Orr, Jessica C; (2023) Higher throughput airway basal cell cultures to identify modulators of repair and regeneration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In human lungs, a pseudostratified epithelium lines the airways from the trachea to the small airways. Restoration of this epithelium is essential following injury and basal cells act as stem cells. However, the mechanisms governing basal cell self-renewal and differentiation are incompletely understood. Screening of small molecules that modulate basal cell proliferation and differentiation could identify compounds for development as pharmaceuticals to improve airway tissue repair. To enable screening, optimisation of primary human bronchial epithelial cells (HBECs) culture conditions was performed. Next, lentiviral promoter-reporter constructs were developed to enable quantification of the promoter activity of genes whose expression is characteristic of the main cell types of the airway epithelium: basal, mucosecretory or ciliated cells. The sensitivity of these constructs was validated using known modulators of differentiation in a 3D ‘tracheosphere’ assay, where HBECs proliferate and differentiate forming polarised spheroid structures that include the major airway epithelial cell types. In the future, this assay could be scaled to enable higher throughput screening. Additionally, promoter activity was observed in 2D cultures for the basal and mucosecretory cell reporter constructs, suggesting the feasibility of 2D screening. A 384-well compound screening system was established in which HBECs were transduced with a pHIV-Luc-ZsGreen lentiviral construct to enable automated quantification of relative cell numbers. Compound libraries (the ENZO and Prestwick Chemical Libraries) were screened for their ability to increase cell number. Several hit compounds were Wnt signalling pathway activators. Compounds were validated in proliferation and differentiation assays. Addition of hit compounds resulted in larger tracheospheres, suggesting increased proliferation. The effects of structurally and functionally related compounds were also investigated, and notably the induced Wnt activity correlated with tracheosphere size. These results suggest that Wnt-activating small molecules could be further developed into a novel treatment to improve airway epithelial repair following airway damage.

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