A lymphatic-stem cell interactome regulates intestinal stem cell activity

Abstract

Barrier epithelia depend on resident stem cells for homeostasis, defense and repair. Intestinal stem cells (ISCs) of the small and large intestines respond to their local microenvironments (niches) to fulfill a continuous demand for tissue turnover, yet the complexity of their niches is still unfolding. Here, we report an extensive lymphatic network that intimately associates with ISCs within these niches. Devising a lymphatic:organoid coculture system, we show that lymphatic-secreted factors maintain ISCs while inhibiting precocious differentiation. Employing a new deconvolution algorithm, BayesPrism, to pair single-cell and spatial transcriptomics, we cartograph the lymphatic ligand:ISC receptor interactomes at high resolution. We unearth crypt lymphatics as a major source of WNT-signaling factors (WNT2, R-SPONDIN-3) known to drive ISC behavior, and REELIN, a hitherto unappreciated ISC regulator secreted by crypt lymphatics. Together, our studies expose lymphatics as a central hub for niche factors that govern the regenerative potential of ISCs.

Endothelial FOXC1 and FOXC2 promote intestinal regeneration after ischemia-reperfusion injury

“…Thus, FOXC1 and FOXC2 are likely to regulate RSPO3 expression directly in LECs. During our manuscript preparation, others have submitted bioRxiv preprints 56,57,58 showing that RSPO3 produced by intestinal LECs is required for maintaining ISCs in homeostasis and regeneration, reinforcing our finding that FOXC transcription factors mediate LEC-derived RSPO3 expression as a critical paracrine regulator in intestinal regeneration after I/R injury 59 .…”

Comparative analysis of cell-cell communication at single-cell resolution

“…Among IP1 ligands were the ephrins EPHB3, whose expression gradient is known to control ISC differentiation 47 , and RSPO3 ( Figure 4E ). Two recent studies have each reported that RSPO3 production by lymphatic endothelial cells (LECs) and GREM1 + fibroblasts is critical for maintaining the ISC niche in mice 45,46 . In this human dataset, we did not observe expression of RSPO3 in LECs ( Supplementary Figure 3 ); however, we did find that GREM1 + fibroblasts expressed RSPO3 as a part of IP1 that was predicted to be sensed primarily by ISCs, thus demonstrating that this interaction pathway may communicate between different cell types in mouse than in human ( Figure 4D-F ).…”

“…Despite the absence of RSPO3 expression in LECs, it remains possible that LECs maintain the ISC niche in human intestinal development, particularly as these cells can reside in close spatial proximity to ISCs 45,46 . We were able to identify a small population of LECs ( Figure 4H ), and we examined CCC between LECs and ISCs.…”

“…Individual cells can be scored for expression of these interaction programs post hoc , enabling downstream comparative analyses with a comprehensive view of CCC structure. We apply this workflow to an atlas-scale dataset of human fetal gut development, where we validate a mode of communication between a fibroblast subset and ISCs that has recently been shown to be important for maintaining the ISC niche 39,40 . Due to the relative scarcity of these cells, it is likely that agglomerative methods may fail to discover these important interactions for downstream mechanistic validation.…”

“…We next examined ways in which our identified interaction programs reflected known biology of intestinal development. Recently, several important interactions have been shown to be critical in maintaining the intestinal stem cell (ISC) niche [38][39][40] . We were able to identify ISCs, defined by expression of LGR5 and SOX9, within the intestinal epithelial cells of this dataset, and discovered a single interaction program (hereafter referred to as IP1) whose receptors were co-expressed with these ISC markers (Figure 4C).…”