Stem Cell-derived Enteroids Research Articles

Co-culturing Human Intestinal Enteroid Monolayers with Innate Immune Cells.

The coordinated interaction between the intestinal epithelium and immune cells is required to maintain proper barrier function and mucosal host defenses to the harsh external environment of the gut lumen. Complementary to in vivo models, there is a need for practical and reproducible in vitro models that employ primary human cells to confirm and advance our understanding of mucosal immune responses under physiologic and pathophysiologic conditions. Here we describe the methods to co-culture human intestinal stem cell-derived enteroids grown as confluent monolayers on permeable supports with primary human innate immune cells (e.g., monocyte-derived macrophages and polymorphonuclear neutrophils). This co-culture model reconstructs the cellular framework of the human intestinal epithelial-immune niche with distinct apical and basolateral compartments to recreate host responses to luminal and submucosal challenges, respectively. Enteroid-immune co-cultures enable multiple outcome measures to interrogate important biological processes such as epithelial barrier integrity, stem cell biology, cellular plasticity, epithelial-immune cellscrosstalk, immune cell effector functions, changes in gene expression (i.e., transcriptomic, proteomic, epigenetic), and host-microbiome interactions.

Respiratory and intestinal epithelial cells exhibit differential susceptibility and innate immune responses to contemporary EV-D68 isolates.

Enterovirus D68 (EV-D68) has been implicated in outbreaks of severe respiratory illness and is associated with acute flaccid myelitis (AFM). EV-D68 is often detected in patient respiratory samples but has also been detected in stool and wastewater, suggesting the potential for both respiratory and enteric routes of transmission. Here, we used a panel of EV-D68 isolates, including a historical pre-2014 isolate and multiple contemporary isolates from AFM outbreak years, to define the dynamics of viral replication and the host response to infection in primary human airway cells and stem cell-derived enteroids. We show that some recent EV-D68 isolates have decreased sensitivity to acid and temperature compared with earlier isolates and that the respiratory, but not intestinal, epithelium induces a robust type III interferon response that restricts infection. Our findings define the differential responses of the respiratory and intestinal epithelium to contemporary EV-D68 isolates and suggest that a subset of isolates have the potential to target both the human airway and gastrointestinal tracts.

New Insights and Enhanced Human Norovirus Cultivation in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are the leading cause of epidemic and sporadic acute gastroenteritis worldwide. We previously demonstrated human intestinal stem cell-derived enteroids (HIEs) support cultivation of several HuNoV strains. However, HIEs did not support virus replication from every HuNoV-positive stool sample, which led us to test and optimize new medium conditions, identify characteristics of stool samples that allow replication, and evaluate consistency of replication over time. Optimization of our HIE-HuNoV culture system has shown the following: (i) a new HIE culture medium made with conditioned medium from a single cell line and commercial media promotes robust replication of HuNoV strains that replicated poorly in HIEs grown in our original culture medium made with conditioned media from 3 separate cell lines; (ii) GI.1, 11 GII genotypes (GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.8, GII.12, GII.13, GII.14, and GII.17), and six GII.4 variants can be cultivated in HIEs; (iii) successful replication is more likely with virus in stools with higher virus titers; (iv) GII.4_Sydney_2012 virus replication was reproducible over 3 years; and (v) HuNoV infection is restricted to the small intestine, based on replication of two viral strains in duodenal and ileal HIEs, but not colonoids, from two susceptible donors. These results improve the HIE culture system for HuNoV replication. Use of HIEs by several laboratories worldwide to study the molecular mechanisms that regulate HuNoV replication confirms the usefulness of this culture system, and our optimized methods for virus replication will advance the development of effective therapies and methods for virus control.IMPORTANCE Human noroviruses (HuNoVs) are highly contagious and cause acute and sporadic diarrheal illness in all age groups. In addition, chronic infections occur in immunocompromised cancer and transplant patients. These viruses are antigenically and genetically diverse, and there are strain-specific differences in binding to cellular attachment factors. In addition, new discoveries are being made on strain-specific differences in virus entry and replication and the epithelial cell response to infection in human intestinal enteroids. Human intestinal enteroids are a biologically relevant model to study HuNoVs; however, not all strains can be cultivated at this time. A complete understanding of HuNoV biology thus requires cultivation conditions that will allow the replication of multiple strains. We report optimization of HuNoV cultivation in human intestinal enteroid cultures to increase the numbers of cultivatable strains and the magnitude of replication, which is critical for testing antivirals, neutralizing antibodies, and methods of virus inactivation.

