Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers

Yin, Jianyi; Tse, Chung Ming; Avula, Leela Rani; Singh, Varsha; Foulke‐Abel, Jennifer; Jonge, Hugo R. de; Donowitz, Mark

doi:10.1016/j.jcmgh.2018.02.002

Cited by 47 publications

(93 citation statements)

References 42 publications

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“…Enteroids were established from crypts containing stem cells isolated from jejunal biopsies from healthy adult subjects using the protocol of Sato et al (21, 22), with minor modifications as previously described (2). Enteroids were maintained in Matrigel (Corning, Tewksbury, MA) in expansion medium composed of advanced Dulbecco’s modified Eagle medium/F12 (Life Technologies, Carlsbad, CA) containing 100 U/mL penicillin/streptomycin (Quality Biological, Gaithersburg, MD), 10 mmol/L HEPES (Life Technologies), and 1X GlutaMAX (Life Technologies), with 50% Wnt3A conditioned medium (produced by L-Wnt3A cell line, ATCC CRL-2647), 15% R-spondin1–conditioned medium (produced by HEK293T cell line stably expressing mouse R-spondin1; kindly provided by Dr Calvin Kuo, Stanford University, Stanford, CA), 10% Noggin conditioned medium (produced by HEK293T cell line stably expressing mouse Noggin), 1X B27 supplement (Life Technologies), 1 mmol/L N-acetylcysteine (Sigma-Aldrich), 50 ng/mL human epidermal growth factor (Life Technologies), 1 μg/mL (Leu-15) gastrin (AnaSpec, Fremont, CA), 500 nmol/L A83-01 (Tocris, Bristol, United Kingdom), 10 μmol/L SB202190 (Sigma-Aldrich), and 100 μg/mL primocin (InvivoGen, San Diego, CA).…”

Section: Methodsmentioning

confidence: 99%

“…The relative fold changes in mRNA levels of MRP4 and MRP5 between static, flow and flow plus stretch were determined using the 2 −ΔΔCT method with human 18S ribosomal RNA as an internal control for normalization (2).…”

Section: Methodsmentioning

confidence: 99%

“…This places limitations on host-pathogen interaction studies. Enteroids grown as monolayers on collagen IV-coated permeable supports overcome the challenges of 3D culture, by permitting direct application of commensal organisms, pathogens, toxins, nutrients, electrolytes and drugs to the apical cell surface and enabling subsequent sampling of exported metabolites from both apical and basolateral surfaces (2, 5–8).…”

Section: Introductionmentioning

confidence: 99%

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Mechanical Stimuli Affect E. Coli Heat Stable Enterotoxin (ST)-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Diarrhea Model

Sunuwar

Yin²,

Kasendra³

et al. 2019

Preprint

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Modeling host-pathogen interactions with human intestinal epithelia using enteroid 20 monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or 21 use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial 22 Introduction: 43Human adult intestinal stem cell-derived enteroids/organoids retain intestinal segment-specific 44 transcriptional as well as phenotypic characteristics, that enable study of untransformed, non-cancer 45 epithelia (1)(2)(3)(4) . Enteroids are generally propagated as 3-dimensional (3D) basement membrane matrix-46 embedded spheroids that are polarized such that the inward facing apical cell surface is not directly 47 accessible. This places limitations on host-pathogen interaction studies. Enteroids grown as monolayers 48 on collagen IV-coated permeable supports overcome the challenges of 3D culture, by permitting direct 49 application of commensal organisms, pathogens, toxins, nutrients, electrolytes and drugs to the apical 50 cell surface and enabling subsequent sampling of exported metabolites from both apical and basolateral 51 surfaces (2, 5-8). 52Static enteroid monolayers do not, however, recreate mechanical forces acting on intestinal epithelia in 53 vivo (4), lacking luminal flow and blood flow (shear stress forces) and the rhythmic contractions that are 54 part of peristalsis. Although multiple efforts have yielded microengineered cell culture systems with flow 55 to recapitulate physical forces of flow and stretch in Organ-on-Chip models (9-12), evidence of discrete 56 advantages associated with inclusion of mechanical forces for modeling host-pathogen interactions 57 within human intestinal epithelia in these systems is not yet clear. In this study, we evaluated the effects 58 of mechanical forces on both baseline characteristics and the responses to heat stable enterotoxin (ST), 59 a virulence factor of enterotoxigenic E. coli (ETEC) infection that is associated with a large burden of 60 diarrheal illness in low-middle income countries (13). The model was a micro-engineered 61 polydimethylsiloxane (PDMS)-based 14, 15). The chip contains two parallel hollow 62 channels separated by a flexible permeable membrane that enables human intestinal epithelial cells to 63 be cultured in the presence of physiologically-scaled fluid shear stress forces and repetitive lateral 64 mechanical deformation (14, 15). 65 5 CA). Enteroids were cultured in a 5% CO2 atmosphere at 37°C and passaged every 7-12 days. Expansion 90 medium was supplemented with 10 μmol/L Y-27632 (Tocris) and 10 μmol/L CHIR99021 (Tocris) during 91 the first 2 days after passaging. Studies were performed with a jeunal enteroid line (passage numbers 92 30-45). 93Intestine-Chip: Organ-Chips were fabricated from PDMS and assembled as described (14, 23, 24) 94 (Emulate Inc.,Boston, MA). The top (1 mm high × 1 mm wide) and bottom (0.2 mm high x 1 mm wide) 95 channels were separated by a thin (50 μm) flexible, PDMS membrane containing 7 μm diam...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Mechanical Stimuli Affect E. Coli Heat Stable Enterotoxin (ST)-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Diarrhea Model

