Human Small Intestinal Tissue Research Articles

Enhancing pre-clinical research with simplified intestinal cell line models

Two-dimensional culture remains widely employed to determine the bioavailability of orally delivered drugs. To gain more knowledge about drug uptake mechanisms and risk assessment for the patient after oral drug admission, intestinal in vitro models demonstrating a closer similarity to the in vivo situation are needed. In particular, Caco-2 cell-based Transwell® models show advantages as they are reproducible, cost-efficient, and standardized. However, cellular complexity is impaired and cell function is strongly modified as important transporters in the apical membrane are missing. To overcome these limitations, primary organoid-based human small intestinal tissue models were developed recently but the application of these cultures in pre-clinical research still represents an enormous challenge, as culture setup is complex as well as time- and cost-intensive. To overcome these hurdles, we demonstrate the establishment of primary organoid-derived intestinal cell lines by immortalization. Besides exhibiting cellular diversity of the organoid, these immortalized cell lines enable a standardized and more cost-efficient culture. Further, our cell line-based Transwell®-like models display an organ-specific epithelial barrier integrity, ultrastructural features and representative transport functions. Altogether, our novel model systems are cost-efficient with close similarity to the in vivo situation, therefore favoring their use in bioavailability studies in the context of pre-clinical screenings.

Alterations in GLP-1 and PYY release with aging and body mass in the human gut

The lining of our intestinal surface contains an array of hormone-producing cells that are collectively our bodies’ largest endocrine cell reservoir. These “enteroendocrine” (EE) cells reside amongst the billions of absorptive epithelial and other cell types that line our gastrointestinal tract and can sense and respond to the ever-changing internal environment in our gut. EE cells release an array of important signalling molecules that can act as hormones, including glucagon-like peptide (GLP-1) and peptide YY (PYY) which are co-secreted from L cells. While much is known about the effects of these hormones on metabolism, insulin secretion and food intake, less is understood about their secretion from human intestinal tissue. In this study we assess whether GLP-1 and PYY release differs across human small and large intestinal tissue locations within the gastrointestinal tract, and/or by sex, body weight and the age of an individual. We identify that the release of both hormones is greater in more distal regions of the human colon, but is not different between sexes. We observe a negative correlation of GLP-1 and BMI in the small, but not large, intestine. Increased aging correlates with declining secretion of both GLP-1 and PYY in human large, but not small, intestine. When the data for large intestine is isolated by region, this relationship with age remains significant for GLP-1 in the ascending and descending colon and in the descending colon for PYY. This is the first demonstration that site-specific differences in GLP-1 and PYY release occur in human gut, as do site-specific relationships of L cell secretion with aging and body mass.

Microbial metabolite mitigates arsenic induced oxidative stress, inflammation, and barrier dysfunction in gut epithelia

The intestinal barrier acts as a physical and immunological defense against luminal microorganisms, viruses, food antigens, and environmental factors. Intestinal inflammation and the pathogenesis of inflammatory bowel diseases (IBD) are closely associated with loss of gut barrier integrity and increased immunological responses. Environmental heavy metal toxins like arsenic (iAs3+) effectively regulates several cellular processes involved in cellular proliferation and homeostasis. Recently, it was shown that gut microbiota is required for full protection against acute arsenic toxicity using mouse models. In current study, we investigated the potential role of microbial metabolites such as urolithins to provide protection against arsenic‐induced gut barrier damage and inflammation. Urolithin A (UroA), a polyphenol derived microbial metabolite has been reported to provide protection against several diseases caused by inflammation and oxidative stress. Previously, our group demonstrated that microbial metabolite reversed the colitis induced defects in epithelial barrier function. In this study, we for the first time reveled the potential of UroA to mitigate arsenite induced toxicity and loss of gut barrier integrity. UroA showed a time‐ and concentration‐ dependent protection against iAs3+‐induced apoptosis and oxidative stress. Moreover, UroA treatment protected against iAs3+‐induced gut epithelial tight junction protein disruption/down regulation. Importantly, treatment with microbial metabolite attenuated arsenic induced barrier dysfunction in pre‐clinical human 3D small intestinal organoid tissue model. Taken together, our study indicates that beneficial microbial metabolites such as UroA intervention is a putative preventive/therapeutic approach against environmental iAs3+ related adverse activities on gut barrier.

A Literature-Derived Knowledge Graph Augments the Interpretation of Single Cell RNA-seq Datasets.

