Gastroenterology

Gastroenterology

Volume 150, Issue 3, March 2016, Pages 638-649.e8
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology

https://doi.org/10.1053/j.gastro.2015.11.047Get rights and content

Background & Aims

Human intestinal crypt-derived enteroids are a model of intestinal ion transport that require validation by comparison with cell culture and animal models. We used human small intestinal enteroids to study neutral Na+ absorption and stimulated fluid and anion secretion under basal and regulated conditions in undifferentiated and differentiated cultures to show their functional relevance to ion transport physiology and pathophysiology.

Methods

Human intestinal tissue specimens were obtained from an endoscopic biopsy or surgical resections performed at Johns Hopkins Hospital. Crypts were isolated, enteroids were propagated in culture, induced to undergo differentiation, and transduced with lentiviral vectors. Crypt markers, surface cell enzymes, and membrane ion transporters were characterized using quantitative reverse-transcription polymerase chain reaction, immunoblot, or immunofluorescence analyses. We used multiphoton and time-lapse confocal microscopy to monitor intracellular pH and luminal dilatation in enteroids under basal and regulated conditions.

Results

Enteroids differentiated upon withdrawal of WNT3A, yielding decreased crypt markers and increased villus-like characteristics. Na+/H+ exchanger 3 activity was similar in undifferentiated and differentiated enteroids, and was affected by known inhibitors, second messengers, and bacterial enterotoxins. Forskolin-induced swelling was completely dependent on cystic fibrosis transmembrane conductance regulator and partially dependent on Na+/H+ exchanger 3 and Na+/K+/2Cl- cotransporter 1 inhibition in undifferentiated and differentiated enteroids. Increases in cyclic adenosine monophosphate with forskolin caused enteroid intracellular acidification in HCO3--free buffer. Cyclic adenosine monophosphate–induced enteroid intracellular pH acidification as part of duodenal HCO3- secretion appears to require cystic fibrosis transmembrane conductance regulator and electrogenic Na+/HCO3- cotransporter 1.

Conclusions

Undifferentiated or crypt-like, and differentiated or villus-like, human enteroids represent distinct points along the crypt–villus axis; they can be used to characterize electrolyte transport processes along the vertical axis of the small intestine. The duodenal enteroid model showed that electrogenic Na+/HCO3- cotransporter 1 might be a target in the intestinal mucosa for treatment of secretory diarrheas.

Section snippets

Human Tissues

Tissues from human subjects were obtained under approval of the Johns Hopkins University School of Medicine Institutional Review Board (protocol NA_00038329). De-identified specimens determined to come from normal intestines were obtained from endoscopic biopsy or surgical resections performed at Johns Hopkins Hospital. Crypt isolation, enteroid propagation, differentiation, immunodetection, lentiviral transduction, and messenger RNA (mRNA) analysis are described in the Supplementary Materials

Enteroid Differentiation Allows Separation of Crypt and Villus Ion Transporter Profiles

We implemented the primary cell culture model based on 3-dimensional propagation of human intestinal crypts2, 12 to compile a bank of enteroids derived from histologically normal duodenal, jejunal, and ileal endoscopic biopsy or surgical specimens. The enteroid model allows study of the undifferentiated and differentiated state separately, starting with the same passage of enteroids from the same subject. Induction of a differentiated phenotype in our enteroid cultures was documented by

Discussion

We describe a functional ion transport study in the human small intestinal enteroid model. We provide novel information concerning electrolyte transporter expression in enteroids and a detailed description of upper small intestinal response to transporter regulation under physiologic and selected pathophysiologic conditions. The strengths and limitations of the enteroid model are discussed in comparison with established models of intestinal ion transport.

Several features make enteroids a

Acknowledgments

The authors acknowledge the Kudsi Imaging Facility of the Conte Digestive Disease Basic and Translational Research Core Center at Johns Hopkins University and the assistance of John Gibas. The authors thank James Potter, Matthew Marcetich, Dr Marc Halushka, and Dr Steven Brant (Hopkins) for their assistance in acquiring patient specimens from which duodenal, jejunal, and ileal enteroid lines were established at Hopkins, and Xi-Lei Zeng (Baylor) for providing two additional jejunal enteroid

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by National Institutes of Health grants U18-TR000552, UH3-TR000503, UH3-TR000504, T32-DK007632, K01-DK088950, R01-DK026523, R01-DK061765, P01-DK072084, P30-DK089502, and P30-DK056338.

    Author names in bold designate shared co-first authorship.

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