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An organoid screening framework to engineer intestinal epithelial composition

ABSTRACT: Barrier epithelia are essential to organismal homeostasis, and changes in their cellular composition are observed in human diseases. Within the small intestine, adult stem cells establish tissue cellularity, and may provide a means to control the abundance and quality of specialized epithelial cells. Nevertheless, we lack suitable approaches to identify biological targets and small molecules to modulate them. Here, we develop an extendable framework utilizing a physiologically-inspired organoid model to identify unknown, druggable regulators of intestinal stem cell differentiation that translate to intended manipulations in vivo. Specifically, we miniaturize and adapt an organoid model of Paneth cell differentiation to enable multiplexed phenotypic screening on the scale of thousands of samples, and employ longitudinal single-cell RNA-sequencing to understand hit biology. Strikingly, we identify that inhibitors of the nuclear exporter XPO1 modulate stem cell fate commitment, significantly increasing the abundance of Paneth cells independent of known differentiation cues. Our framework elucidates small molecules which modulate tissue stem cell biology and their underlying targets without the need for a priori knowledge of in vivo pathway biology.

ORGANISM(S): Mus musculus

PROVIDER: GSE148524 | GEO | 2021/09/16

REPOSITORIES: GEO

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Project description:Mead BE, Ordovas-Montanes J, Braun AP, Levy LE, Bhargava P, Szucs MJ, Ammendolia DA, MacMullan MA, Zheng Y, Yin X, Hughes TK, Wadsworth MH, Ahmad R, Rakoff-Nahoum S, Carr SA, Langer R, Collins JJ, Shalek AK, Karp JM. BMC Biology 2018. Background: Single-cell genomic methods now provide unprecedented resolution for characterizing the component cell types and states of tissues, such as the epithelial subsets of the gastrointestinal tract. Nevertheless, functional studies of these subsets at scale require faithful in vitro models of identified in vivo biology. While intestinal organoids have been invaluable in providing mechanistic insights in vitro, the extent to which organoid-derived cell types recapitulate their in vivo counterparts remains formally untested, with no systematic approach for improving model fidelity. Results: Here, we present a generally applicable framework that utilizes massively-parallel single-cell RNA-seq to compare cell types and states found in vivo to those of in vitro models, such as organoids. Furthermore, we leverage identified discrepancies to improve model fidelity. Using the Paneth cell (PC), which supports the stem cell niche and produces the largest diversity of antimicrobials in the small intestine, as an exemplar, we uncover fundamental gene expression differences in lineage-defining genes between in vivo PCs and those of the current in vitro organoid model. With this information, we nominate a molecular intervention to rationally improve the physiological fidelity of our in vitro PCs. We then perform transcriptomic, cytometric, morphologic, and proteomic characterization, and demonstrate functional (antimicrobial activity, niche support) improvements in Paneth cell physiology. Conclusions: Our systematic approach provides a simple workflow for identifying the limitations of in vitro models and enhancing their physiological fidelity. Using adult stem cell-derived PCs within intestinal organoids as a model system, we successfully benchmark organoid representation relative to in vivo of a specialized cell type and use this comparison to generate a functionally improved in vitro PC population. We predict that the generation of rationally-improved cellular models will facilitate mechanistic exploration of specific disease-associated genes in their respective cell types.

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An organoid screening framework to engineer intestinal epithelial composition

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