Stable production of immortalized hPC-MSC-derived extracellular vesicles: integrated cargo analysis and functional validation.

Extracellular vesicles (EVs) play a critical role in intercellular communication and have shown great potential in disease treatment and tissue repair. However, large-scale production and clinical application of EVs still face challenges such as donor heterogeneity, batch-to-batch variability, and high production costs. This study aimed to establish a stable and reproducible EV production platform using hTERT-immortalized placental mesenchymal stromal cells (PC-MSCs). We demonstrated that stable expression of the TERT gene promotes cell proliferation in vitro while retaining surface marker expression patterns and differentiation capabilities similar to those of primary cells. The EVs produced by immortalized cells maintain similar particle size distributions, protein markers, and protein expression profiles to those of their parental cells, and their therapeutic efficacy was validated in a bleomycin (BLM)-induced pulmonary fibrosis (PF) rat model. In summary, we systematically described the effect of immortalization via ectopic hTERT gene expression on EVs produced from PC-MSCs. These findings provide a new theoretical basis for the sustainable production and scale-up of EVs, as well as a new solution for the treatment of PF.