Technology to generate single cell RNA-sequencing (scRNA-seq) datasets and tools to annotate them have advanced rapidly in the past several years. Such tools generally rely on existing transcriptomic datasets or curated databases of cell type defining genes, while the application of scalable natural language processing (NLP) methods to enhance analysis workflows has not been adequately explored. Here we deployed an NLP framework to objectively quantify associations between a comprehensive set of over 20,000 human protein-coding genes and over 500 cell type terms across over 26 million biomedical documents. The resultant gene-cell type associations (GCAs) are significantly stronger between a curated set of matched cell type-marker pairs than the complementary set of mismatched pairs (Mann Whitney p = 6.15 × 10−76, r = 0.24; cohen’s D = 2.6). Building on this, we developed an augmented annotation algorithm (single cell Annotation via Literature Encoding, or scALE) that leverages GCAs to categorize cell clusters identified in scRNA-seq datasets, and we tested its ability to predict the cellular identity of 133 clusters from nine datasets of human breast, colon, heart, joint, ovary, prostate, skin, and small intestine tissues. With the optimized settings, the true cellular identity matched the top prediction in 59% of tested clusters and was present among the top five predictions for 91% of clusters. scALE slightly outperformed an existing method for reference data driven automated cluster annotation, and we demonstrate that integration of scALE can meaningfully improve the annotations derived from such methods. Further, contextualization of differential expression analyses with these GCAs highlights poorly characterized markers of well-studied cell types, such as CLIC6 and DNASE1L3 in retinal pigment epithelial cells and endothelial cells, respectively. Taken together, this study illustrates for the first time how the systematic application of a literature-derived knowledge graph can expedite and enhance the annotation and interpretation of scRNA-seq data.

New classification of CD4+CD8a+ double positive cells in Crohn’s disease patients

Abstract The gastrointestinal tract absorbs nutrition while providing a defense response to foreign antigens including enteric bacteria or food. Once the antigen enter the intestine, inflammatory mononuclear cells are accumulated quickly, however some tissue resident T cells, such as regulatory T cells (Tregs) block the excessive tissue damage. We previously demonstrated that mouse CD4+CD8a+ double expressing (DP)cells and Tregs complementary play an immuno-suppressive role in IE and LP, and a part of IE DP cells is originally developed from Tregs in LP. However, DP cells are not well characterized in human especially in inflammatory bowel disease (IBD) patients. We obtained human small intestinal tissue from 9 colon cancer patients as normal tissue, and 5 Crohn’s disease(CD) patients, dividing into inflamed and non-inflamed lesion, and analysed by Flow cytometry. We confirmed DP cells are located in both IE and LP in human. First, we demonstrated the ratio of DP cells in normal tissue was as same as in non-inflamed lesion of CD patients. As we expected, the ratio of DP cells in inflamed tissue of CD patients was significantly decreased compared to normal tissue and non-inflamed lesion of CD. Then, we analysed the surface marker of Human DP cells. Surface staining of CD27 and CD45RA showed distinct difference between DP cells and the other CD4 T cells. DP cells expressed more memory T cell marker (CD27−CD45RA−) and less effector T cell marker (CD27+CD45RA−) than the other CD4 subset. Futher more, the more fraction of IE DP cells expressed CD103 than LP DP cells. Conclusively, these data indicated that human DP cell reside both in IE and LP and that DP cells are not a single characterized population, and the DP cells were decreased in inflamed lesion in CD.

Impact of Interleukin-6 on Drug-Metabolizing Enzymes and Transporters in Intestinal Cells.

Inflammatory response is characterized by an increase of several cytokines. Some are known to modify drugs pharmacokinetic by reducing the expression levels of drug-metabolizing enzymes (DMEs) and transporters. This impact of inflammatory signaling is well established in hepatic cells, but not in intestinal cells. EpiIntestinal is a 3D human small intestinal tissue model with epithelial polarity, allowing good evaluation of metabolism and drug transport. This study aimed to analyze the effect of IL-6 on this tissue model. RNA sequencing was performed in cells incubated with 5, 10, or 20 ng/mL IL-6 for 8 h to 72 h to study the impact of IL-6 on drug metabolism and pharmacokinetics gene expression. The influence of IL-6 on the activity of cytochromes P450 (CYPs) was studied by measuring metabolite formation of specific substrates with LC-HRMS. Its impact on ATP-binding cassette (ABC) transport was evaluated by measuring intra- and extracellular substrates using spectrofluorometry. Exposure of EpiIntestinal cells to IL-6 resulted in reduction of some CYP mRNAs, such as CYP2C19, CYP2C9, and CYP3A4, by 40% to 50%. Activities of these CYPs were also decreased in EpiIntestinal cells by 20% to > 75%. IL-6 exposure did not modify ABCB1 and ABCCs transporter activities in this model. This study shows that gene expression levels and activities of drug-metabolizing enzymes and ABC transporters may be altered by the pro-inflammatory cytokine IL-6 in intestinal cells. If these results are confirmed in vivo, it may result in pharmacokinetic modifications, such as pre-systemic metabolism, with clinical effects, and require dosage adaptation.

