Exogenous Sphingomyelinase Mediates MSC-derived EV Biogenesis and Enhances Potency via Repackaging of Molecular Cargo

Mesenchymal stromal cells (MSCs) exert regenerative and immunomodulatory effects largely through secreted paracrine factors and extracellular vesicles (EVs), which transfer proteins, lipids, and nucleic acids to recipient cells. Lipid composition critically influences EV stability, uptake, and bioactivity. Sphingomyelinase (SMase), an enzyme that hydrolyzes sphingomyelin into ceramide, regulates EV biogenesis by inducing membrane curvature and initiating inward membrane budding. Here, MSCs were treated with SMase, and EVs were isolated and characterized by nanoparticle tracking analysis, miRNA sequencing, lipidomics, and proteomics. SMase treatment increased EV yield and altered lipid, protein, and miRNA cargo linked to TNF-α signaling, wound healing, and angiogenesis. Functionally, SMase-EVs suppressed TNF-α in macrophages, showed trending increased HUVEC tubular formation, and altered T-cell populations following local delivery in a critical murine oral wound defect model. These findings highlight how enzymatic lipid remodeling modifies MSC-EVs, enhancing their therapeutic potential and informing strategies for optimized EV-based therapies and scalable production.