Definition of human intestinal mesenchymal origins and of mesenchymal epithelial cross-talk in health and disease

Cells such as fibroblasts and smooth muscle cells provide critical structural support in organs such as the gut. Over the last 10 years it has become apparent that these cells play important and diverse cellular functions during embryonic development and in the steady state adult intestine. In addition to their structural roles these cells send signals that control the level of immune cell activity. They also signal to the cells lining the surface of the gut, epithelial cells, to ensure they grow to provide a healthy barrier between the intestinal tissue and the gut microbes. We previously used new state of the art single cell technologies (single-cell RNA-sequencing) to measure genes expressed within single mesenchymal cells isolated from human or mouse colonic mesenchyme in health or during colitis. This revealed the surprising observation of several new cell types within what was previously thought to be one population. We went onto explore these new mesenchymal cell states and demonstrated differing functions and anatomical location within the colonic tissue. The study enabled us to identify which cells in the mesenchyme act as a niche for intestinal epithelial stem cells producing factors required for their differentiation and formation of the epithelial barrier layer of the colon. In colitis we identified important changes in single mesenchymal cells including in emergence of a highly activated population that expressed factors that attract immune cells and factors that impair epithelial function. Abnormal expansion of these disease-associated cells contributes to inflammation observed in diseases such as inflammatory bowel disease. These structural cells also play important roles in intestinal fibrosis, when they produce too much matrix protein that makes tissues stiff and leads to strictures in the intestine that prevent the passage of food and require surgery. Furthermore they play important roles in cancer, fuelling invasiveness and metastatic behaviour in colon cancer.
Despite their newly identified and important roles in colon physiology and disease little is known of how these cells arise either in human development or adult state. Little is known about how these cells differentiate into different types and how they control anatomical location of different epithelial cells such as stem cells lining the gut. It is important to identify mesenchymal precursor cells and define the molecular determinants that control emergence of pathogenic populations to revert their aberrant behaviour in disease. Furthermore better understanding of mesenchymal progenitors would enable development of organ-on a chip systems to model human intestine during drug development and enable regenerative medicine approaches for gastrointestinal disease. In this work we will use both single-cell RNA-sequencing and spatial imaging to map human foetal development of colonic mesenchymal cells and the associated colonic epithelial layer with a view to defining a mesenchymal progenitor cell and the molecular cross-talk between mesenchymal and epithelia that dictates formation of healthy barrier. We will use the same approach to define how this relationship breaks down to drive pathology in IBD and colorectal cancer. We will use the foetal development data to inform a search for the colonic mesenchymal stem cell in adult tissue and define functionally if such a cell type exists. This work is critical for advancement of treatments targeting the barrier in both developmental and adult colonic diseases.

Here we will use 10x genomics droplet based scRNAseq to sequence upwards of 100,000 mesenchymal and epithelial cells during human foetal development and in specific disease contexts in adults. Data will be analysed using FastQc software, the 10x genomics Cell Ranger software suit, and R for further processing. 'Seurat' R package will be used to normalise expression values. Clustering will be performed prior to dimensionality reduction using PCA and data clustered in the reduced dimension space using "Seurat" package prior to visualisation using tSNE plots and assessments of batch distributions for each dataset visualised to ensure clustering not driven by batch effects. Cluster markers will be recorded together ontology enrichment analysis for each cluster. Cell differentiation trajectories will be reconstructed using R Package "monocle". To identify putative lineage regulators we will identify factors that change between phenotypic branches using branched expression analysis modeling.
We will validate our findings by confirming anatomical segregation of novel populations of cells with differing functions or in differing disease states. Here we will use immunohistochemistry (IHC), single-molecule in situ hybridisation (sm-ISH) and immunofluorescence. The relationship of individual colonic mesenchymal niche cells with specific epithelial cells will be examined using novel multiparameter imaging technologies such as Codex, imaging mass cytometry (IMC) or multiplexed ion beam imaging (MIBI). We will develop methods to isolate/sort newly identified progenitor cells and test how these differentiate into stromal cell states we know to exist in health and disease in the adult colon. We will use CRISPR/Cas9 gene targeting to remove key transcriptional determinants associated with cell differentiation stages to establish their role in shaping mesenchymal architecture in health and disease.

In this work we will define the cellular anatomy of foetal colon development and key molecular determinants of this process. We will obtain spatial information to validate our findings that will reveal key aspects of mesenchyme and epithelial cross talk that facilitate healthy development. This information will act as a resource to understand pathological defects occurring in developmental disorders of the intestine.
We will utilize this developmental data to inform a search for adult human colonic mesenchymal stem cells. This will create new opportunities for regenerative medicine approaches and for creation of better 3D tissue models of colonic disease required for drug target discovery and personalised treatment approaches in inflammatory bowel disease and colorectal cancer. We hope to define key molecular fate switches within the mesenchyme that can be harnessed to prevent emergence of pathogenic populations underpinning IBD.
Our study will use state of the art imaging approaches to map how rare subtypes of mesenchyme and epithelial cells co-exist in health and how their cross-talk breaks to down to drive IBD and cancer in the colon. This spatial mapping will define a new cellular anatomy of the gut that may be used for disease stratification in the future.