Mapping mesenchymal diversity in the human small intestine and organoids

The organization of diverse mesenchymal populations during human small intestinal development is critical for tissue architecture and function yet remains poorly defined. Here, to construct a comprehensive, tissue-scale map of the developing human small intestine at single cell resolution, we leveraged single-cell RNA-sequencing data to build a Xenium spatial transcriptomics gene panel covering the cell diversity of the human small intestine. We defined five subpopulations occupying discrete anatomical locations within the lamina propria and submucosa—the subepithelial cells, lamina propria fibroblasts, submucosal fibroblasts, smooth muscle cells and CXCL13+ fibroblasts. Our data establish molecular markers to distinguish these populations in both sequencing and imaging data. We leverage this high-resolution atlas to interrogate cell–cell signalling, benchmark pluripotent stem cell-derived human intestinal organoids and to demonstrate how this resource can incorporate relative spatial organization into tissue analysis, with broad implications for modelling development, regeneration and disease. Johnson et al. combine single-cell and spatial transcriptomics approaches to build a spatially resolved atlas of the developing human intestine and intestinal organoids, uncovering spatial organization and fibroblast heterogeneity.