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Future lunar missions and beyond will require new and innovative approaches to radiation countermeasures. The Translational Research Institute for Space Health (TRISH) is focused on identifying and supporting unique approaches to reduce risks to human health and performance on future missions beyond low Earth orbit. This paper will describe three funded and complementary avenues for reducing the risk to humans from radiation exposure experienced in deep space. The first focus is on identifying new therapeutic targets to reduce the damaging effects of radiation by focusing on high throughput genetic screens in accessible, sometimes called lower, organism models. The second focus is to design innovative approaches for countermeasure development with special attention to nucleotide-based methodologies that may constitute a more agile way to design therapeutics. The final focus is to develop new and innovative ways to test radiation countermeasures in a human model system. While animal studies continue to be beneficial in the study of space radiation, they can have imperfect translation to humans. The use of three-dimensional (3D) complex in vitro models is a promising approach to aid the development of new countermeasures and personalized assessments of radiation risks. These three distinct and unique approaches complement traditional space radiation efforts and should provide future space explorers with more options to safeguard their short and long-term health.

Background & AimsHuman intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent the infant intestinal anatomy and physiological responses.MethodsWe established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We validated differences in key pathways through functional studies and determined if these cultures recapitulate known features of the infant intestinal epithelium.ResultsRNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed as higher expression of enterocytes, goblet cells and enteroendocrine cells in differentiated infant HIEs, and greater numbers of proliferative cells in undifferentiated cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine.ConclusionsHIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex-vivo model to advance studies of infant-specific diseases and drug discovery for this population.Graphical AbstractCreated with Biorender.com

Diseases of the lung account for more than 5 million deaths worldwide and are a burden to healthcare. Improving clinical outcomes including mortality and quality of life involves a holistic understanding the etiopathogenesis, which can be provided by multi-omics integration of lung data. An enhanced understanding of large comprehensive datasets provides opportunities to mine those datasets for features that contribute to prevention and amelioration of disease. In this review, we evaluate lung disease models including animal models, organoids and single cell lines as mechanisms to study multiomics in lung health and disease. We provide examples of lung diseases where multi-omics investigations have provided a deeper insight into pathogenesis that has resulted in improved preventive and therapeutic interventions.

The human immune system has sites of production, i.e., bone marrow and the thymus; and sites of differentiation and proliferation, i.e., lymph nodes and the spleen. Immune organization also occurs in the lung, gastrointestinal tract, skin, and adipose tissue. Cells can be tissue bound or move to sites during infection via blood or lymphatics. Pluripotent stem cells in bone marrow give rise to progenitors, which differentiate into lymphoid and myeloid cells as well as red blood cells and platelets. Myeloid cells include neutrophils, basophils, and eosinophils with functions in innate immune responses, as well as monocytes, macrophages, and dendritic cells, which are phagocytic and act as an interface with cells of the acquired immune system. Lymphocytes include antigen-specific cells with DNA-rearranged receptors, T cells and B cells, as well as cells termed innate lymphoid cells, which can be cytotoxic (natural killer cells) or noncytotoxic, with functions that mirror classic T-cell functions.

The use of human tissue stem cell-derived organoids has advanced our knowledge of human physiological and pathophysiological processes that are unable to be studied using other model systems. Increased understanding of human epithelial tissues including intestine, stomach, liver, pancreas, lung, and brain have been achieved using organoids. However, it is not yet clear whether these cultures recapitulate in vivo organ-to-organ signaling or communication. In this work, we demonstrate that mature stem cell-derived intestinal and liver organoid cultures each express functional molecules that modulate bile acid uptake and recycling. These organoid cultures can be physically coupled in a Transwell system and display increased secretion of fibroblast growth factor 19 (FGF19) (intestine) and downregulation of P450 enzyme cholesterol 7 α-hydroxylase (CYP7A) (liver) in response to apical exposure of the intestine to bile acids. This work establishes that organoid cultures can be used to study and therapeutically modulate interorgan interactions and advance the development of personalized approaches to medical care. NEW & NOTEWORTHY Interorgan signaling is a critical feature of human biology and physiology, yet has remained difficult to study due to the lack of in vitro models. Here, we demonstrate that physical coupling of ex vivo human intestine and liver epithelial organoid cultures recapitulates in vivo interorgan bile acid signaling. These results suggest that coupling of multiple organoid systems provides new models to investigate interorgan communication and advances our knowledge of human physiological and pathophysiological processes.

