Development of distal lung organoids based on the primary human pulmonary cells as a model to study mechanisms of lung injury and to test new therapeutics

Abstract

Organoids are 3D cell culture models that mimic their representing organ or tissue through: representation of cellular compartments, correct 3D structural organization, and feature some functionality of the representing organ. Although a multitude of pulmonary organoid types have been published, so far none possess endothelial cell presence at organoid maturation, yet the pulmonary endothelium is an important component of pulmonary homeostasis. The use of cellular condensation of adult distal lung cells; HSAECs, HPMECs, and LMSCs, allows for the generation of a distal lung organoid model that is highly comparable with the adult human distal lung microenvironment. At 21 days in maturation, the organoid structure is approximately 1000 μm in size, features hollow bulbous structures composed of alveolar epithelial cells approximately 200 μm in size, and further feature and interacted network of endothelial cells.

These organoid structures were then validated in disease modelling related to COPD/emphysema. It has recently been reported to LMSCs (termed COPD-LMSCs) isolated from emphysema patients are reduced in their expression of paracrine factors associated with normal LMSCs homeostasis, one such factor being HGF which serves function in both endothelial and epithelial proliferation, survival, and maturation. This may indicate that the progressive nature of emphysema can be attributed to epigenetic modification of LMSCs. The use of COPD-LMSCs in distal lung organoid seedings results in organoid structures that present a COPD similar phenotype; destruction of PBSs, loss of endothelial cell presence (representing the loss of pulmonary vasculature in patients), loss of distinct distal lung epithelial cell phenotypes, and deficient in HGF expression. The treatment of normal LMSCs with siRNA to reduce HGF expression allows for the generation of organoid structures that bear resemblance COPD organoid structures. 5 Azacytidine (5AZA) is an epigenetic modifier that is employed in the treatment of myeloid leukaemia’s and has shown benefit in basic model of COPD. The application of 5AZA to COPD-LMSCs has shown an increase in HGF expression.

Next, distal lung organoid structures were applied to the assessment of therapeutic candidates. Bone marrow mesenchymal stromal cell extracellular vesicles (BM-MSCs EVs) have been demonstrated to hold therapeutic benefit in a multitude of preclinical models of IPF/ARDS. One of the barriers to EVs research is the generation of sufficient quantities of EVs to allow standardization across experiments. The utilization of bioreactors in EVs generation can allow mass quantity of EVs generation. A paddle-wheel bioreactor system was employed to generate sufficient quantities of BM-MSCs EVs for use in the assessment of their therapeutic benefit in this organoid model. The application of BM-MSCs EVs generated from bioreactor culture to a bleomycin induced model of IPF in organoids reduced the expression of COL1A1 to healthy levels.

Although this organoid model features endothelial cell presence that is structured in a physiologically relevant manner, it does not feature the tubular structures of the pulmonary endothelium. The initiation of vascular formation within organoid structures is dependent on the use of microfluidic devices that allow the generation of interstitial flow to facilitate endothelial tube formation. The use of a commercially available cell culture device, the AIMbiotech OrganiX device, allows for the generation of vasculature on distal lung organoid models embedded within a vascular matrix. Vasculature matrix develops with organoid maturation, and TEM assessment of vascular ultrastructure’s indicates alteration of the surface of organoid structures consistent with normal vasculature. The channelling of FITC-Dextran through vasculature matrix through the organoid body indicate a level of functionality to these vascular structures.

Together, the findings of this predoctoral research indicate that the use of cellular condensation of adult distal lung cell type is a reliable means to generate and a distal lung organoid that features endothelial cell presence, which can then be utilised in disease modelling and the assessment of therapeutic candidates.

Thesis is embargoed until 31 July 2027.

Date of Award	Jul 2024
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Medical Research Council
Supervisor	Anna Krasnodembskaya (Supervisor) & Cecilia O'Kane (Supervisor)