Development of functional resident macrophages in human pluripotent stem cell-derived colonic organoids and human fetal colon.

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.

[For a good understanding and use of the term "organoids"]. / Pour une bonne compréhension et un bon usage du terme « organoïdes ¼.

Title: Pour une bonne compréhension et un bon usage du terme « organoïdes ¼. Abstract: Depuis une dizaine d'années, des progrès considérables ont été réalisés concernant les conditions qui permettent à des cellules de s'auto-organiser dans l'espace comme elles le font lors des phases précoces du développement embryonnaire ou dans certains tissus adultes. On nomme ainsi « organoïdes ¼ des structures en trois dimensions complexes, organisées et intégrant plusieurs types cellulaires, qui peuvent reproduire in vitro certaines fonctions d'un organe. Toutefois, ces organoïdes ne peuvent actuellement reproduire à l'identique une architecture anatomique et fonctionnelle complète. Bien qu'utilisé pour des raisons de simplification pour la communication, en particulier dans la presse généraliste, il est donc abusif d'utiliser le terme « mini-organes ¼ pour décrire ces structures.

Multi-disciplinary Insights from the First European Forum on Visceral Myopathy 2022 Meeting.

Visceral myopathy is a rare, life-threatening disease linked to identified genetic mutations in 60% of cases. Mostly due to the dearth of knowledge regarding its pathogenesis, effective treatments are lacking. The disease is most commonly diagnosed in children with recurrent or persistent disabling episodes of functional intestinal obstruction, which can be life threatening, often requiring long-term parenteral or specialized enteral nutritional support. Although these interventions are undisputedly life-saving as they allow affected individuals to avoid malnutrition and related complications, they also seriously compromise their quality of life and can carry the risk of sepsis and thrombosis. Animal models for visceral myopathy, which could be crucial for advancing the scientific knowledge of this condition, are scarce. Clearly, a collaborative network is needed to develop research plans to clarify genotype-phenotype correlations and unravel molecular mechanisms to provide targeted therapeutic strategies. This paper represents a summary report of the first 'European Forum on Visceral Myopathy'. This forum was attended by an international interdisciplinary working group that met to better understand visceral myopathy and foster interaction among scientists actively involved in the field and clinicians who specialize in care of people with visceral myopathy.

Gut Microbiota Remodeling and Intestinal Adaptation to Lipid Malabsorption After Enteroendocrine Cell Loss in Adult Mice.

BACKGROUND & AIMS: Enteroendocrine cells (EECs) and their hormones are essential regulators of whole-body energy homeostasis. EECs sense luminal nutrients and microbial metabolites and subsequently secrete various hormones acting locally or at a distance. Impaired development of EECs during embryogenesis is life-threatening in newborn mice and humans due to compromised nutrient absorption. However, the physiological importance of the EEC system in adult mice has yet to be directedly studied. Herein, we aimed to determine the long-term consequences of a total loss of EECs in healthy adults on energy metabolism, intestinal transcriptome, and microbiota. METHODS: We depleted intestinal EECs by tamoxifen treatment of adult Neurog3fl/fl; Villin-CreERT2 male mice. We studied intestinal cell differentiation, food efficiency, lipid absorption, microbiota composition, fecal metabolites, and transcriptomic responses in the proximal and distal small intestines of mice lacking EECs. We also determined the high-fat diet-induced transcriptomic changes in sorted Neurog3eYFP/+ EECs. RESULTS: Induction of EEC deficiency in adults is not life-threatening unless fed with a high-fat diet. Under a standard chow diet, mice lose 10% of weight due to impaired food efficiency. Blood concentrations of cholesterol, triglycerides, and free fatty acids are reduced, and lipid absorption is impaired and delayed in the distal small intestine. Genes controlling lipogenesis, carbohydrate metabolism, and neoglucogenesis are upregulated. Microbiota composition is rapidly altered after EECs depletion and is characterized by decreased α-diversity. Bacteroides and Lactobacillus were progressively enriched, whereas Lachnospiraceae declined without impacting fecal short-chain fatty acid concentrations. CONCLUSIONS: EECs are dispensable for survival in adult male mice under a standard chow diet. The absence of EECs impairs intestinal lipid absorption, leading to transcriptomic and metabolic adaptations and remodeling of the gut microbiota.

