(W1240) CHARACTERIZATION OF EXTRACELLULAR VESICLES (EVS) DERIVED FROM UMBILICAL CORD MESENCHYMAL STROMAL CELLS (UC-MSCS) LOADED WITH MIR-365A-5P ON CD4+ MEMORY T CELLS

Abstract: Autoimmune and inflammatory diseases exhibit an imbalance between pro-inflammatory and regulatory CD4+ T cells, making immune response regulation a persistent challenge. Mesenchymal Stromal Cells (MSCs) are a promising therapeutic option due to their immunosuppressive capacity, mainly attributed to their secretion of extracellular vesicles (EVs). However, clinical outcomes are divergent, leading to proposals for enhancing their therapeutic potential by reprogramming MSCs towards glycolysis. Additionally, studies suggest that miRNAs within EVs may influence immunomodulation. Our studies with glycolysis-reprogrammed UC-MSC-derived EVs (EVs-glyco) demonstrated enhanced immunosuppressive activity on memory CD4+ T cells compared to non-reprogrammed EVs (EVs) and a high expression of miR-365a-5p. Therefore, we characterized EVs internalization through C. elegans miR-39 expression and evaluated the effect of miR-365a-5p on memory CD4+ T cells.
EVs and EVs-glyco were isolated by ultracentrifugation. They were subsequently loaded with miR-39 via electroporation (EVs-miR-39) and characterized by nanoparticle tracking analysis and FACS. The internalization of miR-39 in EVs and treated memory CD4+ T cells was analyzed by qPCR. To assess the effect of miR-365a-5p, EVs were loaded with this miRNA, and its levels were detected by qPCR. Memory CD4+ T cells were treated with EVs, EVs-glyco, and EVs-miR-365a-5p, followed by FACS analysis to determine Th1, Th17, and Treg phenotypes.
The characterization of EVs-miR39 showed no significant differences in size or surface markers CD9/CD63/CD81 when compared to non-electroporated EVs. qPCR confirmed miR-39 presence in EVs-miR-39 and its internalization into memory CD4+ T cells. Analyses showed elevated levels of miR-365a-5p in EVs-miR-365a-5p compared to non-electroporated EVs. In memory CD4+ T cells treated with EVs-miR-365a-5p, a similar trend of immunosuppression was observed as that seen with EVs-glyco on Th1, Th17, and Treg subpopulations.
It is suggested that the electroporation process does not alter the essential characteristics of EVs, and effective transfer of miR-39 as well as miR-365a-5p was demonstrated. This opens possibilities for further investigation into the role of miR-365a-5p in the immunosuppressive function of EVs-UC-MSC.

Funding Source: ANID - Proyect Advanced Technology - IDEA grant N°TA24I10054

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