Beyond Labeling: Differential Ac4ManNAz Dosing as a Tool for Functional Manipulation of Mesenchymal Stem Cells

Abstract Bioorthogonal chemistry technology serves as a powerful tool for 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 DCN expression to alleviate TGF-β inhibition, simultaneously upregulating 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.