In vivo development of immune tissue in human intestinal organoids transplanted into humanized mice.

Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal-immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases.

UNC45A deficiency causes microvillus inclusion disease-like phenotype by impairing myosin VB-dependent apical trafficking.

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

Intestinal Organoids: New Tools to Comprehend the Virulence of Bacterial Foodborne Pathogens.

Foodborne diseases cause high morbidity and mortality worldwide. Understanding the relationships between bacteria and epithelial cells throughout the infection process is essential to setting up preventive and therapeutic solutions. The extensive study of their pathophysiology has mostly been performed on transformed cell cultures that do not fully mirror the complex cell populations, the in vivo architectures, and the genetic profiles of native tissues. Following advances in primary cell culture techniques, organoids have been developed. Such technological breakthroughs have opened a new path in the study of microbial infectious diseases, and thus opened onto new strategies to control foodborne hazards. This review sheds new light on cellular messages from the host-foodborne pathogen crosstalk during in vitro organoid infection by the foodborne pathogenic bacteria with the highest health burden. Finally, future perspectives and current challenges are discussed to provide a better understanding of the potential applications of organoids in the investigation of foodborne infectious diseases.

Intestinal Stem Cell-on-Chip to Study Human Host-Microbiota Interaction.

The gut is a tubular organ responsible for nutrient absorption and harbors our intestinal microbiome. This organ is composed of a multitude of specialized cell types arranged in complex barrier-forming crypts and villi covered by a mucosal layer controlling nutrient passage and protecting from invading pathogens. The development and self-renewal of the intestinal epithelium are guided by niche signals controlling the differentiation of specific cell types along the crypt-villus axis in the epithelium. The emergence of microphysiological systems, or organ-on-chips, has paved the way to study the intestinal epithelium within a dynamic and controlled environment. In this review, we describe the use of organ-on-chip technology to control and guide these differentiation processes in vitro. We further discuss current applications and forthcoming strategies to investigate the mechanical processes of intestinal stem cell differentiation, tissue formation, and the interaction of the intestine with the microbiota in the context of gastrointestinal diseases.

Intestinal multicellular organoids to study colorectal cancer.

Modeling colorectal cancer (CRC) using organoids has burgeoned in the last decade, providing enhanced in vitro models to study the development and possible treatment options for this type of cancer. In this review, we describe both normal and CRC intestinal organoid models and their utility in the cancer research field. Besides highlighting studies that develop epithelial CRC organoid models, i.e. organoids without tumor microenvironment (TME) cellular components, we emphasize on the need for TME in CRC modeling, to help reduce translational disparities in this area. Also, we discuss the utilization of CRC organoids derived from pluripotent stem cells, as well as their potential to be used in cancer research. Finally, limitations and challenges in the current CRC organoids field, are discussed.

Modeling Gastrointestinal Diseases Using Organoids to Understand Healing and Regenerative Processes.

The gastrointestinal tract is a continuous series of organs from the mouth to the esophagus, stomach, intestine and anus that allows digestion to occur. These organs are frequently associated with chronic stress and injury during life, subjecting these tissues to frequent regeneration and to the risk of developing disease-associated cancers. The possibility of generating human 3D culture systems, named organoids, that resemble histologically and functionally specific organs, has opened up potential applications in the analysis of the cellular and molecular mechanisms involved in epithelial wound healing and regenerative therapy. Here, we review how during normal development homeostasis takes place, and the role of the microenvironmental niche cells in the intestinal stem cell crypt as an example. Then, we introduce the notion of a perturbed niche during disease conditions affecting the esophageal-stomach junction and the colon, and describe the potential applications of organoid models in the analysis of human gastrointestinal disease mechanisms. Finally, we highlight the perspectives of organoid-based regenerative therapy to improve the repair of the epithelial barrier.

PGI2 Inhibits Intestinal Epithelial Permeability and Apoptosis to Alleviate Colitis.

