Extracellular Vesicles from Human Induced Pluripotent Stem Cells Reduce Lung Ischemia-Reperfusion Injury via Cytoprotective Mechanisms in both 2D Cultures and a Human Airway Organoid Model

Lung transplantation (LTx) remains the only curative option for patients with end-stage pulmonary diseases. However, long-term outcomes are compromised by ischemia-reperfusion injury (IRI), a major contributor to primary graft dysfunction and chronic lung allograft impairment. Current pharmacological and technological strategies provide only partial protection, highlighting the need for novel therapeutic approaches. In this study, we investigated the therapeutic potential of extracellular vesicles derived from human induced pluripotent stem cells (hiPSC-EV) in a two-dimensional (2D) in vitro model of pulmonary IRI using A549 lung epithelial cells. To better recapitulate the in vivo airway microenvironment, we also employed a human airway organoid (hAO) model. Proteomic analysis of hiPSC-EV identified 1,698 proteins enriched in pathways relevant to apoptosis regulation, oxidative stress responses, and inflammatory signaling. In the 2D IRI model, the administration of hiPSC-EV during reperfusion significantly improved cell viability, reduced lactate dehydrogenase (LDH) release, attenuated reactive oxygen species (ROS) accumulation, and decreased apoptosis and necrosis. Furthermore, hiPSC-EV downregulated pro-inflammatory mediators (NFkB1, TNFα, IL1β, IL8, MCP1) while upregulating anti-inflammatory and anti-apoptotic key factors (IL1RA, BCL2). In the 3D model, hiPSC-EV administration preserved organoid structure and viability under IRI conditions, attenuated transcriptional and protein-level expression of pro-inflammatory and oxidative stress markers (IL1β, IL8, HMGB1, HMOX1), and enhanced anti-inflammatory (IL10) and cytoprotective (HSP27, HSP70) responses. Immunofluorescence further revealed that hiPSC-EV reduced activation of apoptotic and pyroptotic pathways. Moreover, intravenous administration of hiPSC-EV in mice showed no adverse safety signals, including tumorigenicity. Together, these findings demonstrate that hiPSC-EV exert broad cytoprotective effects in pulmonary IRI through coordinated regulation of inflammation, oxidative stress, and cell death. Their scalability, reproducibility, and favorable safety profile underscore their promise as innovative cell-free therapeutics to improve outcomes in LTx.