1. Volume 150, Issue 3, Pages 638-649.e8 (March 2016)
Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology 
Jennifer Foulke-Abel, Julie In, Jianyi Yin, Nicholas C. Zachos, Olga Kovbasnjuk, Mary K. Estes, Hugo de Jonge, Mark Donowitz 
Gastroenterology 
Volume 150, Issue 3, Pages e8 (March 2016)
DOI: /j.gastro
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2. Figure 1
Analysis of differentiation markers in human duodenal enteroids. (A) Transmission electron miscroscope images of the undifferentiated and differentiated enteroid brush border. Scale bar: 1 μm. (B) 5-Ethynyl-2′-deoxyuridine incorporation in whole-mount enteroids during each day of the differentiation time course. ND, undifferentiated; DF1, differentiation day 1, and so forth. Scale bar: 100 μm. Note undetectable 5-ethynyl-2′-deoxyuridine incorporation at DF4 and DF5. (C) Luminal Muc2-positive secretions detected by immunostaining with Muc2 antibody in whole-mount enteroids. Muc2 is present only in differentiated enteroids. Scale bar: 50 μm. (D) mRNA fold change analysis comparing markers of crypt base columnar cells (leucine-rich repeat-containing G-protein–coupled receptor 5 [LGR5], achaete-scute complex homolog 2 [ASCL2], and olfactomedin 4 [OLFM4]), proliferative potential (Ki-67), and lineage commitment (sucrase-isomaltase [SI], trefoil factor 3 [TFF3]) in undifferentiated and differentiated enteroids. Results are means ± SEM. *P < .05, **P < .01, ***P < .001; n = 5 different enteroid lines.
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3. Figure 2
Analysis of major ion transporters in human duodenal enteroids. (A) Representative immunoblots of NHE3, DRA, CFTR, NKCC1, and NBCe1 in undifferentiated (ND) and differentiated (DF) duodenal enteroids. The change in transporter expression after differentiation is normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Graph results are means ± SEM; **P < .01, ***P < .001; n = 4 different enteroid lines. (B–E) Immunofluorescence of NHE3, DRA, CFTR, and NKCC1 in intact human duodenal tissue and undifferentiated or differentiated duodenal enteroids. Similar results were obtained in 3 different enteroid lines. Scale bars: 20 μm.
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4. Figure 3
Na+/H+ exchange in human enteroids. A single donor was used for all experiments. (A) A typical NHE3 activity assay in enteroids as a function of Na+-dependent pHi recovery. (B) NHE3 activity in undifferentiated and differentiated duodenal enteroids. n = 3 enteroids, each taken from a separate passage. (C) Representative traces show the effect of NHE family inhibitors in differentiated duodenal enteroids. (D) Average rate of realkalinization with inhibitors in panel C. Results are means ± SEM; **P < .01; n = 3 enteroids, each taken from a separate passage. (E) Representative immunoblot of NHE3 protein expression in scrambled short hairpin RNA control (Scr), wild-type (WT), and knock-down (KD) confirmed more than 90% knock-down of expressed NHE3 using this approach, and the knock-down persisted for more than 10 passages. n = 3 blots, each taken from a separate passage. (F) NHE3 activity in wild-type (WT) and NHE3 knock-down (KD) differentiated duodenal enteroids. Results are means ± SEM. **P < .01. n = 3 enteroids, each taken from a separate passage. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TMA, tetramethylammonium chloride.
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5. Figure 4
Microbial enterotoxins, cAMP, and cGMP inhibit NHE3 in differentiated enteroids. (A) Effect of cGMP increase by 8-pCPT-cGMP or cAMP increase via 8-Br-cAMP or forskolin in duodenal enteroids. Results are means ± SEM.**P < .01. n = 3 different enteroid lines. (B) Effect of heat-stable enterotoxin A (STa) or cholera toxin (CTX) in jejunal enteroids. Results are means ± SEM. **P < .01. n = 3 different enteroid lines. Fsk, forskolin.
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6. Figure 5
Ion transporters contribute to forskolin-induced apical fluid secretion. (A) Representative FIS time course in undifferentiated or differentiated duodenal enteroids. Results are means ± SEM, n ≥ 10 enteroids for each condition. (B) Contribution to the FIS rate by inhibition of each transporter calculated relative to the inhibitor-free forskolin-stimulated condition. Results are means ± SEM. **P < .01. n = 3 different enteroid lines.
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7. Figure 6
Role of HCO3- and ion transporters in forskolin-induced pHi changes. (A–C) pHi changes in differentiated duodenal enteroids after forskolin treatment in HEPES buffer with (A) CFTRinh-172, H-89, acetazolamide (ATZ), or (B) EIPA, or in (C) HCO3- buffer with DIDS, S0859, ATZ, CFTRinh-172, or bumetanide. Representative traces from individual experiments are shown. (D) Average rate of ΔpHi under conditions in panels A and C. n = 3 different enteroid lines for each condition. (E) Model of stimulated duodenal HCO3- transport based on studies in Figure 6 A-C. HCO3- secretion requires NBCe1 to replace HCO3- lost via CFTR. CA is not essential to maintain intracellular HCO3-, and limiting Cl- uptake by NKCC1 inhibition does not affect the ability of CFTR to conduct HCO3-. Fsk, forskolin.
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8. Supplementary Figure 1
(A) Ki-67 incorporation in human duodenal enteroids indicates an overall decrease in proliferative potential because differentiation occurs over 5 days (DF1, differentiation day 1, and so forth). In undifferentiated (ND) human enteroids, proliferation is distributed evenly among buds and the main body of the enteroid. Images are representative of 3 separate experiments. Scale bars: 100 μm. (B) Alkaline phosphatase (Alk Phos) and sucrase-isomaltase (Suc-Iso) immunofluorescence in human duodenal tissues and in undifferentiated and differentiated duodenal enteroids. Images show that alkaline phosphatase and sucrase-isomaltase are absent in undifferentiated enteroids and that differentiation conditions yield alkaline phosphatase– and sucrase-isomaltase–containing villus-like cells in human enteroids. Scale bars: 50 μm.
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9. Supplementary Figure 2
(A) Immunofluorescence shows that glyceraldehyde-3-phosphate dehydrogenase expression is similar between undifferentiated and differentiated enteroids. Control IgG images to show secondary antibody specificity were obtained using identical microscope settings. Scale bars: 20 μm. (B) Immunofluorescence images showing secondary antibody specificity for each transporter studied in Figure 2. Samples were incubated with the secondary antibody indicated and do not include primary antibody. Image acquisition settings were identical to those used to obtain the experimental images in Figure 2. Scale bars: 50 μm.
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10. Supplementary Figure 3
(A) Pseudocolor multiphoton micrograph illustrates the typical selection of regions of interest in an enteroid for calculation of 640-/580-nm ratio to track pHi at each time point. (B) NHE3 activity in spheroid or budding differentiated duodenal enteroids. Results are means ± SEM, n = 6 enteroids per condition, each taken from a different passage of a single subject. The micrographs show examples of the 2 different enteroid morphologies that can exist in both undifferentiated and differentiated enteroid cultures. Left: Spheroid; right: budding structure. Scale bars: 50 μm. (C) NHE3 activity across 11 passages in a differentiated duodenal enteroid culture derived from a single subject. Results are means ± SEM, n = 3 enteroids per condition. (D) Basal NHE3 activity in differentiated enteroids made from duodenal, jejunal, and ileal segments of the human small intestine. Labels 1D, 1J, 11I, and so forth denote randomly selected individual subjects. Results are means ± SEM, n = 3 enteroids for each line, each taken from a different passage.
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