(W1273) EFFECT OF EXTRACELLULAR VESICLES DERIVED FROM GLYCOLYTIC MESENCHYMAL STEM/STROMAL CELLS ON THE PHENOTYPIC AND METABOLIC REPROGRAMMING OF SYNOVIAL MACROPHAGES IN PATIENTS WITH OSTEOARTHRITIS

Abstract: Osteoarthritis (OA) is a chronic inflammatory disease for which no cure exists; only palliative treatments are available. OA is characterized by an exacerbated inflammatory response primarily driven by the activity of synovial macrophages. Mesenchymal stem/stromal cells (MSCs) are well-known for their therapeutic potential; however, alternative strategies are essential to enhance their immunomodulatory properties. We show that glycolytic MSC (MSCglyco) and their small extracellular vesicles (EVs) holds promise for developing new therapies for OA.

MSCs were isolated from the umbilical cords of healthy donors, with informed consent obtained for the use of these samples. MSCs were treated with oligomycin for 24 hours to induce glycolytic metabolism. EVs were isolated from the MSCglyco (MSCglyco-EVs) using ultracentrifugation, quantified by nanoparticle tracking analysis (NTA), and characterized by transmission electron microscopy (TEM) and flow cytometry. Macrophages were isolated from the synovial membranes of OA patients (OAM), for which informed consent was also obtained. EVs from MSCs or MSCglyco were added to the culture media of the OAM at various doses. After 24 hours, OAM were recovered to evaluate EV internalization using qPCR, surface marker expression through flow cytometry, cytokine secretion via ELISA, and glycolytic flux using SCENITH, a flow cytometry assay.

The metabolic reprogramming of MSCs does not alter the phenotype or size of the released EVs, nor their capacity of internalization. OAMs treated with MSCglyco-EVs internalize these EVs, leading to an increased expression of CD206, which indicates an anti-inflammatory phenotype, while decreasing HLA-DR and CD86 associated with a proinflammatory phenotype. Additionally, OAMs treated with MSCglyco-EVs exhibits a reduction in the secretion of inflammatory mediators.

EVs from metabolically reprogrammed MSC have improved therapeutic properties. We demonstrate that MSCglyco-EVs dose-dependently reduce inflammatory profiles in OAM and increase anti-inflammatory profiles, providing compelling evidence for their enhanced therapeutic effects. This offers a promising approach for the development of novel acellular therapies for OA, focusing on immunomodulation using EVs from metabolically reprogrammed MSCs.

Funding Source: This research was supported by ANID-Chile through Beca Doctorado Nacional folio 21220015; FONDECYT Regular Nº1211353; FONDEF-ID: 21I10194; IMPACT-FB 210024; and TA24I10054

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