Aberrant Protein Phosphorylation in Cancer by Using Raman Biomarkers.

(1) Background: Novel methods are required for analysing post-translational modifications of protein phosphorylation by visualizing biochemical landscapes of proteins in human normal and cancerous tissues and cells. (2) Methods: A label-free Raman method is presented for detecting spectral changes that arise in proteins due to phosphorylation in the tissue of human breasts, small intestines, and brain tumours, as well as in the normal human astrocytes and primary glioblastoma U-87 MG cell lines. Raman spectroscopy and Raman imaging are effective tools for monitoring and analysing the vibrations of functional groups involved in aberrant phosphorylation in cancer without any phosphorecognition of tag molecules. (3) Results: Our results based on 35 fresh human cancer and normal tissues prove that the aberrant tyrosine phosphorylation monitored by the unique spectral signatures of Raman vibrations is a universal characteristic in the metabolic regulation in different types of cancers. Overexpressed tyrosine phosphorylation in the human breast, small intestine and brain tissues and in the human primary glioblastoma U-87 MG cell line was monitored by using Raman biomarkers. (4) We showed that the bands at 1586 cm−1 and 829 cm−1, corresponding to phosphorylated tyrosine, play a pivotal role as a Raman biomarker of the phosphorylation status in aggressive cancers. We found that the best Raman biomarker of phosphorylation is the 1586/829 ratio showing the statistical significance at p Values of ≤ 0.05. (5) Conclusions: Raman spectroscopy and imaging have the potential to be used as screening functional assays to detect phosphorylated target proteins and will help researchers to understand the role of phosphorylation in cellular processes and cancer progression. The abnormal and excessive high level of tyrosine phosphorylation in cancer samples compared with normal samples was found in the cancerous human tissue of breasts, small intestines and brain tumours, as well as in the mitochondria and lipid droplets of the glioblastoma U-87 MG cell line. Detailed insights are presented into the intracellular oncogenic metabolic pathways mediated by phosphorylated tyrosine.

FcRn Expression in Wildtype Mice, Transgenic Mice, and in Human Tissues.

Quantitative real-time PCR and Western blot methods were developed to assess neonatal Fc-receptor (FcRn) mRNA and protein expression in human FcRn transgenic mice, Swiss Webster mice, and in select human tissues. Additionally, FcRn turnover was evaluated via pulse-chase. FcRn mRNA expression was significantly higher in transgenic mice when compared to mouse FcRn mRNA in Swiss Webster mice and it ranged from 184-fold higher in the kidney to 109,000-fold higher in the skin. FcRn protein expression was found to be 13-fold lower in kidney to 5.6-fold higher in lung obtained from transgenic mice compared to FcRn protein expression in lung samples obtained from Swiss Webster mice. FcRn protein expression in human liver and small intestine tissues matched more closely with FcRn expression in Swiss Webster mice but were significantly lower when compared to values found from Swiss Webster and transgenic mice. Although FcRn mRNA expression correlated significantly with protein expression (p < 0.0005), the correlation coefficient was only 0.113. As such, the measurement of FcRn protein may be preferred to FcRn mRNA for quantitative applications. Significant differences were found in FcRn expression in transgenic mice, Swiss Webster mice, and human tissues, which may have implications for the use of mouse models in the assessment of monoclonal antibody disposition, efficacy, and safety.

Human Norovirus Replication in Human Intestinal Enteroids as Model to Evaluate Virus Inactivation.

Human noroviruses are a leading cause of epidemic and endemic acute gastroenteritis worldwide and a leading cause of foodborne illness in the United States. Recently, human intestinal enteroids (HIEs) derived from human small intestinal tissue have been shown to support human norovirus replication. We implemented the HIE system in our laboratory and tested the effect of chlorine and alcohols on human norovirus infectivity. Successful replication was observed for 6 norovirus GII genotypes and was dependent on viral load and genotype of the inoculum. GII.4 viruses had higher replication levels than other genotypes. Regardless of concentration or exposure time, alcohols slightly reduced, but did not completely inactivate, human norovirus. In contrast, complete inactivation of the 3 GII.4 viruses occurred at concentrations as low as 50 ppm of chlorine. Taken together, our data confirm the successful replication of human noroviruses in HIEs and their utility as tools to study norovirus inactivation strategies.

