Telomerase-mediated immortalization preserves the anti-inflammatory activity of dental pulp stem cell extracellular vesicles

Background Extracellular vesicles (EVs) derived from dental pulp stem cells (DPSCs) exhibit immunomodulatory activity in multiple inflammatory models. However, the limited replicative lifespan of primary DPSCs and passage-dependent functional variability restrict scalable and reproducible EV production. Approaches that stabilize EV bioactivity during extended cell expansion are therefore needed. Methods Human primary DPSCs were immortalized via expression of human telomerase reverse transcriptase (hTERT) to mitigate senescence-associated limitations. EVs isolated from naïve and hTERT-immortalized DPSCs were characterized for size distribution, morphology, marker expression, and cellular endocytosis. Immunomodulatory activity of EVs isolated from multiple passages of immortalized DPSCs was evaluated in vitro on lipopolysaccharide (LPS)/interferon-γ–activated primary mouse bone marrow–derived macrophages in comparison with primary DPSC EVs. In vivo efficacy was assessed in a murine model of LPS-induced acute lung injury. Results hTERT expression increased telomerase levels without altering the multi-lineage differentiation potential of the cells. No significant changes in EV size, morphology, or expression of canonical EV markers was observed. EV uptake by macrophages was unchanged between naïve and immortalized DPSC-derived EVs. Both EV sources similarly suppressed pro-inflammatory cytokine expression, including TNF-α, IL-1β, and IL-6, in inflamed primary macrophages. Notably, EVs derived from hTERT-immortalized DPSCs retained immunomodulatory activity through at least passage 15. In vivo , EVs from naïve and immortalized DPSCs comparably reduced pulmonary edema, inflammatory gene expression, and neutrophil accumulation following LPS challenge. Similar preservation of EV activity was observed using EVs derived from immortalized human bone marrow derived mesenchymal stem cells. Conclusion hTERT-mediated immortalization of parent DPSCs retains the immunomodulatory function of derivative EVs during extended culture without altering key physicochemical EV characteristics. Immortalized DPSCs represent a reproducible source of EVs with consistent anti-inflammatory activity, supporting their use as a translationally-relevant platform for EV-based immunomodulatory applications.