Stem cell-derived, organotypic in vitro models, known as organoids, have emerged as superior alternatives to traditional cell culture models due to their unparalleled ability to recreate complex physiological and pathophysiological processes. For this reason, they are attractive targets of tissue-engineering efforts, as constructs that include organoid technology would be expected to better simulate the many functions of the desired tissue or organ. While the 3D spheroidal architecture that is the default architecture of most organoid models may be preferred for some applications, 2D monolayer arrangements remain the preferred organization for many applications in tissue engineering. Therefore, in this work, we present a method to create monolayer organoid cultures on poly(ethylene glycol) (PEG) hydrogel scaffolds, using intestinal epithelial organoids (IEOs) as a proof-of-concept. Our process involves two steps: the hydrogel is first functionalized with a layer of poly(D-lysine) (PDL), which then allows the adsorption of pristine, unmodified basement membrane proteins. This approach successfully mediates the formation of IEO monolayer unlike conventional approaches that rely on covalent modification of the hydrogel surface with cell-adhesive peptides and basement membrane proteins. We show that these IEO monolayers recreate important physiological functions of the native intestinal epithelium, including multilineage differentiation, apical-basal polarization, and the ability to model infections with human norovirus. We also show coating of a scaffold mimicking intestinal villous topography, resulting in a 3D IEO monolayer. We expect that this protocol will be useful to researchers attempting to leverage the increased physiological relevance of organoid models to elevate the potential of their tissue-engineered constructs. IMPACT STATEMENT: While organoids are physiologically superior models of biological functions than traditional cell cultures, their 3D spheroidal architecture is an obstacle to their incorporation in many tissue-engineering applications, which often prefer 2D monolayer arrangements of cells. For this reason, we developed a protocol to establish monolayer cultures of organoids on poly(ethylene glycol) hydrogels and demonstrate its utility using intestinal epithelial organoids as a proof-of-concept. We expect that this protocol will be of use to researchers creating engineered tissues for both regenerative medicine applications, as well as advanced in vitro experimental models.

Background The role of enteropathogenic Escherichia coli (EPEC) as a cause of diarrhea in cancer and immunocompromised patients is controversial. Quantitation of fecal bacterial loads has been proposed as a method to differentiate colonized from truly infected patients. Methods We studied 77 adult cancer and immunosuppressed patients with diarrhea and EPEC identified in stools by FilmArray, 25 patients with pathogen-negative diarrhea, and 21 healthy adults without diarrhea. Stools were studied by quantitative polymerase chain reaction (qRT-PCR) for EPEC genes eaeA and lifA/efa-1 and strains characterized for virulence factors and adherence to human intestinal enteroids (HIEs). Results Patients with EPEC were more likely to have community-acquired diarrhea (odds ratio, 3.82 [95% confidence interval, 1.5–10.0]; P = .008) compared with pathogen-negative cases. Although EPEC was identified in 3 of 21 (14%) healthy subjects by qPCR, the bacterial burden was low compared to patients with diarrhea (≤55 vs median, 6 × 104 bacteria/mg stool; P < .001). Among EPEC patients, the bacterial burden was higher in those who were immunosuppressed (median, 6.7 × 103 vs 55 bacteria/mg; P < .001) and those with fecal lifA/ifa-1 (median, 5 × 104 vs 120 bacteria/mg; P = .015). Response to antimicrobial therapy was seen in 44 of 48 (92%) patients with EPEC as the sole pathogen. Antimicrobial resistance was common and strains exhibited distinct patterns of adherence with variable cytotoxicity when studied in HIEs. Cancer care was delayed in 13% of patients. Conclusions Immunosuppressed cancer patients with EPEC-associated diarrhea carry high burden of EPEC with strains that are resistant to antibiotics, exhibit novel patterns of adherence when studied in HIEs, and interfere with cancer care.

Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis worldwide; yet currently, no vaccines or FDA-approved antiviral drugs are available to counter these pathogens. To understand HuNoV biology and the epithelial response to infection, we performed transcriptomic analyses, RT-qPCR, CRISPR-Cas9 modification of human intestinal enteroid (HIE) cultures, and functional studies with two virus strains (a pandemic GII.4 and a bile acid-dependent GII.3 strain). We identified a predominant type III interferon (IFN)-mediated innate response to HuNoV infection. Replication of both strains is sensitive to exogenous addition of IFNs, suggesting the potential of IFNs as therapeutics. To obtain insight into IFN pathway genes that play a role in the antiviral response to HuNoVs, we developed knockout (KO) HIE lines for IFN alpha and lambda receptors and the signaling molecules, MAVS, STAT1, and STAT2. An unexpected differential response of enhanced replication and virus spread was observed for GII.3, but not the globally dominant GII.4 HuNoV in STAT1-knockout HIEs compared to parental HIEs. These results indicate cellular IFN responses restrict GII.3 but not GII.4 replication. The strain-specific sensitivities of innate responses against HuNoV replication provide one explanation for why GII.4 infections are more widespread and highlight strain specificity as an important factor in HuNoV biology. Genetically modified HIEs for innate immune genes are useful tools for studying immune responses to viral or microbial pathogens.

Intestinal epithelial damage is associated with most digestive diseases and results in detrimental effects on nutrient absorption and production of hormones and antimicrobial defense molecules. Thus, understanding epithelial repair and regeneration following damage is essential in developing therapeutics that assist in rapid healing and restoration of normal intestinal function. Here we used a well-characterized enteric virus (rotavirus) that damages the epithelium at the villus tip but does not directly damage the intestinal stem cell, to explore the regenerative transcriptional response of the intestinal epithelium at the single-cell level. We found that there are specific Lgr5(+) cell subsets that exhibit increased cycling frequency associated with significant expansion of the epithelial crypt. This was accompanied by an increase in the number of immature enterocytes. Unexpectedly, we found rotavirus infects tuft cells. Transcriptional profiling indicates tuft cells respond to viral infection through interferon-related pathways. Together these data provide insights as to how the intestinal epithelium responds to insults by providing evidence of stimulation of a repair program driven by stem cells with involvement of tuft cells that results in the production of immature enterocytes that repair the damaged epithelium.

Infectious diseases affect individual health and have widespread societal impacts. New ex vivo models are critical to understand pathogenesis, host response, and features necessary to develop preventive and therapeutic treatments. Pluripotent and tissue stem cell–derived organoids provide new tools for the study of human infections. Organoid models recapitulate many characteristics of in vivo disease and are providing new insights into human respiratory, gastrointestinal, and neuronal host–microbe interactions. Increasing culture complexity by adding the stroma, interorgan communication, and the microbiome will improve the use of organoids as models for infection. Organoid cultures provide a platform with the capability to improve human health related to infectious diseases.

Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the human intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from 29 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and gene set enrichment analysis (GSEA) were used to identify differentially expressed genes and enriched pathways. PERMANOVA, Pearson’s correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3-D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect; and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell versus monolayer cultures and E2F target genes enriched in collagen versus Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Surprisingly, large differences in organoid transcriptome were driven by variations in culture methods such as format, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal epithelial organoids.