BACKGROUND & AIMS: Inflammatory bowel diseases (IBDs) that encompass both ulcerative colitis and Crohn's disease are a major public health problem with an etiology that has not been fully elucidated. There is a need to improve disease outcomes and preventive measures by developing new effective and lasting treatments. Although polyunsaturated fatty acid metabolites play an important role in the pathogenesis of several disorders, their contribution to IBD is yet to be understood. METHODS: Polyunsaturated fatty acids metabolite profiles were established from biopsy samples obtained from Crohn's disease, ulcerative colitis, or control patients. The impact of a prostaglandin I2 (PGI2) analog on intestinal epithelial permeability was tested in vitro using Caco-2 cells and ex vivo using human or mouse explants. In addition, mice were treated with PGI2 to observe dextran sulfate sodium (DSS)-induced colitis. Tight junction protein expression, subcellular location, and apoptosis were measured in the different models by immunohistochemistry and Western blotting. RESULTS: A significant reduction of PGI2 in IBD patient biopsies was identified. PGI2 treatment reduced colonic inflammation, increased occludin expression, decreased caspase-3 cleavage and intestinal permeability, and prevented colitis development in DSS-induced mice. Using colonic explants from mouse and human control subjects, the staurosporine-induced increase in paracellular permeability was prevented by PGI2. PGI2 also induced the membrane location of occludin and reduced the permeability observed in colonic biopsies from IBD patients. CONCLUSIONS: The present study identified a PGI2 defect in the intestinal mucosa of IBD patients and demonstrated its protective role during colitis.

In Vivo Human PSC-Derived Intestinal Organoids to Study Stem Cell Maintenance.

Human intestinal organoids (HIOs), derived from pluripotent stem cells, are a new tool to gain insights in gastrointestinal development, physiology, and associated diseases. Herein, we present a method for renal transplantation of HIOs in immunocompromised mice and subsequent analysis to study intestinal epithelial cell proliferation. In addition, we describe how to generate enteroids from transplanted HIOs. The method highlights the specific steps to successful engraftment and provides insight into the study of human intestinal stem cells.

Pluripotent stem cell derived intestinal organoids with an enteric nervous system.

The use of human pluripotent stem cells (hPSCs) and differentiation techniques offer new ways to generate specific tissue. It is now possible to differentiate hPSC into human intestinal organoids that include an enteric nervous system. Using step-wise differentiation processes, we generate innervated intestinal organoids that form three-dimensional structures bearing an epithelium, neurons and glial cells embedded in a supporting mesenchyme. Innervated organoids further develop to a complex structure with similar organization and cellular differentiation as the developing intestine. These tools open up new fields of application in the study of the development and pathophysiology of enteric neuropathies. Herein, we describe the generation of both human intestinal organoids and vagal neural crest cells from hPSC and their combination into an innervated organoid. We also discuss technical considerations for these experiments, and highlight advantages and limitations of the system.

Secretion of Acid Sphingomyelinase and Ceramide by Endothelial Cells Contributes to Radiation-Induced Intestinal Toxicity.

Ceramide-induced endothelial cell apoptosis boosts intestinal stem cell radiosensitivity. However, the molecular connection between these two cellular compartments has not been clearly elucidated. Here we report that ceramide and its related enzyme acid sphingomyelinase (ASM) are secreted by irradiated endothelial cells and act as bystander factors to enhance the radiotoxicity of intestinal epithelium. Ceramide and the two isoforms of ASM were acutely secreted in the blood serum of wild-type mice after 15 Gy radiation dose, inducing a gastrointestinal syndrome. Interestingly, serum ceramide was not enhanced in irradiated ASMKO mice, which are unable to develop intestinal failure injury. Because ASM/ceramide were secreted by primary endothelial cells, their contribution was studied in intestinal epithelium dysfunction using coculture of primary endothelial cells and intestinal T84 cells. Adding exogenous ASM or ceramide enhanced epithelial cell growth arrest and death. Conversely, blocking their secretion by endothelial cells using genetic, pharmacologic, or immunologic approaches abolished intestinal T84 cell radiosensitivity. Use of enteroid models revealed ASM and ceramide-mediated deleterious mode-of-action: when ceramide reduced the number of intestinal crypt-forming enteroids without affecting their structure, ASM induced a significant decrease of enteroid growth without affecting their number. Identification of specific and different roles for ceramide and ASM secreted by irradiated endothelial cells opens new perspectives in the understanding of intestinal epithelial dysfunction after radiation and defines a new class of potential therapeutic radiomitigators. SIGNIFICANCE: This study identifies secreted ASM and ceramide as paracrine factors enhancing intestinal epithelial dysfunction, revealing a previously unknown class of mediators of radiosensitivity.

