Effect of constant inflammation on in vitro expanded adipose-derived mesenchymal stromal cells

Human adipose-derived mesenchymal stromal cells (AD-MSCs) are known for their immunomodulatory responses to inflammation, a key trait for their therapeutic use in tissue injury and chronic inflammatory diseases. However, the effects of constant inflammation on their transcriptomic profile, proliferative capacity, and immunosuppressive potential remain largely unexplored. To investigate this, we evaluated the in vitro response of human AD-MSCs from three donors expanded across seven passages under, non-inflamed (DMEM), acute (IFN?-24h) and constant (IFN?-C) inflammatory conditions. Bulk RNA-sequencing results demonstrated that AD-MSCs respond significantly to inflammatory stimuli. Both acute and constant inflammation induced extensive transcriptomic alterations, with differentially expressed genes related to cell cycle regulation, DNA replication, metabolism, and immunomodulation. Notably, constant inflammation was associated with a transcriptomic shift toward oxidative phosphorylation (OXPHOS). Moreover, microscopy analysis revealed a significant reduction in AD-MSCs proliferative capacity under constant inflammation, as evidenced by lower cumulative population doubling and the appearance of senescence-like morphological changes in early passages when compared to non-inflamed (DMEM) and acute inflammatory (IFN?-24h) conditions. Despite proliferation was impaired, co-culture assays with PBMCs demonstrated that under constant inflammation AD-MSCs immunosuppressive potential was maintained in early and late passages. Therefore, our results provide novel insights into the dual effects of constant inflammation on AD-MSCs, impairing cell proliferation while maintaining their immunosuppressive capacity. Understanding AD-MSCs' behaviour in chronically inflamed microenvironments is essential for optimizing AD-MSCs' clinical applications. Overall design: Adipose-derived mesenchymal stromal cells (AD-MSCs) were isolated from human subcutaneous adipose tissue of three independent donors and expanded from passage 0 to passage 7 under three conditions: non-inflamed medium (DMEM), 24-hour interferon-gamma (IFN?) exposure, or continuous IFN? treatment. At each passage, cells were grown to approximately 80% confluency, harvested by trypsinization, and pelleted for RNA extraction using the RNeasy Mini Kit (Qiagen). RNA purity and concentration were assessed by NanoDrop, and RNA integrity was confirmed by TapeStation (RIN =7). High-quality RNA was used for cDNA library preparations, which were sequenced at BGI using a paired-end protocol (100-bp reads). Raw FASTQ files were produced for downstream differential expression and pathway analyses, and pertinent metadata, including donor identity, passage number, and treatment condition, were recorded for each sample.