Assessment of stromal cell diversity and development in the mouse intestinal mucosa

The intestine comprises anatomically distinct segments, largely divided into the small intestine and colon. These segments perform essential and diverse physiological tasks, such as food absorption and digestion in the small intestine or water reabsorption, waste disposal and provision of a niche for beneficial microbes in the colon.

The luminal side of the intestinal tissue is composed of a single layer of heterogeneous epithelial cells covering the underlying lamina propria (LP). The LP accommodates diverse subsets of mesenchymal stromal cells (MSCs), immune cells, endothelia and cells of the enteric nervous system. MSCs were historically thought to mainly provide structure through extracellular matrix secretion, whereas evidence of other vital tissue functions have recently been reported.

For example, MSCs support the intestinal epithelial stem cell nice, support the vasculature, guide intestinal development, interact with immune cells, and are involved in several pathological conditions, which have fostered an increased attention in recent years. Nevertheless, the diversity of small intestinal and colon MSCs as well as distinct subset-specific functionalities and locations have remained unclear.

Here, we provide novel insight into small intestine and colon MSC diversity through single cell RNA sequencing (scRNA-seq) of both tissues. We have used the generated data to identify subset-specific markers, which provide evidence for distinct subset locations within both the small intestine and colon, locations that were largely reflected in the differential expressions of epithelial support genes between subsets.

It has remained elusive how intestinal MSCs originate during ontogeny, and through grafting studies of embryonic intestine under the kidney capsule of adult mice, we show that embryonic intestinal-resident precursors develop into the highly heterogeneous adult MSCs in both small intestine and colon.

By lineage-tracing of such embryonic precursors into adulthood, we find an embryonic Gli1+ precursor that gives rise to the diverse MSCs. This was further supported by trajectory analysis of integrated embryonic colon scRNA-seq and adult colon scRNA-seq data, which when combined with the lineage-tracing data, provide evidence that embryonic serosal mesothelial cells give rise to all adult intestinal MSC subsets.

Interestingly, we find the embryonic trajectory to separate into smooth muscle cell (SMC) and fibroblast (FB) lineages from the mesothelial start point, suggesting different developmental requirements. The FB branch of the trajectory show mesothelial cells to give rise to adult FBs through an adult submucosal FB subset, which we identify as the FB precursor in adult steady state intestine.

The trajectory analysis further revealed several distinct colonic subepithelial FB subsets and displays unprecedented diversity and complexity within the subepithelial FB compartment. Finally, correlating our murine intestinal MSC subsets with their counterparts in humans, our data highlights the translational potential of studying murine MSCs.

In conclusion, our data amends the current knowledge of intestinal MSC subsets, functions and origins by providing a comprehensive roadmap of these enigmatic cells in the murine intestine, work we are confident will pave the way for future studies required to unravel the full functionality of individual intestinal MSCs in health and disease.