Analysing the properties of mesothelial cells: Applications in tissue engineering.
Lead Research Organisation:
University of Liverpool
Department Name: Cellular and Molecular Physiology
Abstract
The mesothelium is a serous membrane that lines the surfaces of the serosal cavities. It enables frictionless movement between adjacent viscera, preventing pain and damage. Wilms' tumour suppressor 1 (Wt1) is commonly used as a marker of mesothelial cells. The mesothelium has been investigated thoroughly due to its ability to contribute to peritoneal and visceral homeostasis, wound repair, antigen presentation and ultrafiltration. Indeed, Wt1 has been suggested to directly regulate genes that are involved in epithelial to mesenchymal transition (EMT) and its reverse, mesenchymal to epithelial transition (MET). Recent findings from our lab suggest that not all mesothelial cells express Wt1. The main aim of this project is to identify whether Wt1 is required for mesothelial cell identity and ability to maintain mesothelial epithelial characteristics. This will be achieved by performing single cell RNAseq of mesothelial cells to determine whether lack of Wt1 expression in mesothelial cells results in an altered expression profile, especially with respect to key EMT signalling pathways and adhesion molecules. We will also test different surface modifications to explore the relationship between Wt1 expression in mesothelial cells and their interaction with extracellular domains and cell-basement membrane adhesion.
Our current findings indicate that the percentage Wt1 expression was 91.8% and 91.7% in the parietal and visceral peritoneal mesothelia, respectively, and 74% in the omentum. We are now aiming to optimise the extraction of peritoneal mesothelial cells by injecting the peritoneal cavity with 10x trypsin/EDTA solution. After isolation of mesothelial cells we will perform numerous quality assurance assays including qPCR, flow cytometry and immunofluorescence to both determine their suitability for RNAseq and their characteristics following shorter (3 days) and longer (7 days) culture. We will utilise RNAseq analysis to determine relationships between the Wt1-expression statuses of mesothelial cells with other biochemical pathways; potentially providing greater insight into the link between Wt1 and mesothelial pathobiology.
In addition to the investigation between Wt1 and mesothelial cell biology; we are investigating the behaviour of mesothelial cells derived from murine omental explants. Omental explants have been touted as a source of stable mesothelial cultures. Our aim is to further investigate the behaviour of these cells during outgrowth and migration away from the explant. Initial analyses suggest that there are both mesothelial and fibroblastic cell types originating from the explants, with fibroblasts being considerably more mobile than mesothelial cells. ImageJ analysis of live cell imaging of the explants taken over a 48 hour period can determine the speed, displacement and morphology of cells. These data can be analysed to determine relationships between these variables and any distinctions between the cells. We will explore any significant signalling pathways that arise from the RNAseq study, as well as known regulators of mesothelial cell behaviour, including BMP4 and TGFbeta, using this live cell imaging model.
Greater understanding of the behaviour of mesothelial cells in culture will contribute to increased understanding of mesothelial pathology and may pave the way for therapeutic models.
Our current findings indicate that the percentage Wt1 expression was 91.8% and 91.7% in the parietal and visceral peritoneal mesothelia, respectively, and 74% in the omentum. We are now aiming to optimise the extraction of peritoneal mesothelial cells by injecting the peritoneal cavity with 10x trypsin/EDTA solution. After isolation of mesothelial cells we will perform numerous quality assurance assays including qPCR, flow cytometry and immunofluorescence to both determine their suitability for RNAseq and their characteristics following shorter (3 days) and longer (7 days) culture. We will utilise RNAseq analysis to determine relationships between the Wt1-expression statuses of mesothelial cells with other biochemical pathways; potentially providing greater insight into the link between Wt1 and mesothelial pathobiology.
In addition to the investigation between Wt1 and mesothelial cell biology; we are investigating the behaviour of mesothelial cells derived from murine omental explants. Omental explants have been touted as a source of stable mesothelial cultures. Our aim is to further investigate the behaviour of these cells during outgrowth and migration away from the explant. Initial analyses suggest that there are both mesothelial and fibroblastic cell types originating from the explants, with fibroblasts being considerably more mobile than mesothelial cells. ImageJ analysis of live cell imaging of the explants taken over a 48 hour period can determine the speed, displacement and morphology of cells. These data can be analysed to determine relationships between these variables and any distinctions between the cells. We will explore any significant signalling pathways that arise from the RNAseq study, as well as known regulators of mesothelial cell behaviour, including BMP4 and TGFbeta, using this live cell imaging model.
Greater understanding of the behaviour of mesothelial cells in culture will contribute to increased understanding of mesothelial pathology and may pave the way for therapeutic models.
