Analysing the molecular and cellular mechanisms involved in tissue homeostasis and scar formation in the peritoneum using novel photo-activated photos

Summary:

Tissue homeostasis and repair are important processes in life. While we have a good understanding of the cells and molecular processes involved in maintaining the function and integrity of some organs, such as the skin or lungs, it is not clear how this is regulated in others. For example, it is not clear how the peritoneum is maintained and repaired, and progenitor cells that facilitate tissue maintenance and respective molecular mechanisms have yet to be identified.

The peritoneum comprises an epithelial barrier of mesothelial cells (MCs), and a submesothelial layer of connective tissue containing a range of cells including mesenchymal/fibroblast cells. Through genetic lineage tracing studies we showed that MCs expressing the transcription factor Wt1 divide slowly during homeostasis, but following injury multiply and become migratory, contributing to fibrotic scar formation.

TGFb and PDGF-BB are factors previously shown to stimulate a more migratory mesenchymal behaviour in MCs. However, retinoic acid (RA), released by peritoneal MCs, stimulates more epithelial characteristics.

We demonstrated that MCs emerge from mouse peritoneal explant cultures in serum-rich conditions. However, a group of emerging cells display more migratory behaviour and mesenchymal characteristics while still expressing the mesothelial marker Wt1, suggesting that peritoneal cells undergo highly dynamic changes to complex signalling cocktails.

The project consists of two work-packages (WPs):

WP1: Molecular phenotyping of MCs in response to modulators of RA, Notch and Wnt. The student will learn peritoneal explant culture methods, and characterisation of emerging cells over an 8-10-day period using live cell imaging and end-point immunofluorescence and confocal microscopy.

WP2: Specific ablation of mesenchymal or epithelial cells emerging from peritoneal explants under conditions identified in WP1. This involves using confocal-guided photo-ablation of explants treated with diphenylacetylene-based photosensitisers which lead to ROS formation and subsequent cell death. Remaining cells of the explants will be assessed for their migratory or epithelial characteristics and cell proliferation, to determine progenitor cell status.

The ablation experiments will be carried out with support of the industrial partner, LightOx, who developed the photosensitiser technologies.