Intestinal epithelial cells derive from stem cells at the crypt base and travel along the crypt-villus axis to die at the villus tip. The two dominant villus epithelial cell types, absorptive ...enterocytes and mucous-secreting goblet cells, are mature when they exit crypts. Murine enterocytes switch functional cell states during migration along the villus. Here, we ask whether this zonation is driven by the bone morphogenetic protein (BMP) gradient, which increases toward the villus. Using human intestinal organoids, we show that BMP signaling controls the expression of zonated genes in enterocytes. We find that goblet cells display similar zonation involving antimicrobial genes. Using an inducible Bmpr1a knockout mouse model, we confirm that BMP controls these zonated genes in vivo. Our findings imply that local manipulation of BMP signal strength may be used to reset the enterocyte “rheostat” of carbohydrate versus lipid uptake and to control the antimicrobial response through goblet cells.
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•Human intestinal lineages display divergent gene expression along the crypt-villus axis•Top villus cell functions are not accurately recapitulated in conventional organoids•BMP activation drives the expression of villus tip genes in human organoid cultures•Loss of Bmpr1a receptor in human organoids and mice reduces top villus gene expression
Beumer et al. show that the bone morphogenetic protein (BMP) signaling pathway controls functional zonation of the major human intestinal epithelial lineages along the crypt-villus axis. A gene lipid uptake gene signature is enriched in the villus tip and could be therapeutically exploited with BMP inhibitors.
Intestinal organoids are three-dimensional cultures that resemble key aspects of the epithelium of origin. Here, we describe how to differentiate human small intestinal organoids by combining growth ...media variations and genetic engineering. We detail the differentiation of human intestinal organoids in the presence and absence of BMP agonists to recapitulate a broader scope of functional cell states found in vivo. Using transient overexpression of the transcription factor Neurogenin-3, we describe the enhancement of differentiation toward rare enteroendocrine cells.
For complete details on the use and execution of this protocol, please refer to Beumer et al. (2022).
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•Differentiation of human small intestinal organoids by growth media variations•BMP-mediated functional specialization of intestinal lineages•CRISPR/Cas9-mediated loss of function experiments in human small intestinal organoids•Generation of enteroendocrine-cell enriched organoids via NEUROG3 overexpression
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Intestinal organoids are three-dimensional cultures that resemble key aspects of the epithelium of origin. Here, we describe how to differentiate human small intestinal organoids by combining growth media variations and genetic engineering. We detail the differentiation of human intestinal organoids in the presence and absence of BMP agonists to recapitulate a broader scope of functional cell states found in vivo. Using transient overexpression of the transcription factor Neurogenin-3, we describe the enhancement of differentiation toward rare enteroendocrine cells.
Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host-microbe interactions with great experimental control. This protocol comprises methods to ...coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.
Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of ...human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.
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•A human organoid biobank combines hormone labeling and enteroendocrine cell generation•Transcriptomic profiling of human enteroendocrine cells uncovers differences with mice•Functional validation of EEC receptors and transcription factors•Secretome analysis reveals the repertoire of enteroendocrine secreted products
An organoid-based platform for studying human enteroendocrine cells, which sense intestinal content and release hormones to regulate many processes throughout the body, is developed by Beumer et al. and used to describe the landscape of mRNA expression and secreted products.
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