Activation of Hedgehog Signaling Promotes Development of Mouse and Human Enteric Neural Crest Cells, Based on Single-Cell Transcriptome Analyses.

BACKGROUND & AIMS: It has been a challenge to develop fully functioning cells from human pluripotent stem cells (hPSCs). We investigated how activation of hedgehog signaling regulates derivation of enteric neural crest (NC) cells from hPSCs. METHODS: We analyzed transcriptomes of mouse and hPSC-derived enteric NCs using single-cell RNA sequencing (scRNA-seq) to identify the changes in expression associated with lineage differentiation. Intestine tissues were collected from Tg(GBS-GFP), Sufuf/f; Wnt1-cre, Ptch1+/-, and Gli3Δ699/Δ699 mice and analyzed by flow cytometry and immunofluorescence for levels of messenger RNAs encoding factors in the hedgehog signaling pathway during differentiation of enteric NCs. Human NC cells (HNK-1+p75NTR+) were derived from IMR90 and UE02302 hPSC lines. hPSCs were incubated with a hedgehog agonist (smoothened agonist [SAG]) and antagonists (cyclopamine) and analyzed for differentiation. hPSC-based innervated colonic organoids were derived from these hPSC lines and analyzed by immunofluorescence and neuromuscular coupling assay for expression of neuronal subtype markers and assessment of the functional maturity of the hPSC-derived neurons, respectively. RESULTS: Single-cell RNA sequencing analysis showed that neural fate acquisition by human and mouse enteric NC cells requires reduced expression of NC- and cell cycle-specific genes and up-regulation of neuronal or glial lineage-specific genes. Activation of the hedgehog pathway was associated with progression of mouse enteric NCs to the more mature state along the neuronal and glial lineage differentiation trajectories. Activation of the hedgehog pathway promoted development of cultured hPSCs into NCs of greater neurogenic potential by activating expression of genes in the neurogenic lineage. The hedgehog agonist increased differentiation of hPSCs into cells of the neuronal lineage by up-regulating expression of GLI2 target genes, including INSM1, NHLH1, and various bHLH family members. The hedgehog agonist increased expression of late neuronal markers and neuronal activities in hPSC-derived neurons. CONCLUSIONS: In enteric NCs from humans and mice, activation of hedgehog signaling promotes differentiation into neurons by promoting cell-state transition, expression of genes in the neurogenic lineage, and functional maturity of enteric neurons.

[From human pluripotent stem cells to custom-made intestinal organoids]. / Organoïdes (2) - Façonner l'intestin à partir des cellules souches pluripotentes humaines.

The study of gut diseases is often limited by the access to human biological tissues and animal models that do not faithfully mimic the human pathologies. In this context, the development of intestinal organoids from human pluripotent stem cells is paving the way of gastrointestinal physiology and digestive disease study. In this review, we recall the embryonic development of the digestive tract and its translation to human pluripotent stem cell differentiation. We also present the different types of intestinal organoids that can be generated, as well as their applications in research.

TITLE: Façonner l'intestin à partir des cellules souches pluripotentes humaines. ABSTRACT: L'étude des maladies digestives est parfois limitée par l'accès aux tissus de patients et les modèles précliniques ne sont pas toujours fidèles aux pathologies observées chez l'homme. Dans ce contexte, le développement d'organoïdes intestinaux à partir de cellules souches pluripotentes humaines représente une avancée importante dans l'étude des processus physiologiques et des pathologies digestives. Dans cette revue, nous rappelons les étapes majeures du développement du tractus digestif chez l'homme et décrivons le rationnel de la différenciation dirigée des cellules souches pluripotentes humaines. Nous faisons également un état des lieux sur les différents types d'organoïdes intestinaux existants et leurs applications en recherche fondamentale et préclinique. Enfin, nous discutons des opportunités offertes par les organoïdes intestinaux humains dans un contexte de médecine de précision et de médecine réparatrice.

