MechanicalTension Regulates Mesenchymal Stem Cell Paracrine Signaling on DermalFibroblasts via microRNA- and lincRNA-enriched Exosomes
Natalie Templeman1, Hui Li1, Emily Steen1, Xinyi Wang1, Alexander Blum1, Paul Bollyky2, Sundeep Keswani1, Swathi Balaji1.
1Baylor College of Medicine, Houston, TX, USA, 2Stanford University, Stanford, CA, USA.
Background: Mesenchymal stem cells(MSCs) have a huge therapeutic potential in wound healing. Cues from the extracellular environment affect MSC secretome, but the role of mechanical tension on the bioactive extracellular vesicles(EVs), namely exosomes released by MSCs is not known. We hypothesized that mechanical tension regulates MSC exosome production and influences wound healing via paracrine effects on dermal fibroblasts. Methods: Human MSCs were cultured on silicone membranes +/-10% static/cyclic strain for 24h and analyzed for phenotypic changes(alpha-SMA, and inflammation PCR-array) and genes encoding exosome synthesis(RAb27a-b;SMPD3). Exosomes were isolated and analyzed for size and quantity(Zetasizer). The exosome protein level was quantified(BCA Assay) and Westernblotting(CD63,HSP70,CD9) and Next-Gen Sequencing(Exo-RNA) were performed. Exosomes were labeled by Exo-Glow before use in a primary human dermal fibroblast(FB) migration assay. p-values by ANOVA;(n=3/group). Results: Tension induced morphologic changes and increased alpha-SMA staining in MSCs. There was a significant change(>5-fold) in ~ 30/84 inflammatory genes with tension. Tension downregulated the expression of RAb27a-b and SMPD3(p<0.01) in MSCs, but more exosomes with increase in size distribution and protein levels were produced by tension(p<0.05). The three exosome surface markers were verified by Westernblotting. Tension induced significant changes in abundance(>100) of several lincRNAs and miRNAs in exosomes, which are being evaluated using ingenuity analysis. MSC-derived exosome uptake by FB was tracked using fluorescent imaging. Interestingly, MSC-derived exosomes under static conditions slowed the migration of FB in a scratch wound assay, whereas those derived under tension increased FB migration(p<0.05), but there was no effect of the complete MSC-conditioned media from either static or tension conditions on FB migration. Conclusions: Tension induces a fibrogenic and inflammatory phenotype in MSCs. Exosomes are a likely target for extracellular communication, as their production/cargo in MSC are regulated by tension and can influence FB behavior. The novel insight of how tension effects MSC paracrine activity will play a pivotal role in clinical MSC therapies.
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