Immortalization strategies for human mesenchymal stromal cells for large scale production of extracellular vesicles

Abstract

Background & Aim Mesenchymal stromal cells (MSCs) provide therapeutic activities and have been applied in many different clinical trials. Apparently, their therapeutic effect does not depend on direct intercellular interactions, but rather is mediated by paracrine factors, especially by Extracellular Vesicles (EVs). Indeed, we and others have used MSC-EVs to improve diseases symptoms in several different disease models and in a patient suffering from steroid refractory Graft-versus-Host Disease. Thus, MSC-EVs provide a promising therapeutic agent for the future. For now, the scalability of the MSC-EV production is limited by the lifespan of MSCs. Since immortalization of MSCs might overcome this issue, we aim to create immortalized MSC lines and test whether their EVs are still therapeutically active. Methods, Results & Conclusion We compared six different lentiviral plasmid based immortalization strategies for MSCs and obtained some immortalized MSC lines. Cells of these cell lines retained their characteristic MSC morphology and molecular cell surface phenotype, as well as their ability to differentiate along the osteogenic and adipogenic lineage. To learn whether the immortalization affects the quality of released EVs, the immune modulatory capabilities of their EVs were analyzed in a mixed lymphocyte reaction assay. Like EVs harvested from supernatants of the original MSCs, EVs isolated from immortalized MSC supernatants retained their ability to modulate immune responses in the MLR assay.