archives@tulane.edu / Human adipose-derived stem cells (ASCs) efficiently modulate the inflammatory microenvironment, making them ideal for the treatment of inflammatory, autoimmune, and neurodegenerative diseases like multiple sclerosis (MS). Clinical translation of ASC therapies has been limited, making strategies to improve ASC post-transplant immunosuppressive capabilities especially important. Autophagy, a stress-induced degradative pathway, plays a crucial role in the paracrine signaling of ASCs, which drives their therapeutic action. Therefore, I investigated the modulatory effect of autophagy preconditioning in ASC physiology and therapeutic potential using the autophagy inducer Rapamycin (Rapa-ASCs) or the inhibitor 3-methyladenine (3MA-ASCs). Following 4 and 24-hour preconditioning, ASC stemness and immunomodulatory capacity were examined. Results demonstrate that neither Rapa nor 3-MA altered morphology or surface marker expression, indicating preservation of stemness. RT-qPCR analysis revealed that 4-hour preconditioning with Rapa-ASCs and 3MA-ASCs alone upregulated transcription of cyclooxygenase-2 (COX2), but not secretion of its downstream effector molecule prostaglandin E2 (PGE2). Following stimulation with interferon-gamma (IFNγ) to mimic a pathological microenvironment, both 4-hour Rapa-ASCs and 3MA-ASCs upregulated COX2 transcription and PGE2 secretion. 4-hour Rapa-ASCs also upregulated expression of the cytokines transforming growth factor beta (TGF-β) and interleukin-6 (IL-6). As each of these molecules have demonstrated therapeutic effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, I hypothesized that 4-hour Rapamycin preconditioning may bestow the greatest improvement to ASC immunomodulatory potential in EAE. To test this, EAE mice were treated at peak disease severity with control ASCs (EAE-ASCs), Rapa-preconditioned ASCs (EAE-Rapa-ASCs), or a vehicle control (EAE). Results revealed that EAE-ASCs enhanced rotarod locomotor activity, improved clinical disease scores, and elevated intact myelin in the spinal cord compared to both EAE and EAE-Rapa-ASC animals. This correlated with augmented CD4+ T helper (Th) and T regulatory (Treg) cells in the spinal cord, and significantly increased interleukin-10 (IL-10) transcripts. Conversely, EAE-Rapa-ASC mice showed no clinical or motor function improvement, reduced myelin levels, and significantly less Th and Treg cells in the spinal cord. These findings suggest that short-term Rapamycin preconditioning diminishes the therapeutic efficacy of ASCs when applied to late-stage EAE and highlights the importance of investigating novel therapeutic strategies in vivo in physiologically relevant disease models. / 1 / Rachel Wise

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