IL-13-induced intestinal secretory epithelial cell antigen passages are required for IgE-mediated food-induced anaphylaxis.

BACKGROUND: Food-induced anaphylaxis (FIA) is an IgE-dependent immune response that can affect multiple organs and lead to life-threatening complications. The processes by which food allergens cross the mucosal surface and are delivered to the subepithelial immune compartment to promote the clinical manifestations associated with food-triggered anaphylaxis are largely unexplored. OBJECTIVE: We sought to define the processes involved in the translocation of food allergens across the mucosal epithelial surface to the subepithelial immune compartment in FIA. METHODS: Two-photon confocal and immunofluorescence microscopy was used to visualize and trace food allergen passage in a murine model of FIA. A human colon cancer cell line, RNA silencing, and pharmacologic approaches were used to identify the molecular regulation of intestinal epithelial allergen uptake and translocation. Human intestinal organoid transplants were used to demonstrate the conservation of these molecular processes in human tissues. RESULTS: Food allergens are sampled by using small intestine (SI) epithelial secretory cells (termed secretory antigen passages [SAPs]) that are localized to the SI villous and crypt region. SAPs channel food allergens to lamina propria mucosal mast cells through an IL-13-CD38-cyclic adenosine diphosphate ribose (cADPR)-dependent process. Blockade of IL-13-induced CD38/cADPR-dependent SAP antigen passaging in mice inhibited induction of clinical manifestations of FIA. IL-13-CD38-cADPR-dependent SAP sampling of food allergens was conserved in human intestinal organoids. CONCLUSION: We identify that SAPs are a mechanism by which food allergens are channeled across the SI epithelium mediated by the IL-13/CD38/cADPR pathway, regulate the onset of FIA reactions, and are conserved in human intestine.

Transplantation of human intestinal organoids into the mouse mesentery: A more physiologic and anatomic engraftment site.

BACKGROUND: We previously described the development of human intestinal organoids from pluripotent stem cells, as well as their in vivo maturation when transplanted into the mouse kidney capsule. While sufficient for certain aspects of study, this model has limitations. Herein, we describe an alternative model of human intestinal organoids transplantation into the mouse mesentery. We hypothesize that efficient engraftment and marked differentiation of human intestinal organoids will be similar to our kidney model yet in a more anatomically appropriate location allowing for improved in vivo modeling. METHODS: Human intestinal organoids were generated by directed differentiation of H1 embryonic stem cells. Human intestinal organoids were then transplanted into the mesentery of immunosuppressed mice. Gross and histologic analysis of tissue was performed. RESULTS: Human intestinal organoids were transplanted into the mouse mesentery and allowed to grow for 10 weeks. Mouse survival was 85%, and among the surviving mice, 82% of transplanted human intestinal organoids successfully engrafted. Upon graft harvest, transplanted HIOs were larger than in vitro human intestinal organoids (1.75 mm vs 6.27 mm, P < .0001) and grew along a vascular pedicle, allowing for interventions and reconstructive surgeries to access the human intestinal organoid lumen. Histologic analyses of transplanted human intestinal organoids confirmed the presence of major cell types, as well as stem cell activity. CONCLUSIONS: The mouse mesentery is a viable location for the transplantation of human intestinal organoids, yielding grafts of reproducible size and quality. This improved model serves to advance functional and translational studies of human intestinal organoids.

Mechanically induced development and maturation of human intestinal organoids in vivo.

The natural ability of stem cells to self-organize into functional tissue has been harnessed for the production of functional human intestinal organoids. Although dynamic mechanical forces play a central role in intestinal development and morphogenesis, conventional methods for the generation of intestinal organoids have relied solely on biological factors. Here, we show that the incorporation of uniaxial strain, by using compressed nitinol springs, in human intestinal organoids transplanted into the mesentery of mice induces growth and maturation of the organoids. Assessment of morphometric parameters, transcriptome profiling, and functional assays of the strain-exposed tissue revealed higher similarities to native human intestine, with regards to tissue size and complexity, and muscle tone. Our findings suggest that the incorporation of physiologically relevant mechanical cues during the development of human intestinal tissue enhances its maturation and enterogenesis.