Abstract
Although cellular therapy has been proposed for inflammation-related disorders such as periodontitis for decades, clinical application has been unsuccessful. One explanation for these disappointing results is that the functions of stem cells are substantially compromised when they are transplanted into an inflammatory in vivo milieu. Considering the previous finding that P2X7 receptor (P2X7R) gene modification is able to reverse inflammation-mediated impairment of periodontal ligament stem cells (PDLSCs), we further hypothesized that cells subjected to P2X7R gene transduction also exert influences on other cells within an in vivo milieu via an exosome-mediated paracrine mechanism. To define the paracrine ability of P2X7R gene-modified cells, P2X7R gene-modified stem cell-derived conditional medium (CM-Ad-P2X7) and exosomes (Exs-Ad-P2X7) were used to incubate PDLSCs. In an inflammatory osteogenic microenvironment, inflammation-mediated changes in PDLSCs were substantially reduced, as shown by quantitative real-time PCR (qRT-PCR) analysis, Western blot analysis, alkaline phosphatase (ALP) staining/activity assays, and Alizarin red staining. In addition, the Agilent miRNA microarray system combined with qRT-PCR analysis revealed that miR-3679-5p, miR-6515-5p, and miR-6747-5p were highly expressed in Exs-Ad-P2X7. Further functional tests and luciferase reporter assays revealed that miR-3679-5p and miR-6747-5p bound directly to the GREM-1 protein, while miR-6515-5p bound to the GREM-1 protein indirectly; these effects combined to rescue inflammation-compromised PDLSCs from dysfunction. Thus, in addition to maintaining their robust functionality under inflammatory conditions, P2X7R gene-modified stem cells may exert positive influences on their neighbors via a paracrine mechanism, pointing to a novel strategy for modifying the harsh local microenvironment to accommodate stem cells and promote improved tissue regeneration.