Sphingomyelinase Licensing of Mesenchymal Stromal Cells Alters Lipid and Protein Metabolites for Immunomodulation

Mesenchymal stromal cells (MSCs) are widely studied for their immunomodulatory and tissue reparative capabilities, but clinical translation has been hampered by inconsistent efficacy and limited standardization in manufacturing. While cytokine-based priming methods, such as interferon-gamma (IFN-γ) stimulation, have shown promise in enhancing MSC potency, alternative approaches targeting distinct biological metabolism integral to secretome and membrane architecture have not been explored in MSCs. In this study, we investigate sphingomyelinase (SMase), an enzyme that generates ceramide from sphingomyelin, as a novel lipid-based priming strategy to modulate MSC function. Here, human MSCs were treated with SMase and high-content imaging and morphological profiling revealed that SMase-treated cells adopted a phenotype overlapping with IFN-γ–licensed MSCs, including increased cell compactness and solidity. Lipidomic analysis showed broad alterations in sphingolipid species, and dynamic flux estimation (DFE) modeling predicted distinct metabolic shifts in SMase-treated cells compared to untreated controls. These changes were sustained up to 35 hours post-stimulation, indicating stable metabolic reprogramming. SMase priming also altered the MSC secretome, enriching for factors implicated in immune regulation. Functionally, SMase-primed MSCs retained the ability to suppress T-cell activation and promote anti-inflammatory macrophage phenotypes. Collectively, these findings demonstrate that SMase stimulation induces a durable, immunomodulatory-like state in MSCs through coordinated changes in lipid metabolism and secretory activity. This lipid-centric priming approach represents a promising alternative to cytokine-based licensing strategies and may support therapeutic MSC products.