A Holistic Analysis of the Intestinal Stem Cell Niche Network

Abstract

Although many studies into the intestinal stem cell (ISC) niche have been carried out, they have focused on the role of a single cell type or molecular signal. However, no holistic comparisons of the predominant cell types and signals present within the intestinal mucosa have been conducted to date. We utilize bulk RNA sequencing to profile 20 different mucosal cell types covering four major cell categories: epithelial, stromal, endothelial and immune. We further examined the stromal signaling environment using scRNAseq to provide a more comprehensive view of the signaling microenvironment within the intestinal mucosa. We identified the primary signals for the major ISC regulatory pathways and their respective cellular sources. Our analysis suggests that a 'niche network' exists, with no single cell type being responsible for ISC self-renewal, proliferation, or differentiation; rather, each cell type within the network carries out specific functions in a highly cooperative and coordinated manner. KeywordsStem Cell Niche, Intestinal Stem Cells, ISC, RNA-Sequencing As expected, principle component analysis of the various transcriptomes resulted in the cell types clustering according to their categorical distinctions ( Fig. 1G). To briefly summarize, Grem1 CreErt2 + cells adopted a unique position between stromal and epithelial cells. Nestin GFP + and Ncad tdT + cells clustered together, consistent with their overlap and distribution pattern. Intriguingly, Ng2 dsR + and Dmp1 Cre + cells were transcriptomically similar, yet showed differences in several genes important to cellular function ( Fig. S1B). Innate lymphoid type 3 cells (ILC3) were positioned between adaptive T Cells and Innate Immune Dendritic cells and Macrophages (Fig. 1G). Characterization of Genetic Models and Stromal PopulationsBoth Nestin GFP + and Ncad CreErt2 + stromal cells were present from the smooth muscle to the villus tips ( Fig. 2A-B) and possessed somewhat similar distributions and morphologies to the niche cells described within the bone marrow. Though there was considerable overlap and similarities between these genetic markers, several differences were apparent. Ncad CreErt2 + cells were more likely to encircle the vasculature with a flattened morphology, while Nestin GFP + cells frequently laid on top of these cells and possessed long filopodia which connect with one another ( Fig. S2A-E) Ncad CreErt2 + cells were more likely to be present at the lower "ring", while Nestin GFP + were more likely to localize to the isthmus. Ng2 dsR + cells also overlapped with Nestin GFP + cells but were the only cell type examined that exhibited a strong spatial bias within the mucosa, being almost exclusively found within the villi (Fig. 2C,S2B).Unfortunately, their morphological characteristics could not be determined due to the tendency of the dsRed protein to form aggregates, preventing even cytosolic distribution.Dmp1 Cre + cells showed the greatest amount of morphological and positional diversity, likely due to their constitutively active Cre, wh...