Beyond labeling: differential Ac₄ManNAz dosing as a tool for functional manipulation of mesenchymal stem cells

Bioorthogonal chemistry technology serves as a powerful tool for mesenchymal stem cell (MSC) transplantation by utilizing metabolic engineering for direct cell modification either in vivo or in vitro. However, the unclear effects of varying concentrations of non-natural metabolic monosaccharides on labeling efficiency, functional impact and safety pose significant challenges for clinical translation. Herein, we screened the concentration-dependent effects of the metabolic labeling agent Ac₄ManNAz (10-100 µM) on the functionality of MSC. Visualizing azido groups on the cell surface through bioorthogonal reactions confirmed that labeling efficiency positively correlated with the concentration of Ac₄ManNAz. Interestingly, treatment of MSC with 50-µM Ac₄ManNAz enhances their immune regulatory function. Mechanistically, 50-µM Ac₄ManNAz remodels the extracellular matrix of MSC by downregulating decorin (DCN) expression to alleviate TGF-β inhibition, simultaneously upregulating serglycin (SRGN) expression to activate and stabilize TGF-β. This dual action results in increased secretion of TGF-β, thereby augmenting the immune regulatory capacity of MSC. Nevertheless, at the level of cellular motility, treatment with 50-µM Ac₄ManNAz significantly impairs the migratory capacity of MSC. Overall, this study clearly defined the concentration effect of non-natural monosaccharides on MSC biological functionality for clinical applications of this technology.