Hyperglycemia minimally alters primary self-renewing human colonic epithelial cells while TNFα-promotes severe intestinal epithelial dysfunction.

Hyperglycemia is thought to increase production of inflammatory cytokines and permeability of the large intestine. Resulting intestinal inflammation is then often characterized by excess secretion of tumor necrosis factor alpha (TNFα). Thus, hyperglycemia in hospitalized patients suffering from severe trauma or disease is frequently accompanied by TNFα secretion, and the combined impact of these insults on the intestinal epithelium is poorly understood. This study utilized a simple yet elegant model of the intestinal epithelium, comprised of primary human intestinal stem cells and their differentiated progeny, to investigate the impact of hyperglycemia and inflammatory factors on the colonic epithelium. When compared to epithelium cultured under conditions of physiologic glucose, cells under hyperglycemic conditions displayed decreased mucin-2 (MUC2), as well as diminished alkaline phosphatase (ALP) activity. Conditions of 60 mM glucose potentiated secretion of the cytokine IL-8 suggesting that cytokine secretion during hyperglycemia may be a source of tissue inflammation. TNFα measurably increased secretion of IL-8 and IL-1ß, which was enhanced at 60 mM glucose. Surprisingly, intestinal permeability and paracellular transport were not altered by even extreme levels of hyperglycemia. The presence of TNFα increased MUC2 presence, decreased ALP activity, and negatively impacted monolayer barrier function. When TNFα hyperglycemia and ≤30 mM glucose and were combined, MUC2 and ALP activity remained similar to that of TNFα alone, although synergistic effects were seen at 60 mM glucose. An automated image analysis pipeline was developed to assay changes in properties of the zonula occludens-1 (ZO-1)-demarcated cell boundaries. While hyperglycemia alone had little impact on cell shape and size, cell morphologic properties were extraordinarily sensitive to soluble TNFα. These results suggest that TNFα acted as the dominant modulator of the epithelium relative to glucose, and that control of inflammation rather than glucose may be key to maintaining intestinal homeostasis.

Primary Cell-Derived Intestinal Models: Recapitulating Physiology.

The development of physiologically relevant intestinal models fueled by breakthroughs in primary cell-culture methods has enabled successful recapitulation of key features of intestinal physiology. These advances, paired with engineering methods, for example incorporating chemical gradients or physical forces across the tissues, have yielded ever more sophisticated systems that enhance our understanding of the impact of the host microbiome on human physiology as well as on the genesis of intestinal diseases such as inflammatory bowel disease and colon cancer. In this review we highlight recent advances in the development and usage of primary cell-derived intestinal models incorporating monolayers, organoids, microengineered platforms, and macrostructured systems, and discuss the expected directions of the field.

Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform.

In vitro models of the human intestinal epithelium derived from primary stem cells are much needed for the study of intestinal immunology in health and disease. Here, we describe an intestinal monolayer cultured on a porous membrane with accessible basal and apical surfaces for assay of intestinal cytokine production in response to stimuli. The system was composed of a differentiated, confluent epithelial monolayer derived from human primary stem cells obtained from small or large intestine. Interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) were the most abundant inflammatory cytokines produced by the intestinal epithelium. The epithelium from all five tested regions of the intestine preferentially secreted into the apical reservoir of the monolayer, with a 26-fold greater concentration of IL-8 present in the apical reservoir of the colonic monolayer relative to that in the basal reservoir. Upon application of tumor-necrosis factor α (TNF-α) to the basal surface of the colonic monolayer, the IL-8 concentration significantly increased in the basal, but not the apical, reservoir. A dose-dependent elevation of IL-8 in the basal reservoir was observed for TNF-α-stimulation of the monolayer but not for an organoid-based platform. To demonstrate the utility of the monolayer system, 88 types of dietary metabolites or compounds were screened for their ability to modulate IL-8 production in the basal reservoir of the intestinal monolayer in the absence or presence of TNF-α. No dietary metabolite or compound caused an increase in IL-8 in the basal reservoir in the absence of TNF-α. After addition of TNF-α to the monolayer, two compounds (butyrate and gallic acid) suppressed IL-8 production, suggesting their potential anti-inflammatory effects, whereas the dietary factor forskolin significantly increased IL-8 production. These results demonstrate that the described human-intestinal-monolayer platform has the potential for assays and screening of metabolites and compounds that alter the inflammatory response of the intestine.

Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells.

BACKGROUND & AIMS: Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished. METHODS: The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified. RESULTS: The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells. CONCLUSIONS: This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies.

In Vitro Generation of Mouse Colon Crypts.

Organoid culture has had a significant impact on in vitro studies of the intestinal epithelium; however, the exquisite architecture, luminal accessibility, and lineage compartmentalization found in vivo has not been recapitulated in the organoid systems. We have used a microengineered platform with suitable extracellular matrix contacts and stiffness to generate a self-renewing mouse colonic epithelium that replicates key architectural and physiological functions found in vivo, including a surface lined with polarized crypts. Chemical gradients applied to the basal-luminal axis compartmentalized the stem/progenitor cells and promoted appropriate lineage differentiation along the in vitro crypt axis so that the tissue possessed a crypt stem cell niche as well as a layer of differentiated cells covering the luminal surface. This new approach combining microengineered scaffolds, native chemical gradients, and biophysical cues to control primary epithelium ex vivo can serve as a highly functional and physiologically relevant in vitro tissue model.

Trans-3,4-dideoxyglucone-3-ene (trans-3,4-DGE), a most reactive glucose degradation product in freshly heat sterilized glucose solutions.

In our study, one or more glucose degradation products (GDPs) in freshly heat sterilized dextrose 5% in water (D5W) were found to react with a drug candidate having a ß-keto amide group (Compound A) to form several drug related compounds with the same molecular weight. However the previously identified GDPs did not react with Compound A to produce the observed adducts, indicating that unidentified GDP(s) reacted with Compound A to form these adducts. Our investigation by reaction-directed fractionation of the reactive D5W with HPLC led to the identification of the reactive GDP, trans-3,4-dideoxyglucosone-3-ene (trans-3,4-DGE), responsible for producing these reaction products. The trans-3,4-DGE was identified from its derivatives of dinitrophenylhydrazine (DNPH) and acetoacetanilide and confirmed by (1) admixing Compound A with authentic trans-3,4-DGE to produce the identical impurities as admixing with freshly heat sterilized D5W, and (2) NMR analysis of the reactive fraction of glucose solutions.