Examining the potential for human amnion epithelial cells to rescue lung injury in an experimental model of bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) remains a major complication of preterm birth despite the improvements in perinatal care and clinical management. The development of BPD is multifactorial and characteristic pathology includes impaired alveolar and vascular growth, airway injury and lung inflammation. Although current interventions may provide symptomatic relief, functional improvement and reduction in short-term mortality, there is no cure for established BPD. Cell therapy is being explored as a potential therapeutic modality for a variety of diseases including BPD, and human amnion epithelial cells (hAECs) are an attractive source of cell therapy. The therapeutic and regenerative potential of these stem-like cells has been associated with their multipotent differentiation potential, anti-inflammatory and anti-fibrotic effects that have been shown in in vitro experiments and in various animal models of injury.
   
   We have previously shown in several animal models of lung diseases that hAEC administration results in improved lung structure, reduced lung inflammation and fibrosis, and prevention of declined lung function. Specifically, we showed that hAECs attenuate pulmonary inflammation and improve lung architecture in several large and small animal models of neonatal lung injury. These findings suggest that hAECs may be a viable approach for the treatment of BPD.
   
   Since angiogenesis is essential for normal alveolar development, and dysregulated pulmonary vascular development is a hallmark of BPD, my first study investigated the angiogenic effects of hAECs. Here I showed that both term and preterm hAECs expressed angiogenic factors, which significantly increased after TNFα and IFNγ stimulation. And the impact of hAECs on angiogenesis may be complicated by inflammation in vitro. For example, in the presence of TNFα and IFNγ, co-culture with term hAECs reduced gene transcription of Tie2 and Foxc1 in huVECs, while co-culture with preterm hAECs increased gene transcription of PDGFRα and β and reduced gene transcription of FOXC1 in huVECs. I also raised the point that angiogenesis may be a mechanism through which hAECs elicit their protective and/or reparative effects. This was demonstrated in Chapter 2 where I showed that term hAECs, but not preterm hAECs, inhibited the excessive angiogenesis in bleomycin-induced mouse lung fibrosis model. However, term hAECs increased angiogenesis in a neonatal model of hyperoxia induced lung injury. The findings in lung fibrosis model are consistent with our previous findings that preterm hAECs exert significantly less protective effects in vivo compared to term hAECs. The conclusions drawn from this study suggest that, angiogenesis may be one mechanism through which hAECs augment lung repair, and that the angiogenic potential differs between term and preterm hAECs. These findings led to the application of only term hAECs in the subsequent animal work.
   
   Next, I assessed the therapeutic effects of hAECs in a more clinically relevant murine BPD model with antenatal inflammation induced by intra-amniotic LPS injection and oxidative stress caused by postnatal hyperoxia exposure. I showed that there was a dose-effect response to hAEC treatment, intra-tracheal and intravenous administrations of hAECs were equally efficacious, and both early and late hAEC treatment rescued lung damage at neonatal stage. Further, I investigated long-term outcomes of the injury and treatment. Surprisingly, a single dose of early hAEC administration improved lung structure and lung function, as well as mitigated pulmonary hypertension and right ventricular hypertrophy in adolescent and adult stages. Additionally, hAEC treatment appeared to decrease lung inflammation, adjust immune cell population, promote angiogenesis and activate the bronchioalveolar stem cell niche.
   
   Finally, I investigated the effects of hAEC-derived extracellular vesicles (EVs) in the same experimental BPD model in order to assess the potential for a cell-free treatment for BPD. I showed that the isolated EVs were mostly of exosome size and shape, and they express some typical exosome markers. EV administration improved lung morphology and decreased lung inflammation. Additionally, EVs exerted therapeutic effect partly through the activation of type II alveolar cell population.
   
   In summary, the work reported in this thesis provide insights into the mechanisms through which hAECs and their derived EVs may augment endogenous repair during neonatal lung injury. While there is a continued need to better understand specific cell signalling pathways triggered by the hAECs and their EVs that mediate these effects, it is also important to assess their efficacy in other disease settings, particularly other diseases of prematurity such as necrotizing enterocolitis where mortality rates are high and clinical interventions limited.