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Compound screening in primary human airway basal cells identifies Wnt pathway modulators as potential pro-regenerative therapies

J C Orr(1) Y Ishii(1) D Bairros de Pilger(1) K Lazarus(1) R Hughes(2) M -B El Mdawar(1) M Z Nikolic(1) R Ketteler(1) N O Carragher(2) S M Janes(1) R E Hynds(1)

1:University College London; 2:University of Edinburgh

Airway basal cells are multipotent stem cells that can differentiate to replace epithelial cell types of the airway epithelium. This makes them a suitable target cell for cell and gene therapies for genetic diseases that affect the airways, such as cystic fibrosis or primary ciliary dyskinesia.

The addition of compounds that increase basal cell proliferation to a cell product could improve engraftment success by giving the modified basal cells an initial competitive advantage over host cells.

Primary human bronchial epithelial cells (HBECs) were transduced with the pHIV-Luc-ZsGreen construct to enable automated quantification of relative cell numbers. Using a 2D, 384-well cell culture assay, compounds from the ENZO chemical libraries and the Prestwick Chemical Library (FDA-approved) were screened for their ability to increase cell number.

Several hit compounds were activators of Wnt signalling. We validated the screen results in concentration-response and colony forming assays using additional HBEC donors to assess effects on cell proliferation and clonogenicity. Compounds were tested in the ‘tracheosphere’ assay, in which HBECs proliferate and differentiate to form a polarised spheroid structure containing the major cell types of the airway epithelium. Addition of hit compounds resulted in larger spheroid structures, suggesting increased proliferation, and did not inhibit differentiation to the major luminal cell types.

We additionally investigated the effects of structurally and functionally related compounds in these assays, finding that the induced Wnt activity correlated with organoid size. We envisage that Wnt activating small molecules could be developed into novel treatments to improve airway epithelial cell engraftment.

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