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Enteroendocrine cells (EECs) are gut epithelial cells that respond to intestinal contents by secreting hormones, including the incretins glucagon-like peptide 1 (GLP-1) and gastric inhibitory protein (GIP), which regulate multiple physiological processes. Hormone release is controlled through metabolite-sensing proteins. Low expression, interspecies differences, and the existence of multiple EEC subtypes have posed challenges to the study of these sensors. We describe differentiation of stomach EECs to complement existing intestinal organoid protocols. CD200 emerged as a pan-EEC surface marker, allowing deep transcriptomic profiling from primary human tissue along the stomach-intestinal tract. We generated loss-of-function mutations in 22 receptors and subjected organoids to ligand-induced secretion experiments. We delineate the role of individual human EEC sensors in the secretion of hormones, including GLP-1. These represent potential pharmacological targets to influence appetite, bowel movement, insulin sensitivity, and mucosal immunity.

Enteroendocrine cells (EECs) secrete hormones in response to ingested nutrients to control physiological processes such as appetite and insulin release. EEC hormones are synthesized as large proproteins that undergo proteolytic processing to generate bioactive peptides. Mutations in EEC-enriched proteases are associated with endocrinopathies. Due to the relative rarity of EECs and a paucity of in vitro models, intestinal prohormone processing remains challenging to assess. Here, human gut organoids in which EECs can efficiently be induced are subjected to CRISPR-Cas9-mediated modification of EEC-expressed endopeptidase and exopeptidase genes. We employ mass spectrometry-based analyses to monitor peptide processing and identify glucagon production in intestinal EECs, stimulated upon bone morphogenic protein (BMP) signaling. We map the substrates and products of major EECs endo- and exopeptidases. Our studies provide a comprehensive description of peptide hormones produced by human EECs and define the roles of specific proteases in their generation.

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)., Competing Interests: H.C. is head of Roche’s Pharma Research and Early Development in Basel, Switzerland since March 2022. J.B. and H.C. are inventors on several patents related to organoid technology. H.C.’s full disclosure is given at https://www.uu.nl/staff/JCClevers/., (© 2022 The Author(s).)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

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. [Display omitted] • 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. [ABSTRACT FROM AUTHOR]

Co-culture of intestinal organoids with a colibactin-producing pks + E. coli strain (EcC) revealed mutational signatures also found in colorectal cancer (CRC). E. coli Nissle 1917 (EcN) remains a commonly used probiotic, despite harboring the pks operon and inducing double strand DNA breaks. We determine the mutagenicity of EcN and three CRC-derived pks + E. coli strains with an analytical framework based on sequence characteristic of colibactin-induced mutations. All strains, including EcN, display varying levels of mutagenic activity. Furthermore, a machine learning approach attributing individual mutations to colibactin reveals that patients with colibactin-induced mutations are diagnosed at a younger age and that colibactin can induce a specific APC mutation. These approaches allow the sensitive detection of colibactin-induced mutations in ∼12% of CRC genomes and even in whole exome sequencing data, representing a crucial step toward pinpointing the mutagenic activity of distinct pks + E. coli strains. [Display omitted] • Detection of specific mutations induced by pks+ E. coli strains, including Nissle 1917 • Mutation classifier indicates 12% of CRC display colibactin mutagenesis • Colibactin-associated CRC cases have APC mutations at colibactin motifs • Colibactin-CRC cases have a younger age of onset in multiple cohorts In this study, Rosendahl Huber et al. show the mutagenic properties of pks+ E. coli strains, including probiotic E. coli Nissle 1917, using the extended target sequence context of colibactin and with a machine-learning model. These approaches allow for better distinguishing of colibactin-associated colorectal cancer cases, which are younger and are enriched for APC mutations matching the colibactin motif. [ABSTRACT FROM AUTHOR]

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. • 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. [ABSTRACT FROM AUTHOR]