There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition driven by diverse genetic and nongenetic programs that converge to disrupt immune homeostasis in the intestine. We have reported that, in murine intestinal epithelium with telomere dysfunction, DNA damage-induced activation of ataxia-telangiectasia mutated (ATM) results in ATM-mediated phosphorylation and activation of the YAP1 transcriptional coactivator, which in turn up-regulates pro-IL-18, a pivotal immune regulator in IBD pathogenesis. Moreover, individuals with germline defects in telomere maintenance genes experience increased occurrence of intestinal inflammation and show activation of the ATM/YAP1/pro-IL-18 pathway in the intestinal epithelium. Here, we sought to determine the relevance of the ATM/YAP1/pro-IL-18 pathway as a potential driver of IBD, particularly older-onset IBD. Analysis of intestinal biopsy specimens and organoids from older-onset IBD patients documented the presence of telomere dysfunction and activation of the ATM/YAP1/precursor of interleukin 18 (pro-IL-18) pathway in the intestinal epithelium. Employing intestinal organoids from healthy individuals, we demonstrated that experimental induction of telomere dysfunction activates this inflammatory pathway. In organoid models from ulcerative colitis and Crohn's disease patients, pharmacological interventions of telomerase reactivation, suppression of DNA damage signaling, or YAP1 inhibition reduced pro-IL-18 production. Together, these findings support a model wherein telomere dysfunction in the intestinal epithelium can initiate the inflammatory process in IBD, pointing to therapeutic interventions for this disease.

Background & AimsHuman intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity.MethodsRNA-sequencing datasets from 251 experiments performed on 35 human enteroid and colonoid lines from 28 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and Gene Set Enrichment Analysis (GSEA) was used to identify differentially expressed genes and enriched of pathways.ResultsPERMANOVA, Pearson correlations, and dendrogram analysis of all data indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3D, transwell, and monolayer) had the largest effect (7,271-1,305 differentially expressed genes-DEGs); segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect (5,977-420 DEGs), and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell vs. monolayer cultures, and cholesterol biosynthesis genes enriched in Matrigel vs. collagen cultures.ConclusionsSurprisingly large differences in organoid transcriptome were driven by variations in culture methods such as format and substrate, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal organoids.

Objective The 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. Methods We 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. Results Four 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. Conclusions Obesity 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., Highlights • Human stem cell-derived enteroids preserve the heterogeneity of obesity-related phenotypes. • Four phenotypes representing the relationship between patients' BMI and intestinal dietary glucose absorption were found. • Glucose absorption and gluconeogenesis were elevated in enteroids from a cohort of obese patients. • Elevated glucose absorption was associated with increased expression of SGLT1 and GLUT2 in enteroids. • Elevated gluconeogenesis was associated with increased expression of GLUT5, PEPCK1, and G6Pase in enteroids.

Enteroaggregative Escherichia coli (EAEC) is a significant cause of acute and chronic diarrhea, foodborne outbreaks, infections of the immunocompromised, and growth stunting in children in developing nations. There is no vaccine and resistance to antibiotics is rising. Unlike related E. coli pathotypes that are often associated with acute bouts of infection, EAEC is associated with persistent diarrhea and subclinical long-term colonization. Several secreted virulence factors have been associated with EAEC pathogenesis and linked to disease in humans, less certain are the molecular drivers of adherence to the intestinal mucosa. We previously established human intestinal enteroids (HIEs) as a model system to study host-EAEC interactions and aggregative adherence fimbriae A (AafA) as a major driver of EAEC adherence to HIEs. Here, we report a large-scale assessment of the host response to EAEC adherence from all four segments of the intestine across at least three donor lines for five E. coli pathotypes. The data demonstrate that the host response in the duodenum is driven largely by the infecting pathotype, whereas the response in the colon diverges in a patient-specific manner. Major pathways altered in gene expression in each of the four enteroid segments differed dramatically, with responses observed for inflammation, apoptosis and an overwhelming response to different mucin genes. In particular, EAEC both associated with large mucus droplets and specific mucins at the epithelial surface, binding that was ameliorated when mucins were removed, a process dependent on AafA. Pan-screening for glycans for binding to purified AafA identified the human ligand as heparan sulfate proteoglycans (HSPGs). Removal of HSPG abrogated EAEC association with HIEs. These results may mean that the human intestine responds remarkably different to distinct pathobionts that is dependent on the both the individual and intestinal segment in question, and uncover a major role for surface heparan sulfate proteoglycans as tropism-driving factor in adherence and/or colonization., Author summary E. coli is a significant cause of worldwide diarrhea and systemic infection. We previously established human intestinal enteroids as a model system to study E. coli-host interactions. We report the first large-scale assessment of the host response to infection from all four segments of the intestine across different donor lines for five different pathotypes. Whereas the host response in the duodenum is driven by the infecting pathotype, the response in the colon diverges by donor. We observed a major upregulation of host mucins, particularly for EAEC, which led to the identification of heparan sulfate proteoglycans (HSPGs) as the receptor on human colonoids. Our research highlights a new role for HSPGs for EAEC infection.