Intestinal stem cell-derived enteroids from morbidly obese patients preserve obesity-related phenotypes: Elevated glucose absorption and gluconeogenesis

ObjectiveThe mechanisms behind the efficacy of bariatric surgery (BS) for treating obesity and type 2 diabetes, particularly with respect to the influence of the small bowel, remain poorly understood. In vitro and animal models are suboptimal with respect to their ability to replicate the human intestinal epithelium under conditions induced by obesity. Human enteroids have the potential to accelerate the development of less invasive anti-obesity therapeutics if they can recapitulate the pathophysiology of obesity. Our aim was to determine whether adult stem cell-derived enteroids preserve obesity-characteristic patient-specific abnormalities in carbohydrate absorption and metabolism. MethodsWe established 24 enteroid lines representing 19 lean, overweight, or morbidly obese patients, including post-BS cases. Dietary glucose absorption and gluconeogenesis in enteroids were measured. The expression of carbohydrate transporters and gluconeogenic enzymes was assessed and a pharmacological approach was used to dissect the specific contribution of each transporter or enzyme to carbohydrate absorption and metabolism, respectively. ResultsFour phenotypes representing the relationship between patients’ BMI and intestinal dietary sugar absorption were found, suggesting that human enteroids retain obese patient phenotype heterogeneity. Intestinal glucose absorption and gluconeogenesis were significantly elevated in enteroids from a cohort of obese patients. Elevated glucose absorption was associated with increased expression of SGLT1 and GLUT2, whereas elevated gluconeogenesis was related to increased expression of GLUT5, PEPCK1, and G6Pase. ConclusionsObesity phenotypes preserved in human enteroids provide a mechanistic link to aberrant dietary carbohydrate absorption and metabolism. Enteroids can be used as a preclinical platform to understand the pathophysiology of obesity, study the heterogeneity of obesity mechanisms, and identify novel therapeutics.

Stem cell-derived enteroid cultures as a tool for dissecting host-parasite interactions in the small intestinal epithelium.

Toxoplasma gondii and Cryptosporidium spp. can cause devastating pathological effects in humans and livestock, and in particular to young or immunocompromised individuals. The current treatment plans for these enteric parasites are limited due to long drug courses, severe side effects or simply a lack of efficacy. The study of the early interactions between the parasites and the site of infection in the small intestinal epithelium has been thwarted by the lack of accessible, physiologically relevant and species-specific models. Increasingly, 3D stem cell-derived enteroid models are being refined and developed into sophisticated models of infectious disease. In this review, we shall illustrate the use of enteroids to spearhead research into enteric parasitic infections, bridging the gap between cell line cultures and in vivo experiments.

Aminopeptidase N Expression, Not Interferon Responses, Determines the Intestinal Segmental Tropism of Porcine Deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an economically important enteropathogen of swine with worldwide distribution. PDCoV primarily infects the small intestine instead of the large intestine in vivo However, the underlying mechanism of PDCoV tropism to different intestinal segments remains poorly understood as a result of the lack of a suitable in vitro intestinal model that recapitulates the cellular diversity and complex functions of the gastrointestinal tract. Here, we established the PDCoV infection model of crypt-derived enteroids from different intestinal segments. Enteroids were susceptible to PDCoV, and multiple types of different functional intestinal epithelia were infected by PDCoV in vitro and in vivo We further found that PDCoV favorably infected the jejunum and ileum and restrictedly replicated in the duodenum and colon. Mechanistically, enteroids from different intestinal regions displayed a distinct gene expression profile, and the differential expression of primary viral receptor host aminopeptidase N (APN) instead of the interferon (IFN) responses determined the susceptibility of different intestinal segments to PDCoV, although PDCoV substantially elicited antiviral genes production in enteroids after infection. Additional studies showed that PDCoV infection significantly induced the expression of type I and III IFNs at the late stage of infection, and exogenous IFN inhibited PDCoV replication in enteroids. Hence, our results provide critical inputs to further dissect the molecular mechanisms of PDCoV-host interactions and pathogenesis.IMPORTANCE The zoonotic potential of the PDCoV, a coronavirus efficiently infecting cells from a broad range species, including porcine, chicken, and human, emphasizes the urgent need to further study the cell and tissue tropism of PDCoV in its natural host. Herein, we generated crypt stem cell-derived enteroids from porcine different intestinal regions, which well recapitulated the events in vivo of PDCoV infection that PDCoV targeted multiple types of intestinal epithelia and preferably infected the jejunum and ileum over the duodenum and colon. Mechanistically, we demonstrated that the expression of APN receptor rather than the IFN responses determined the susceptibility of different regions of the intestines to PDCoV infection, though PDCoV infection markedly elicited the IFN responses. Our findings provide important insights into how the distinct gene expression profiles of the intestinal segments determine the cell and tissue tropism of PDCoV.

Human Norovirus Cultivation in Nontransformed Stem Cell-Derived Human Intestinal Enteroid Cultures: Success and Challenges.

Noroviruses, in the genus Norovirus, are a significant cause of viral gastroenteritis in humans and animals. For almost 50 years, the lack of a cultivation system for human noroviruses (HuNoVs) was a major barrier to understanding virus biology and the development of effective antiviral strategies. This review presents a historical perspective of the development of a cultivation system for HuNoVs in human intestinal epithelial cell cultures. Successful cultivation was based on the discovery of genetically-encoded host factors required for infection, knowledge of the site of infection in humans, and advances in the cultivation of human intestinal epithelial cells achieved by developmental and stem cell biologists. The human stem cell-derived enteroid cultivation system recapitulates the multicellular, physiologically active human intestinal epithelium, and allows studies of virus-specific replication requirements, evaluation of human host-pathogen interactions, and supports the pre-clinical assessment of methods to prevent and treat HuNoV infections.

