An evaluation of the therapeutic potential of human amniotic epithelial cells during ex-vivo donor lung perfusion

Abstract

Ph. D. Thesis.Introduction: Ex Vivo Lung Perfusion (EVLP) provides a normothermic isolated environment for the evaluation and reconditioning of donor lungs deemed unsuitable for immediate transplantation and offers a unique opportunity to administer advanced therapeutics, such as cell-based therapies. Human Amniotic Epithelial Cells (hAECs) have been shown to have immunomodulatory properties that could reduce injury in donor lungs. Our aim was to assess the anti-inflammatory actions of hAECs when administered during EVLP to lungs declined for transplant due to poor organ function. Methods: hAECs were isolated from term placenta through enzymatic digestion. In in vitro studies, THP-1 derived macrophage phagocytosis and activation was determined after treatment with hAECs for 6 hours. Neutrophils were migrated through an IL-1b activated Human Microvascular Endothelial Cells (HMEC)-1 monolayer, after treatment of hAECs. In ex vivo perfusion studies; human lungs declined for transplant were split, with 150 x 106 hAECs or the HTR-8/SVneo cell line administered to each single lung and perfused concurrently for up to 4 hours (n=3). Serial samples of perfusate and tissue biopsies were collected for ELISA, qPCR and immunofluorescence (IF). Results: hAECs were isolated with 94 ± 4% purity, with an average isolation yielding 134.2 x 106 with viability >90%. hAECs reduced neutrophil transendothelial migration (p=0.0128). hAEC treatment of macrophages led to an increase in phagocytosis observed in vitro and a decrease in CXCL8 (p=0.0465) and TNFa (p=0.0158) expression. hAEC-treated lungs had significantly reduced TNFa expression in the tissue (p=0.0415). IF staining demonstrated reduced expression of CXCL8 and 3- nitrotyrosine in the hAEC-treated lungs compared to the HTR cell treated lungs. Conclusion: In vitro assays demonstrated the potential of hAECs to minimise proinflammatory macrophage activation and neutrophil migration. hAEC-treated lungs led to a reduction in pro-inflammatory cytokine production and oxidative stress. hAECs may offer a therapeutic approach to reduce inflammation in donor lungs during EVLP.NIHR Blood and Transplant Research Unit in Organ Donation and Transplantation (BTRU-ODT