Secretome enriched with small extracellular vesicles derived from human gingiva-derived mesenchymal stem cells enhances rat tongue muscle regeneration.

Background: Accumulating evidence demonstrates that the therapeutic effects of stem cells are most likely attributed to their secretome, composed of a myriad of bioactive factors, including small extracellular vesicles (EVs). Due to the potential benefits over cells in term of handling, preservation, stability, and safety, MSC-derived secretome is emerging as a novel cell-free therapeutic for regenerative therapy of various diseases. The purpose of this study is to optimize the xeno-free culture conditions to improve the secretome production by human gingiva-derived mesenchymal stem cells (GMSCs) and test their regenerative potential using an experimental rat model of tongue muscle defect.

Methods: Next-generation mRNA sequencing was performed to compare the gene expression profiles between GMSCs cultured under the defined xeno-free induction culture conditions (iGMSCs) and their 2D-cultured counterparts under regular serum-free conditions. The conditioned media (CM) from iGMSCs and 2D-GMSCs were harvested and concentrated through ultrafiltration to obtain secretomes. The EVs and soluble protein/peptide factor fractions (SPs) from the concentrated CM/secretome were separated using the 35 nm qEVoriginal size exclusion columns. The EVs were confirmed by Nanoparticle Tracking Analysis (NTA), Western blot, and transmission electron microscopy (TEM). The functional effects of secretomes derived from iGMSCs and 2D-GMSCs on macrophage polarization and skeletal muscle progenitor cells were compared both in vitro and in vivo using a rat tongue defect model.

Results: Next-generation mRNA sequencing showed profound transcriptomic changes in iGMSCs compared to their 2D counterparts. Further Gene Ontology (GO)-term annotation and Gene Set Enrichment Analysis (GSEA) revealed significant upregulation of a panel of differentially expressed genes (DEGs) related to EVs and secreted cellular components (GO_CCs) and enriched pathways in oxidative phosphorylation, Wnt/β-catenin signaling, Notch signaling, and inflammatory responses in iGMSCs compared to 2D-GMSCs. iGMSC-derived CM/secretome showed a significant enrichment of both EVs and SPs compared to that derived from 2D-GMSCs, as confirmed by Nanoparticle Tracking Analysis (NTA), Western blot, and transmission electron microscopy (TEM). In vitro functional assays revealed a markedly enhanced secretion of IL-10, whilst suppressed LPS-stimulated secretion of TNF-α in macrophages treated with iGMSC-derived CM/secretome in comparison with that from 2D-GMSCs. In addition, iGMSC-derived CM/secretome potently induced the expression of myogenic transcriptional factors in both murine myoblasts and human skeletal muscle progenitors in comparison with 2D-GMSC-derived CM/secretome. Notably, in vivo studies using a rat tongue wound defect model, iGMSC-derived CM/secretome applied topically at the excised wound bed promoted rapid tissue repair/regeneration without fibrosis/scar and shape deformity.

Conclusion: Secretome derived from GMSCs cultured under optimized xeno-free induction displayed enrichment of EVs and SPs and enhanced pro-myogenic potentials and anti-inflammatory effect on macrophages. These findings have shed light on the potential applications of the optimized iGMSC-derived secretome as cell-free therapeutics for regenerative therapy of tongue wound defects and other muscular diseases.