Prematurity negatively impacts therapeutic effect of human amnion epithelial cells in experimental bronchopulmonary dysplasia

Background \u0026 Aim Extracellular vesicles derived from human amniotic epithelial cells (hAEC-EVs) have been previously shown to be beneficial in a preclinical model of bronchopulmonary dysplasia (BPD) when obtained from full-term, healthy pregnancies. In this study, we compare their therapeutic potential against preterm, complicated pregnancies. Methods, Results \u0026 Conclusion Methods: Lipopolysaccharide (LPS) was introduced to C57bl6 mouse fetuses intra-amniotically at E16 prior to exposure to 65% oxygen (hyperoxia) at birth. hAEC-EVs were injected intravenously on postnatal day (PND) 4. One cohort of mice was culled on either postnatal day PND7 or PND14, while another cohort were kept in hyperoxia until PND 28, following which they were recovered under normoxia conditions for a further 2 weeks for cardiovascular assessment. Results: The EVs isolated from either term or preterm hAEC had distinct cup shaped morphology with average size of 40-120nm. All hAEC-EV samples presented epithelial marker CD326, and EV markers CD9, CD63, CD81 and CD105. Term EVs had significant higher level of CD142 and CD133 compared to preterm EV. Mass spectrometry showed that all samples contained CD9, CD81, Alix and Grp94. Compared to preterm hAEC-EV, 47 proteins were significantly enriched in term hAEC-EVs and 38 proteins significantly reduced by more than 20-fold. In mouse model of BPD-like lung injury, we observed that term but not preterm hAEC-EV improved tissue-to-airspace ratio and septal crest density in a dose-dependent manner. Although both term and preterm hAEC-EV reduced the levels of inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α) on PND7, the improvement of lung injury was associated with the increase of the percentage of type II alveolar cells only observed in term EV treatment group. Furthermore, only neonatal term hAEC-EV delivery reduced airway hyper-responsiveness, mitigated pulmonary hypertension and prevented right ventricle hypertrophy that associated with BPD-like lung injury until 6 weeks of age. Conclusions: Both term and preterm hAEC-EVs bore exosome characteristics based on size distribution, morphology under EM and exosomal markers, however, EV cargo and functional potency were significantly different. Term hAEC-EVs were more efficient at repairing the BPD-like lung injury compared to preterm EVs, and this impact of donor criteria should be considered when applying perinatal cells derived EV therapy for clinical use.