Single-cell RNA sequencing of small intestinal organoid self-organization [scRNA-Seq]

The mouse small intestinal epithelium is composed of diverse cell types that interact to regulate signaling, cell patterning, and tissue form and function. The self-organization of small intestinal organoids, starting from a single cell (0 h) to a budded crypt-villus structure (96–108 h), recapitulates aspects of such multicellular intestinal morphogenesis. To characterize the cell states and lineage trajectories underlying organoid self-organization, we performed single-cell RNA sequencing of intestinal organoids in a high-resolution time course. Organoids derived from mouse duodenal epithelium were dissociated to single cells and seeded to initiate organoid formation, followed by sampling for sequencing at 12-hour intervals from 24 to 108 h. This dataset captures dynamic changes in cell state composition during organoid development. Early time points (24–36 h) reveal a population of reprogramming cells characterized by loss of mature lineage markers, followed by the emergence and differentiation of major intestinal lineages, including absorptive, secretory, and stem cell populations. A secretory progenitor population emerges by 48 h, which gives rise to both exocrine (Paneth and goblet cells) and endocrine lineages (enteroendocrine progenitors and differentiated enteroendocrine cell types). Absorptive and stem cell lineages continue their differentiation and maturation following their emergence at 48–72 h. Overall, this dataset provides a comprehensive resource for studying cell state transitions and lineage specification during intestinal organoid self-organization and recapitulates key features of epithelial differentiation observed in vivo.