Effectiveness of extracellular vesicles derived from hiPSCs in repairing hyperoxia-induced injury in a fetal murine lung explant model

Abstract

Background Despite advances in neonatal care, the incidence of Bronchopulmonary Dysplasia (BPD) remains high among preterm infants. Human induced pluripotent stem cells (hiPSCs) have shown promise in repairing injury in animal BPD models. Evidence suggests they exert their effects via paracrine mechanisms. We aim herein to assess the effectiveness of extracellular vesicles (EVs) derived from hiPSCs and their alveolar progenies (diPSCs) in attenuating hyperoxic injury in a preterm lung explant model. Methods Murine lung lobes were harvested on embryonic day 17.5 and maintained in air–liquid interface. Following exposure to 95% O2 for 24 h, media was supplemented with 5 × 10^6 particles/mL of EVs isolated from hiPSCs or diPSCs by size-exclusion chromatography. On day 3, explants were assessed using Hematoxylin–Eosin staining with mean linear intercept (MLI) measurements, immunohistochemistry, VEGFa and antioxidant gene expression. Statistical analysis was conducted using one-way ANOVA and Multiple Comparison Test. EV proteomic profiling was performed, and annotations focused on alveolarization and angiogenesis signaling pathways, as well as anti-inflammatory, anti-oxidant, and regenerative pathways. Results Exposure of fetal lung explants to hyperoxia induced airspace enlargement, increased MLI, upregulation of anti-oxidants Prdx5 and Nfe2l2 with decreased VEGFa expression. Treatment with hiPSC-EVs improved parenchymal histologic changes. No overt changes in vasculature structure were observed on immunohistochemistry in our in vitro model. However, VEGFa and anti-oxidant genes were upregulated with diPSC-EVs, suggesting a pro-angiogenic and cytoprotective potential. EV proteomic analysis provided new insights in regard to potential pathways influencing lung regeneration. Conclusion This proof-of-concept in vitro study reveals a potential role for hiPSC- and diPSC-EVs in attenuating lung changes associated with prematurity and oxygen exposure. Our findings pave the way for a novel cell free approach to prevent and/or treat BPD, and ultimately reduce the global burden of the disease.

1. Introduction

Bronchopulmonary Dysplasia, or BPD, is a developmental lung disease characterized by a disruption or arrest of the normal processes of lung alveolarization and angiogenesis. Extreme preterm infants, born before 28 weeks’ gestation during the late canalicular or early saccular stages of their lung development, are mostly affected [1–3]. Gentle ventilation strategies, fluid restriction, caffeine, diuretics, and steroids, have all been used to prevent or attenuate lung injury; however, the incidence of BPD remains greater than 40% among extreme preterm infants [4], and there is still no effective therapy to restore normal alveolarization and angiogenesis.

Various types of stem cells, including mesenchymal stem cells (MSCs) and human induced pluripotent stem cells (hiPSCs), have shown promise in reducing inflammatory changes and restoring lung morphogenesis in experimental animal BPD models [5–9]. However, their potential immunogenicity remains a major limitation to their clinical applicability [10–12]. Tumorigenicity is also a concern, mostly with hiPSC-based treatments [13, 14]. The differentiation of hiPSCs into distal lung phenotypes (diPSCs, or differentiated hiPSCs) before their administration mitigates this risk [15, 16]; it might however reduce their effectiveness [5].

Recent evidence suggests that extracellular vesicles (EVs) secreted by stem cells play a significant role in mediating their therapeutic effects [7, 17–21]. EVs are nanoparticles secreted by most cells. They are capable of transferring biological components such as proteins, growth factors, RNAs and miRNAs to host cells or tissues, thus playing an important role in cellular communication and cell signaling. EVs have been shown to contribute to vital physiological functions, including homeostasis, immune regulation, and tissue regeneration and repair. In several diseases processes, including lung disease of prematurity, it has been shown that stem cell-conditioned media is as effective as stem cell therapy itself.