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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE329635 | GEO | 2026/06/30

REPOSITORIES: GEO

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Project description:Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating sub-populations of cells are based on mRNA or protein expression of only few known marker genes. The unequivocal identification of a specific marker gene, however, poses a major challenge, particularly if this cell type is rare. Identifying rare cell types, such as stem cells, short-lived progenitors, cancer stem cells, or circulating tumor cells is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids, cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbor stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances. Since available computational methods can only resolve more abundant cell types, we developed RaceID, an algorithm for rare cell type identification in complex populations of single cells. We demonstrate that this algorithm can resolve cell types represented by only a single cell in a population of randomly sampled organoid cells. We use this algorithm to identify Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone producing intestinal cells. Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. Reassuringly, RaceID confirmed the existence of known enteroendocrine lineages, and moreover, discovered novel subtypes, which we subsequently validated in vivo. Having validated RaceID by this proof-of-principle experiment we then apply the algorithm to ex vivo isolated LGR5 positive cells and their direct progeny and demonstrate homogeneity of the stem cell pool. We envision broad applicability of our method for discovering rare cell types and the corresponding marker genes in healthy and diseased organs.

Project description:In Rspondin-based three-dimensional cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. Here we report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely resemble the original tumor. The spectrum of genetic changes within the 'living biobank' agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell line- and xenograft-based drug studies and allow personalized therapy design. Self-renewal of the intestinal epithelium is driven by Lgr5 stem cells located in crypts. We have recently developed a long-term culture system that maintains basic crypt physiology. Wnt signals are required for the maintenance of active crypt stem cells. Indeed, the Wnt agonist R-spondin1 induces dramatic crypt hyperplasia in vivo. R-spondin-1 is the ligand for Lgr5. Epidermal growth factor (EGF) signaling is associated with intestinal proliferation, while transgenic expression of Noggin induces a dramatic increase in crypt numbers. The combination of R-spondin-1, EGF, and Noggin in Matrigel sustains ever-expanding small intestinal organoids, which display all hallmarks of the original tissue in terms of architecture, cell type composition, and self-renewal dynamics. We adapted the culture condition for long-term expansion of human colonic epithelium and primary colonic adenocarcinoma, by adding nicotinamide, A83-01 (Alk inhibitor), Prostaglandin E2 and the p38 inhibitor SB202190. Of note, a two-dimensional culture method for cells from normal and malignant primary tissue has been described by Schlegel and colleagues. Here, we explore organoid technology to routinely establish and phenotypically annotate ‘paired organoids’ derived from adjacent tumor and healthy epithelium from CRC patients.

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

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