In Vitro Replication of Human Norovirus.

Human norovirus (HuNoV) infection is a major cause of gastroenteritis all over the world. Despite this, these non-enveloped RNA viruses are poorly characterized due to the lack of robust and widely available HuNoV culture systems. The two published systems (B cell line and stem cell-derived enteroids) support replication of HuNoVs but the levels of replication are not sufficient for the generation of highly purified virus stocks or the development of culture-based quantification assays. Therefore, improvement of HuNoV in vitro replication is still needed. Murine norovirus and other caliciviruses have provided insights into norovirus replication that paved the way for the development of the current HuNoV culture systems and may also aid in the improvement of these systems. This review will highlight ways in which previous research guided and impacted the development of HuNoV culture systems and discuss ways in which more recent discoveries might be utilized to improve the quality of the HuNoV in vitro replication.

Inhibition of sodium/hydrogen exchanger 3 in the gastrointestinal tract by tenapanor reduces paracellular phosphate permeability.

Hyperphosphatemia is common in patients with chronic kidney disease and is increasingly associated with poor clinical outcomes. Current management of hyperphosphatemia with dietary restriction and oral phosphate binders often proves inadequate. Tenapanor, a minimally absorbed, small-molecule inhibitor of the sodium/hydrogen exchanger isoform 3 (NHE3), acts locally in the gastrointestinal tract to inhibit sodium absorption. Because tenapanor also reduces intestinal phosphate absorption, it may have potential as a therapy for hyperphosphatemia. We investigated the mechanism by which tenapanor reduces gastrointestinal phosphate uptake, using in vivo studies in rodents and translational experiments on human small intestinal stem cell-derived enteroid monolayers to model ion transport physiology. We found that tenapanor produces its effect by modulating tight junctions, which increases transepithelial electrical resistance (TEER) and reduces permeability to phosphate, reducing paracellular phosphate absorption. NHE3-deficient monolayers mimicked the phosphate phenotype of tenapanor treatment, and tenapanor did not affect TEER or phosphate flux in the absence of NHE3. Tenapanor also prevents active transcellular phosphate absorption compensation by decreasing the expression of NaPi2b, the major active intestinal phosphate transporter. In healthy human volunteers, tenapanor (15 mg, given twice daily for 4 days) increased stool phosphorus and decreased urinary phosphorus excretion. We determined that tenapanor reduces intestinal phosphate absorption predominantly through reduction of passive paracellular phosphate flux, an effect mediated exclusively via on-target NHE3 inhibition.

A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions

Integration of the intestinal epithelium and the mucosal immune system is critical for gut homeostasis. The intestinal epithelium is a functional barrier that secludes luminal content, senses changes in the gut microenvironment, and releases immune regulators that signal underlying immune cells. However, interactions between epithelial and innate immune cells to maintain barrier integrity and prevent infection are complex and poorly understood. We developed and characterized a primary human macrophage-enteroid co-culture model for in-depth studies of epithelial and macrophage interactions. Human intestinal stem cell-derived enteroid monolayers co-cultured with human monocyte-derived macrophages were used to evaluate barrier function, cytokine secretion, and protein expression under basal conditions and following bacterial infection. Macrophages enhanced barrier function and maturity of enteroid monolayers as indicated by increased transepithelial electrical resistance and cell height. Communication between the epithelium and macrophages was demonstrated through morphological changes and cytokine production. Intraepithelial macrophage projections, efficient phagocytosis, and stabilized enteroid barrier function revealed a coordinated response to enterotoxigenic and enteropathogenic E. coli infections. In summary, we have established the first primary human macrophage-enteroid co-culture system, defined conditions that allow for a practical and reproducible culture model, and demonstrated its suitability to study gut physiology and host responses to enteric pathogens.

Enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner

Enteroviruses are among the most common viral infectious agents of humans and are primarily transmitted by the fecal-oral route. However, the events associated with enterovirus infections of the human gastrointestinal tract remain largely unknown. Here, we used stem cell-derived enteroids from human small intestines to study enterovirus infections of the intestinal epithelium. We found that enteroids were susceptible to infection by diverse enteroviruses, including echovirus 11 (E11), coxsackievirus B (CVB), and enterovirus 71 (EV71), and that contrary to an immortalized intestinal cell line, enteroids induced antiviral and inflammatory signaling pathways in response to infection in a virus-specific manner. Furthermore, using the Notch inhibitor dibenzazepine (DBZ) to drive cellular differentiation into secretory cell lineages, we show that although goblet cells resist E11 infection, enteroendocrine cells are permissive, suggesting that enteroviruses infect specific cell populations in the human intestine. Taken together, our studies provide insights into enterovirus infections of the human intestine, which could lead to the identification of novel therapeutic targets and/or strategies to prevent or treat infections by these highly clinically relevant viruses.