Sunuwar

Yin²,

Kasendra³

et al. 2019

Preprint

Self Cite

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show abstract

“…One of the important limitations of using 3D enteroids to study fluid and electrolyte transport is that the enclosed apical surface fails to allow normal transport of nutrients . Monolayers of enteroids successfully addressed this issue and were used to investigate transepithelial ion transport and the effect of mutations on the function of CFTR, the major intestinal anion channel that is mutated in cystic fibrosis patients . The sodium‐dependent glucose transporter SGLT1/SLC5A1, proton‐coupled peptide transporters PEPT1/SLC15A1, and bile acid receptor TGR5 were found at the brush border locus of the enteroids, and glucose transporter GLUT2 was found on the basolateral side of the enteroids, which were in line with the in vivo scenario, indicating that enteroids are appropriate models to study nutrient transport .…”

Section: Enteroids As Models In Nutrition‐focused Studiesmentioning

confidence: 99%

Enteroids for Nutritional Studies

Yin

Jonge

et al. 2019

Molecular Nutrition Food Res

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Nutritional studies are greatly hampered by a paucity of proper models. Previous studies on nutrition have employed conventional cell lines and animal models to gain a better understanding of the field. These models lack certain correlations with human physiological responses, which impede their applications in this field. Enteroids are cultured from intestinal stem cells and include enterocytes, enteroendocrine cells, goblet cells, Paneth cells, and stem cells, which mimic hallmarks of in vivo epithelium and support long‐term culture without genetic or physiological changes. Enteroids have been used as models to study the effects of diet and nutrients on intestinal growth and development, ion and nutrient transport, secretory and absorption functions, the intestinal barrier, and location‐specific functions of the intestine. In this review, the existing models for nutritional studies are discussed and the importance of enteroids as a new model for nutritional studies is highlighted. Taken together, it is suggested that enteroids can serve as a potential model system to be exploited in nutritional studies.

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“…Intestinal enteroid culture is useful in order to understand developmental epithelial processes of the intestine and to model disease (Yin et al, 2018;Zou et al, 2017). However, because this model is exclusively composed of epithelium (Rookmaaker, Schutgens, Verhaar, & Clevers, 2015), the contributions of adjacent mesenchymal elements, including intestinal subepithelial myofibroblasts, muscle, neurons and glia, to the developing epithelium are not demonstrated.…”

Section: Introductionmentioning

confidence: 99%

Short‐term and long‐term human or mouse organoid units generate tissue‐engineered small intestine without added signalling molecules

Hou

Chang

Trecartin

et al. 2018

Experimental Physiology

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Mouse and human postnatal and fetal organoid units (OUs) maintained in either short-term culture (2 weeks) or long-term culture (from 4 weeks up to 3 months) without adding exogenous growth factors were implanted in immunocompromised mice to form tissue-engineered small intestine (TESI) in vivo. Intestinal epithelial stem and neuronal progenitor cells were maintained in long-term OU cultures from both humans and mice without exogenous growth factors, and these cultures were successfully used to form TESI. This was enhanced with OUs derived from human fetal tissues. Organoid unit culture is different from enteroid culture, which is limited to epithelial cell growth and requires supplementation with R-Spondin, noggin and epidermal growth factor. Organoid units contain multiple cell types, including epithelial, mesenchymal and enteric nervous system cells. Short- and long-term cultured OUs derived from mouse and human intestine develop into TESI in vivo, which contains key components of the small intestine similar to native intestine.

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Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers

Cited by 47 publications

References 42 publications

Mechanical Stimuli Affect E. Coli Heat Stable Enterotoxin (ST)-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Diarrhea Model

Mechanical Stimuli Affect E. Coli Heat Stable Enterotoxin (ST)-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Diarrhea Model

Enteroids for Nutritional Studies

Short‐term and long‐term human or mouse organoid units generate tissue‐engineered small intestine without added signalling molecules

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