Pathologies affecting the small intestine contribute significantly to the disease burden of both the developing and the developed world, which has motivated investigation into the disease mechanisms through in vitro models. Although existing in vitro models recapitulate selected features of the intestine, various important aspects have often been isolated or omitted due to the anatomical and physiological complexity. The small intestine's intricate microanatomy, heterogeneous cell populations, steep oxygen gradients, microbiota, and intestinal wall contractions are often not included in in vitro experimental models of the small intestine, despite their importance in both intestinal biology and pathology. Known and unknown interdependencies between various physiological aspects necessitate more complex in vitro models. Microfluidic technology has made it possible to mimic the dynamic mechanical environment, signaling gradients, and other important aspects of small intestinal biology. This review presents an overview of the complexity of small intestinal anatomy and bioengineered models that recapitulate some of these physiological aspects. IMPACT STATEMENT: There is a need for improved cell culture models of the small intestine. To develop such models, it is necessary to give engineers a better understanding of intestinal biology, and conversely, to give biologists an idea of what is possible with microfluidic devices and biomaterial platforms. This work provides a detailed overview of aspects of intestinal biology that are important for tissue engineers to understand when designing models of the small intestine and provides a summary of current approaches. This review highlights both unique and common features of various strategies for modeling the intestinal cell environment, not only using microfluidic chip-based systems but also several elegant designs that use a materials-based static culture approach.

Obesity and obesity-related comorbidities are significant health care challenges. Bariatric surgery (BS) is the most effective therapy for treating obesity and type 2 diabetes. A barrier in the development of therapeutic alternatives is incomplete mechanistic understanding of the benefits of BS and the lack of human intestinal models that recapitulate the pathophysiology of obesity. Using adult intestinal stem cell-derived enteroid cultures established from healthy lean subjects and morbidly obese patients, including post-BS cases, four phenotypes correlating patient BMI and intestinal glucose absorption were identified suggesting that enteroids retain patient phenotype heterogeneity associated with healthy and diseased state. In a sub-population of obese patients, increased dietary glucose absorption and gluconeogenesis was due to significantly higher expression of intestinal carbohydrate transporters (SGLT1, GLUT2 and GLUT5) and gluconeogenic enzymes (PEPCK1 and G6Pase) compared to enteroids from lean subjects that demonstrated low glucose absorption and lacked gluconeogenesis. Enteroids established from successful BS cases exhibited low glucose absorption similar to that observed in lean subjects. These data show that human enteroids preserve the patient phenotype in long-term cultures and represent a reliable preclinical model to study the heterogeneity of the obesity mechanisms, which is necessary to determine the efficacy of therapeutic interventions.

