-treated PDLSCs. Error bars represent the standard deviation from the mean values. *P < 0.05. **P < 0.01. ***P < 0.005.

Figure 2.

Resveratrol elevated periodontal ligament stem cell (PDLSC) proliferation. (A) Gene Ontology (GO) enrichment analysis showed resveratrol treatment significantly activated cell cycle and cell proliferation pathways in PDLSCs. (B) Next-generation RNA sequencing (RNA-seq) data of resveratrol-treated PDLSCs were interrogated by gene set enrichment analysis to identify enriched biologic pathways. Cell proliferation pathways, including DNA replication, RNA polymerase, and biosynthesis of amino acids, were significantly activated after resveratrol treatment in PDLSCs. (C) Ki67 immunofluorescence staining showed resveratrol treatment elevated Ki67⁺ cells compared to the vehicle-treated group. Scale bar: 25 µm. (D) Assessment of cell viability by the MTT assay showed that resveratrol promoted PDLSC viability. (E) Quantitative polymerase chain reaction assay showed the levels of cell cycle genes with or without resveratrol treatment in PDLSCs. Error bars represent the standard deviation from the mean values. **P < 0.01. ***P < 0.005.

Figure 3.

Resveratrol-activated ERK/WNT crosstalk through olfactory and growth factor pathways. (A) Gene set enrichment analysis assay showed resveratrol treatment elevated olfactory receptor signaling in periodontal ligament stem cells (PDLSCs). (B) Heatmap of RNA sequencing (RNA-seq) expression data showing the growth factor genes that were differentially regulated following resveratrol treatment in PDLSCs. (C) Quantitative polymerase chain reaction assay further confirmed that the expression levels of olfactory receptors (ORs) and growth factors were significantly increased after resveratrol treatment in PDLSCs. (D) Western blotting analysis showed the expression levels of FASL, active β-catenin, β-catenin, p-ERK, and ERK in PDLSCs with or without resveratrol treatment. ERK small interfering RNA (siRNA) transfection was also performed in resveratrol-treated PDLSCs to knock down ERK expression level. (E) Alizarin red staining showed the capacity to form mineralized nodules under osteoinductive conditions in siControl- and siERK-transfected PDLSCs with resveratrol treatment. (F) Western blotting analysis showed the expression levels of the osteogenic genes RUNX2 and ALP under osteoinductive conditions in siControl- and siERK-transfected PDLSCs with resveratrol treatment. β-Actin was used as a protein loading control. Error bars represent the standard deviation from the mean values. *P < 0.05. **P < 0.01. ***P < 0.005.

Figure 4.

Resveratrol improved periodontal ligament stem cell (PDLSC) immunomodulation in vitro and in vivo in a DSS-induced experimental colitis mouse model. (A) PDLSCs induced annexinV⁺7AAD⁺ double-positive apoptotic CD3⁺ T cells in a PDLSC/T-cell direct coculture system in vitro. Resveratrol treatment further increased annexinV⁺7AAD⁺ double-positive apoptotic CD3⁺ T cells. (B) Schema showing PDLSC transplantation in dextran sulfate sodium (DSS)–induced experimental colitis mice. (C) Colitis mice showed significantly reduced body weight from 8 to 12 d after DSS induction. The PDLSC transplantation, resveratrol-treated PDLSC transplantation, and 0.1× resveratrol-treated PDLSC transplantation groups showed inhibition of body weight loss compared to the colitis group at 12 d after DSS induction (n = 6 per group). (D) Disease activity index (DAI) was significantly increased in colitis mice compared to C57BL6 mice at 12 d after DSS induction. PDLSC transplantation, resveratrol-treated PDLSC transplantation, and 0.1× resveratrol-treated PDLSC transplantation significantly reduced DAI score. (E, G) Hematoxylin and eosin staining showed the infiltration of inflammatory cells (white arrows) in colon with destruction of epithelial layer in colitis mice. PDLSC transplantation, resveratrol-treated PDLSC transplantation, and 0.1× resveratrol-treated PDLSC transplantation rescued disease phenotype in colon and reduced histological activity index (G). Scale bar: 25 µm. (F, H) Immunofluorescence staining showed CD4⁺IL17⁺ Th17 cell infiltration in colitis. PDLSC transplantation, resveratrol-treated PDLSC transplantation, and 0.1× resveratrol-treated PDLSC transplantation dramatically reduced Th17 infiltration in colon. Scale bar: 25 µm. (I) Flow cytometry analysis further confirmed that Th17 cell level was significantly elevated in colitis mice compared to C57BL6 mice after DSS induction. PDLSC transplantation, resveratrol-treated PDLSC transplantation, and 0.1× resveratrol-treated PDLSC transplantation reduced the levels of Th17 cells in colitis mice. (J) Activated T cells were capable of inducing significant PDLSC death in a PDLSC/T-cell direct coculture system. Resveratrol treatment protected PDLSC apoptosis. Error bars represent the standard deviation from the mean values. *P < 0.05. ***P < 0.005.

Figure 5.

Resveratrol treatment partially rescued disease phenotypes in a periodontitis mouse model. (A) Schematic illustration for ligature-induced periodontitis model and local administration of resveratrol. A 5-0 silk ligature was tied around the maxillary second molar in C57BL/6 mice on day 0, and either placebo (PBS) or resveratrol was simultaneously injected as illustrated. (B) Periodontal bone resorption analysis. The distance from the cementoenamel junction (CEJ) to the pinnacle of the alveolar bone (AB) was determined to assess periodontal bone loss. (C) Immunofluorescence (IF) staining revealed Ki67⁺Sca1⁺ cells were reduced in periodontitis mice. Resveratrol treatment significantly elevated Ki67⁺Sca1⁺ cells. (D) IF staining showed increased CD4⁺TNFα⁺ inflammatory T-cell infiltration in periodontitis mice. After resveratrol administration, CD4⁺TNFα⁺ inflammatory T-cell infiltration was largely reduced. Scale bar: 25 µm. (E, F) Cell viability and apoptosis were measured in periodontal ligament stem cells (PDLSCs) after treatment with various concentrations of lipopolysaccharide (LPS) for 12 h by using the MTT assay and flow cytometry analysis. Cell viability (G) and apoptosis (H) were measured in PDLSCs treated with resveratrol during 10 µg/mL LPS stimulation. Error bars represent the standard deviation from the mean values. *P < 0.05. **P < 0.01. ***P < 0.005.