Melatonin primes mesenchymal stem cells via glutathione dynamics to enhance therapeutic efficacy in asthma

Mesenchymal stem cells (MSCs) offer therapeutic potential for inflammatory diseases, but their efficacy is often compromised during in vitro expansion. Here, we identified melatonin (MT) as a safe and effective priming agent that enhances MSC functionality by reinforcing intracellular glutathione (GSH) dynamics. Using FreSHtracer, a live-cell GSH-sensitive imaging probe, we established a high-throughput screening platform and developed a set of quantitative GSH indices to assess redox capacity. Screening over 50 flavonoids and vitamins, we identified MT as a clinically relevant compound that robustly activates the CREB1–NRF2 pathway, leading to improved antioxidant defense. MT-primed MSCs (MT-MSCs) exhibited enhanced clonogenicity, migration, angiogenesis, and immunosuppressive properties via JAK–STAT3–PD-L1 signaling. Transcriptomic analysis revealed molecular signatures associated with immune modulation and tissue regeneration. In a murine model of allergic asthma, MT-MSCs significantly alleviated Th2-mediated inflammation and improved lung engraftment. Additionally, we implemented a deep learning-based convolutional neural network for automated bronchoalveolar lavage fluid analysis, enabling objective and efficient inflammatory cell profiling. This study introduces MT as a pharmacopeia-compliant priming molecule and presents an integrated approach combining functional priming and AI-assisted diagnostics to advance the development of high-quality MSC-based therapies for asthma and other inflammatory diseases Overall design: RNA-seq data of naïve hES-MMSC cells and hES-MMSC primed with melatonin (200 µM) for 24 hours.