Immortalization of mesenchymal stromal cells by hTERT does not affect the functional properties of secreted extracellular vesicles

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have emerged as promising and safe therapeutic agents, however, donor heterogeneities, limited replicative life span and changes in the cellular phenotype throughout in vitro cultivation remain major hurdles for scalable EV production. For these reasons, this study aims to investigate the use of hTERT immortalized (‘telomerized’) MSCs as a potential source for efficient, standardized, reliable MSC-EVs production by comparing parental primary to their telomerized MSC counterparts. We observed that hTERT expression does not affect cell morphology or cellular doubling time, while ensuring unlimited, stable in vitro propagation. In addition, telomerized WJ-MSCs maintained the canonical expression profile of surface markers and the tri-lineage differentiation potential of their primary counterparts. In terms of EV characteristics, the immortalization by hTERT expression did not affect size, number, cargo composition or biological activity regarding anti-inflammatory, anti-fibrotic and wound healing properties in vitro. In summary, the use of hTERT to immortalize MSCs leads to the creation of cell lines that continuously produce MSC-EVs without altering any key functionalities of the cells or resulting EVs. This suggests that telomerization of human cells from single donors is a promising strategy for generating cell factories that can produce EVs in standardized conditions and at scale and with standardization. • hTERT immortalization alone ensures stable, unlimited in vitro propagation of MSCs • MSC-markers and differentiation are maintained upon hTERT immortalization • Immortalization by hTERT expression do not affect size, number or miRNA content of EVs • Biological activity of MSC-EVs is not affected by hTERT immortalization • Safety profile of MSCs and EVs is not affected by hTERT immortalization