Sphingolipid Metabolism Links Morphological Signatures to Immunomodulatory Potency in Human Mesenchymal Stromal Cells.

Interest in human mesenchymal stromal cells (MSCs) as an immune therapeutic has been on the increase over the past two decades with preclinical research yielding promising results, but so far only a singular MSC-based immunotherapy has been approved by the U.S. Food and Drug Administration (FDA). A major barrier for MSC clinical translation is the lack of reliable potency metrics and sufficient understanding of the mechanisms of action, as reported by the National Cell Manufacturing Consortium (NCMC). We integrated high-content morphological profiling and targeted sphingolipid analysis across MSC donors exhibiting a range of IFN-γ induced indoleamine-2,3-deoxygenase (IDO) activities to investigate the role of cell membrane components in MSC immunomodulatory function. High- and low IDO potency cells have distinct morphological signatures that are also reflected in the sphingolipid (SL) activity, with low IDO potency cell lines having low sphingomyelinase activity and high IDO potency cell lines having high sphingomyelinase activity. Perturbation of SL metabolism through the addition of exogenous neutral sphingomyelinase not only shifted morphological signatures to mimic those of high potency, but also significantly increased IDO activity within both high and low IDO potency donors. Additionally, these perturbations resulted in increased extracellular vesicle production with rich SL content and a stark reduction of TNF-α following EV treatment of activated macrophages. Together, these findings link morphology features from potent MSCs to sphingolipid remodeling and suggest a tractable strategy to tune MSC immunomodulatory potency. Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s12195-026-00933-x.