Gastrointestinal diseases are a significant health care and economic burden. Prevention and treatment of these diseases have been limited by the available human biologic models. Microphysiological systems comprise organ-specific human cultures that recapitulate many structural, biological, and functional properties of the organ in smaller scale including aspects of flow, shear stress and chemical gradients. The development of intestinal microphysiological system platforms represents a critical component in improving our understanding, prevention, and treatment of gastrointestinal diseases. This minireview discusses: shortcomings of classical cell culture models of the gastrointestinal tract; human intestinal enteroids as a new model and their advantages compared to cell lines; why intestinal microphysiological systems are needed; potential functional uses of intestinal microphysiological systems in areas of drug development and modeling acute and chronic diseases; and current challenges in the development of intestinal microphysiological systems. Impact statement The development of a gastrointestinal MPS has the potential to facilitate the understanding of GI physiology. An ultimate goal is the integration of the intestinal MPS with other organ MPS. The development and characterization of nontransformed human intestinal cultures for use in MPS have progressed significantly since the inception of the MPS program in 2012, and these cultures are a key component of advancing MPS. Continued efforts are needed to optimize MPS to comprehensively and accurately recapitulate the complexity of the intestinal epithelium within intestinal tissue. These systems will need to include peristalsis, flow, and oxygen gradients, with incorporation of vascular, immune, and nerve cells. Regional cellular organization of crypt and villus areas will also be necessary to better model complete intestinal structure.

The recent development of stem cell-derived, organotypic in vitro models, known as organoids, has revolutionized our ability to study important biological processes in vitro. However, their continued development is limited by the failure of the hydrogel matrices in which they are grown to adequately replicate the tissue-specific ECM cues they experience in their native in vivo environment. Here, we present a highly customizable, modular hydrogel scaffold that can incorporate tissue-specific cues from the extracellular matrix. We demonstrate that these scaffolds can be functionalized with a wide variety of cell adhesion molecules, including peptides and full-length proteins, and can support the attachment and growth of intestinal epithelials organoids, a model organoid system. We also found that these scaffolds can be patterned with large, high-aspect ratio topographical features that mimic anatomical structures (such as intestinal villi) found in vivo. Finally, we show that organoids cultured on these hydrogel scaffolds retain their capacity for multi-lineage differentiation and their ability to model enteric infections. Together, these findings are an excellent proof-of-concept that such hydrogel scaffolds can facilitate the development of organoid models of many organ systems and improve our ability to study a variety of important developmental and pathological processes.

Intestinal organoid cultures provide an in vitro model system for studying pathways and mechanisms involved in epithelial damage and repair. Derived from either embryonic or induced pluripotent stem cells or adult intestinal stem cells or tissues, these self-organizing, multicellular structures contain polarized mature cells that recapitulate both the physiology and heterogeneity of the intestinal epithelium. These cultures provide a cutting-edge technology for defining regenerative pathways that are induced following radiation or chemical damage, which directly target the cycling intestinal stem cell, or damage resulting from viral, bacterial, or parasitic infection of the epithelium. Novel signaling pathways or biological mechanisms identified from organoid studies that mediate regeneration of the epithelium following damage are likely to be important targets of preventive or therapeutic modalities to mitigate intestinal injury. The evolution of these cultures to include more components of the intestinal wall and the ability to genetically modify them are key components for defining the mechanisms that modulate epithelial regeneration.

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer’s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-α to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