Pre-validation of human small intestinal tissue model system to screen drug induced gastrointestinal toxicity and wound healing

Pre-validation of human small intestinal tissue model system to screen drug induced gastrointestinal toxicity and wound healing

Development of an Advanced Primary Human In Vitro Model of the Small Intestine.

Intestinal in vitro models are valuable tools in drug discovery and infection research. Despite several advantages, the standard cell line-based Transwell(®) models based for example on colonic epithelial Caco-2 cells, lack the cellular complexity and transport activity associated with native small intestinal tissue. An additional experimental set-back arises from the most commonly used synthetic membranes, on which the cells are routinely cultured. These can lead to an additional barrier activity during in vitro testing. To overcome these limitations, we developed an alternative primary human small intestinal tissue model. This novel approach combines previously established gut organoid technology with a natural extracellular matrix (ECM) based on porcine small intestinal scaffold (SIS). Intestinal crypts from healthy human small intestine were expanded as gut organoids and seeded as single cells on SIS in a standardized Transwell-like setting. After only 7 days on the ECM scaffold, the primary cells formed an epithelial barrier while a subpopulation differentiated into intestinal specific cell types such as mucus-producing goblet cells or hormone-secreting enteroendocrine cells. Furthermore, we tested the influence of subepithelial fibroblasts and dynamic culture conditions on epithelial barrier function. The barrier integrity was stabilized by coculture in the presence of gut-derived fibroblasts. Compared to static or dynamic culture on an orbital shaker, dynamic culture in a defined perfusion bioreactor had an additional significant impact on epithelial cell differentiation, indicated by high prismatic cell morphology and upregulation of CYP3A4 enzyme and Mdr1 transporter activity. In summary, more physiological tissue models as presented in our study might be useful tools in preclinical research and development.

Organotypic in vitro human small intestinal tissue models to assess drug toxicity and permeation

Organotypic in vitro human small intestinal tissue models to assess drug toxicity and permeation

Novel 3-D human small intestinal tissue model to assess drug permeation, inflammation, and wound healing

Novel 3-D human small intestinal tissue model to assess drug permeation, inflammation, and wound healing

Developmental trajectory of intestinal MDR1/ABCB1 mRNA expression in children

Developmental trajectory of intestinal <scp>MDR1/ABCB1</scp> mRNA expression in children

Cross-sectional and longitudinal associations between parents’ and preschoolers’ physical activity and TV viewing

colonic tissue mRNA libraries and validated an in vitro human enteroendocrine I-cell model (Hutu80) using a variety of known TAS2R bitter agonists. We show that mRNAs for bitter taste receptors are expressed in human gastric, small intestinal and colonic tissue as well as the Hutu-80 cellline. In addition, Hutu-80 cells respond to a subset of known bitter agonists via a G-protein coupled phospholipase C mediated pathway, elevated cytoplasmic calcium concentrations and subsequent release of CCK. In conclusion, the capability to sense luminal bitter compounds may occur throughout the GI tract. However, not all known lingual bitter agonists stimulate hormone secretion in Hutu-80 cells, suggesting key differences between lingual and GI bitter sensing that may have important implications for design of functional foods to modulate appetite through bitter sensing.

Human small intestinal and colonic tissue mounted in the Ussing chamber as a tool for characterizing the intestinal absorption of drugs

The purpose of this study was to validate human small intestinal and colonic tissue mounted in the Ussing chamber as a tool for predicting the oral drug absorption in humans with the main focus on moderately and poorly permeable compounds. The obtained apparent permeability coefficient (Papp) of eleven test compounds was compared to their fraction absorbed (Fa) in humans taken from the literature. Beside the conventional Papp a new parameter, the apparent permeability coefficient total (Papp,total), involving both the apical-to-basolateral permeability and the time-dependent compound accumulation in the tissue was established. The permeability of lucifer yellow (LY), a fluorescent marker of the paracellular pathway and the test compounds showed no obvious differences between small intestine and colon. Furthermore, small intestinal and colonic tissue from a single donor showed similar permeability of both LY and a transcellularly transported compound metoprolol. All test compounds including low molecular weight hydrophilic compounds such as metformin, atenolol, sulpiride and famotidine showed adequate permeability reflecting human Fa values (R2=0.87). The Papp values of digoxin, a P-glycoprotein (P-gp) substrate, were not significantly affected by the addition of verapamil, a P-gp inhibitor. In contrast, the Papp,total values of digoxin increased approximately threefold in the presence of verapamil. In conclusion, both small intestinal and colonic tissue mounted in the Ussing chamber provide a good opportunity to predict the oral drug absorption rate in humans even for moderately and poorly absorbed compounds. The novel calculation of Papp,total allows the study of the carrier-mediated drug–drug interactions in human intestine.

Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium

We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo.

Campylobacter jejuni 81-176 forms distinct microcolonies on in vitro-infected human small intestinal tissue prior to biofilm formation

Human small and large intestinal tissue was used to study the interaction of Campylobacter jejuni with its target tissue. The strain used for the study was 81-176 (+pVir). Tissue was processed for scanning and transmission electron microscopy, and by immunohistochemistry for light microscopy. Organisms adhered to the apical surface of ileal tissues at all time points in large numbers, in areas where mucus was present and in distinct groups. Microcolony formation was evident at 1-2&emsp14;h, with bacteria adhering to mucus on the tissue surface and to each other by flagellar interaction. At later time points (3-4&emsp14;h), biofilm formation on ileal tissue was evident. Flagellar mutants did not form microcolonies or biofilms in tissue. Few organisms were observed in colonic tissue, with organisms present but not as abundant as in the ileal tissue. This study shows that C. jejuni 81-176 can form microcolonies and biofilms on human intestinal tissue and that this may be an essential step in its ability to cause diarrhoea in man.

Expansion and Differentiation of Neural Progenitors Derived From the Human Adult Enteric Nervous System

Neural stem and progenitor cells from the enteric nervous system have been proposed for use in cell-based therapies against specific neurogastrointestinal disorders. Recently, enteric neural progenitors were generated from human neonatal and early postnatal (until 5 years after birth) gastrointestinal tract tissues. We investigated the proliferation and differentiation of enteric nervous system progenitors isolated from human adult gastrointestinal tract. Human enteric spheroids were generated from adult small and large intestine tissues and then expanded and differentiated, depending on the applied cell culture conditions. For implantation studies, spheres were grafted into fetal slice cultures and embryonic aganglionic hindgut explants from mice. Differentiating enteric neural progenitors were characterized by 5-bromo-2-deoxyuridine labeling, in situ hybridization, immunocytochemistry, quantitative real-time polymerase chain reaction, and electrophysiological studies. The yield of human neurosphere-like bodies was increased by culture in conditional medium derived from fetal mouse enteric progenitors. We were able to generate proliferating enterospheres from adult human small or large intestine tissues; these enterospheres could be subcultured and maintained for several weeks in vitro. Spheroid-derived cells could be differentiated into a variety of neuronal subtypes and glial cells with characteristics of the enteric nervous system. Experiments involving implantation into organotypic intestinal cultures showed the differentiation capacity of neural progenitors in a 3-dimensional environment. It is feasible to isolate and expand enteric progenitor cells from human adult tissue. These findings offer new strategies for enteric stem cell research and future cell-based therapies.

Improved visualization of GFP‐labeled HIV‐1 virions in human mucosa using confocal microscopy and image analysis

Mucosal tissues play a key role in the immunopathogenesis of HIV-1 infection. The goal of this study was to improve identification of GFP-labeled HIV-1 virions in our mucosal explant model. Fresh explanted human small intestinal tissue was inoculated in vitro by GFP-labeled HIV-1 virions for 1 hr, then frozen, fixed, blocked and stained with fluorescent antibodies against GFP and either mAb to dendritic cells (CD83) or macrophages (HAM56) followed by Cy3. Confocal images were analyzed by image analysis software IPLab. Results: (1) Virions labeled with anti-GFP antibody conjugated with far-red fluorochrome (emission at 647 nm) were easily identified using confocal microscopy. Resolution was enhanced further by yellow colorization of virions. Controls showed no far-red staining. (2) Taking images in multiple optical sections increased visualization of rare virions within cells. (3) Colocalization of virions within CD83- and HAM56-positive cells was confirmed by overlapping line intensity histograms using image analysis software. Conclusion: The described technique, widely applicable to small GFP-labeled particles, significantly improves visualization of HIV virions and their colocalization within specific cell types in intestinal mucosa. Supported by NIH grants AI 67854 and DK74033.