Background There is a significant and unmet need for pre-clinical models to predict responsiveness of immunotherapies to both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) infection. Airway organoid models have been recently developed to study respiratory viruses; however, the current methods rely on invasive or biopsy derived samples to generate lung or airway organoids. Objective To establish human nose organoids (HNOs) as a model to study SARS-CoV-2 and RSV pathogenesis and test therapeutics. Methods We developed a non-invasive method to establish HNOs using stem cells isolated from nasal-wash and mid-turbinate samples. We made air liquid interface (ALI) cultures from undifferentiated 3-dimensional HNOs and differentiated for 21 days to form differentiated nasal epithelium. We inoculated the apical epithelium and assessed SARS-CoV-2 and RSV infection on the apical compartment using real time-polymerase chain reaction, plaque assays and immunofluorescence techniques. We then evaluated the feasibility of HNO-ALI model system to test the efficacy of serum antibodies to prevent SARS-CoV-2 infection and palivizumab monoclonal antibodies to prevent infection using palivizumab sensitive and resistant RSV strains. We introduced the antibodies in the basolateral compartment and monitored its neutralizing capacity on the apical side mimicking the neutralizing effects of antibodies in circulation. Results Our HNO-ALI cultures consist of well-differentiated, pseudostratified, ciliated, and mucosal respiratory epithelial cells and are susceptible to SARS-CoV-2, RSV A and B infection. SARS-CoV-2 and RSV replicates in the apical ciliated cells of the HNO-ALI cultures, peaks at 4 days, and plateaus at 8 days post infection. Infected HNO-ALI recapitulates aspects of SARS-CoV-2 and RSV disease, including viral shedding, asynchronous cilia beating/ciliary damage, and mucus hyper-secretion. Our model effectively showed protection to infection in a concentration dependent manner of the antibodies used. Conclusion We established a non-invasive method to generate HNO-ALI epithelial model as an authentic and an alternative model to 1-D cell culture systems. Our ex-vivo HNO-ALI infection model provides a novel approach for testing therapeutic interventions.

The coronavirus disease-19 (COVID-19) pandemic underscores the threat posed by newly emerging viruses. Understanding the biology of novel viruses rests in large part on in vitro models that allow viral replication. Human and animal organoids are now proving their value as an experimental virology platform., Hans Clevers discusses the value of organoids as an experimental platform for understanding the biology of novel viruses.

SUMMARYIntestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagicE. colisecreted cytotoxin, EspP to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.

Studies of human infectious diseases have been limited by the paucity of functional models that mimic normal human physiology and pathophysiology. Recent advances in the development of multicellular, physiologically active organotypic cultures produced from embryonic and pluripotent stem cells, as well as from stem cells isolated from biopsies and surgical specimens are allowing unprecedented new studies and discoveries about host-microbe interactions. Here, we summarize recent developments in the use of organoids for studying human viral pathogens, including intestinal infections with human rotavirus, norovirus, enteroviruses and adenoviruses (intestinal organoids and enteroids), neuronal infections with Zika virus (cerebral organoids) and respiratory infections with respiratory syncytial virus in (lung bud organoids). Biologic discovery of host-specific genetic and epigenetic factors affecting infection, and responses to infection that lead to disease are possible with the use of organoid cultures. Continued development to increase the complexity of these cultures by including components of the normal host tissue microenvironment such as immune cells, blood vessels and microbiome, will facilitate studies on human viral pathogenesis, and advance the development of platforms for pre-clinical evaluation of vaccines, antivirals and therapeutics.

Intestinal pathogens encounter the microbiota as they seek their host target, potentially impacting the infection. In a recent Cell paper, Shi et al. (2019) show that unique segmented filamentous bacteria prevent rotavirus from infecting the intestinal epithelium. Proposed mechanisms include direct effects on the virus and stimulation of epithelial proliferation.

Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes and goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these nontransformed, three-dimensional HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs, as they generally replicate poorly in transformed cell lines, and host range restriction prevents their replication in many animal models, whereas many animal rotaviruses (ARVs) exhibit a broader host range and replicate in mice. Using HRVs, including the Rotarix RV1 vaccine strain, and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than do ARVs. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused physiological lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model that will allow the study of HRV biology, including host restriction, cell type restriction, and virus-induced fluid secretion. IMPORTANCE Our research establishes HIEs as nontransformed cell culture models to understand human intestinal physiology and pathophysiology and the epithelial response, including host restriction of gastrointestinal infections such as HRV infection. HRVs remain a major worldwide cause of diarrhea-associated morbidity and mortality in children ≤5 years of age. Current in vitro models of rotavirus infection rely primarily on the use of animal rotaviruses because HRV growth is limited in most transformed cell lines and animal models. We demonstrate that HIEs are novel, cellularly diverse, and physiologically relevant epithelial cell cultures that recapitulate in vivo properties of HRV infection. HIEs will allow the study of HRV biology, including human host-pathogen and live, attenuated vaccine interactions; host and cell type restriction; virus-induced fluid secretion; cell-cell communication within the epithelium; and the epithelial response to infection in cultures from genetically diverse individuals. Finally, drug therapies to prevent/treat diarrheal disease can be tested in these physiologically active cultures.

Human rotavirus (HRV) and human norovirus (HuNoV) infections are recognized as the most common causes of epidemic and sporadic cases of gastroenteritis worldwide. The study of these two human gastrointestinal viruses is important for understanding basic virus-host interactions and mechanisms of pathogenesis and to establish models to evaluate vaccines and treatments. Despite the introduction of live-attenuated vaccines to prevent life-threatening HRV-induced disease, the burden of HRV illness remains significant in low-income and less-industrialized countries, and small animal models or ex vivo models to study HRV infections efficiently are lacking. Similarly, HuNoVs remained non-cultivatable until recently. With the advent of non-transformed human intestinal enteroid (HIE) cultures, we are now able to culture and study both clinically relevant HRV and HuNoV in a biologically relevant human system. Methods described here will allow investigators to use these new culture techniques to grow HRV and HuNoV and analyze new aspects of virus replication and pathogenesis.

Organ interactions resulting from drug, metabolite or xenobiotic transport between organs are key components of human metabolism that impact therapeutic action and toxic side effects. Preclinical animal testing often fails to predict adverse outcomes arising from sequential, multi-organ metabolism of drugs and xenobiotics. Human microphysiological systems (MPS) can model these interactions and are predicted to dramatically improve the efficiency of the drug development process. In this study, five human MPS models were evaluated for functional coupling, defined as the determination of organ interactions via an in vivo-like sequential, organ-to-organ transfer of media. MPS models representing the major absorption, metabolism and clearance organs (the jejunum, liver and kidney) were evaluated, along with skeletal muscle and neurovascular models. Three compounds were evaluated for organ-specific processing: terfenadine for pharmacokinetics (PK) and toxicity; trimethylamine (TMA) as a potentially toxic microbiome metabolite; and vitamin D3. We show that the organ-specific processing of these compounds was consistent with clinical data, and discovered that trimethylamine-N-oxide (TMAO) crosses the blood-brain barrier. These studies demonstrate the potential of human MPS for multi-organ toxicity and absorption, distribution, metabolism and excretion (ADME), provide guidance for physically coupling MPS, and offer an approach to coupling MPS with distinct media and perfusion requirements.

The intestinal epithelium can limit enteric pathogens by producing antiviral cytokines, such as IFNs. Type I IFN (IFN-α/β) and type III IFN (IFN-λ) function at the epithelial level, and their respective efficacies depend on the specific pathogen and site of infection. However, the roles of type I and type III IFN in restricting human enteric viruses are poorly characterized as a result of the difficulties in cultivating these viruses in vitro and directly obtaining control and infected small intestinal human tissue. We infected nontransformed human intestinal enteroid cultures from multiple individuals with human rotavirus (HRV) and assessed the host epithelial response by using RNA-sequencing and functional assays. The dominant transcriptional pathway induced by HRV infection is a type III IFN-regulated response. Early after HRV infection, low levels of type III IFN protein activate IFN-stimulated genes. However, this endogenous response does not restrict HRV replication because replication-competent HRV antagonizes the type III IFN response at pre- and posttranscriptional levels. In contrast, exogenous IFN treatment restricts HRV replication, with type I IFN being more potent than type III IFN, suggesting that extraepithelial sources of type I IFN may be the critical IFN for limiting enteric virus replication in the human intestine.