Human enteroids as an ex-vivo model of host-pathogen interactions in the gastrointestinal tract

Abstract

Currently, 9 out of 10 experimental drugs fail in clinical studies. This has caused a 40% plunge in the number of drugs approved by the US Food and Drug Administration (FDA) since 2005. It has been suggested that the mechanistic differences between human diseases modeled in animals (mostly rodents) and the pathophysiology of human diseases might be one of the critical factors that contribute to drug failure in clinical trials. Rapid progress in the field of human stem cell technology has allowed the in-vitro recreation of human tissue that should complement and expand upon the limitations of cell and animal models currently used to study human diseases and drug toxicity. Recent success in the identification and isolation of human intestinal epithelial stem cells (Lgr5(+)) from the small intestine and colon has led to culture of functional intestinal epithelial units termed organoids or enteroids. Intestinal enteroids are comprised of all four types of normal epithelial cells and develop a crypt-villus differentiation axis. They demonstrate major intestinal physiologic functions, including Na(+) absorption and Cl(-) secretion. This review discusses the recent progress in establishing human enteroids as a model of infectious diarrheal diseases such as cholera, rotavirus, and enterohemorrhagic Escherichia coli, and use of the enteroids to determine ways to correct the diarrhea-induced ion transport abnormalities via drug therapy.

Keywords: Enteroids; host–pathogen interaction; intestinal pathophysiology; intestinal physiology.

Figure 1

Na⁺/H⁺ transport assays were performed using the cell-permeable pH-sensitive fluorophore SNARF-4 F acetoxymethyl ester and multi-photon laser scanning microscopy. Differentiated human duodenal enteroids were first incubated in the presence of NH₄Cl buffer, then pre-pulsed with Na⁺-free buffer (tetramethylammonium chloride, TMA) to induce intracellular pH acidification, and finally transferred to Na⁺ buffer to follow the rate of alkalinization. Specific inhibition of NHE3 with S3226 (20 μmol/L) dramatically decreased the rate of Na⁺ driven alkalinization (NHE activity) in 3-D differentiated human duodenal enteroids relative to specimens treated with HOE-694 (50 μmol/L) to block activity of other NHE isoforms. Please note the similarity of Na⁺-dependent alkalinization in the presence of HOE-694 plus S3226 and S3226 alone, supporting that nearly all NHE activities are related to NHE3. Graph depicts a representative experiment for each condition; each condition was evaluated in triplicate. (A color version of this figure is available in the online journal.)

Figure 2

(a) Differentiated human duodenal enteroids treated with cholera toxin (0.1 μg/mL, 2 h) have markedly inhibited NHE3 activity, which is similar to observations in human disease. The graph shows a representative experiment, n =3. (b) Exposure of differentiated human duodenal enteroids to rotavirus (1 h) inhibited NHE3 activity by >65%. NHE3 activity was measured in the presence of HOE694 (50 μmol/L) to inhibit all other NHE isoforms (bar graphs are mean ± SEM of three experiments). (c) Rotavirus infects human enteroids. (Left panel) Human jejunal enteroids were metabolically labeled with fluorescent-tagged BODIPY-fatty acid for 1 h prior to mock or rotavirus infection. Enteroids imaged by epifluorescence at 16 h post infection show BODIPY-fatty acid is retained in the endoplasmic reticulum lipid bilayer of mock-infected enteroids. In contrast, rotavirus infection induces lipid droplet formation indicated by the round, red lipid droplets. The inset on the right is a magnification of the boxed area. (Right panel) Electron micrograph of a rotavirus-infected human jejunal enteroid shows that rotavirus infects and replicates in the enteroids. (Inset) Magnification of the boxed area show viroplasms (V), sites of immature particle assembly and genome replication, and rotavirus particles (R). (d) The calcium/calmodulin kinase kinase 2 (CAMKK2) inhibitor STO-609 reduces the yield of rotavirus. Mock- and rotavirus-infected human jejunal enteroids were cultured in the absence or presence of STO-609 (STO). The enteroids were harvested at the indicated times. (Left panel) qRT-PCR was used to quantify the level of rotavirus VP7 gene, normalized to GAPDH, and expressed relative to 0 h post infection. (Right panel) Infectious virus was quantified in the enteroids by fluorescent focus assay. No infectious virus was observed in mock-infected enteroids and no drug toxicity was observed (data not shown). (e) Normal enterocytes in enteroids adopt a columnar shape with a clear brush border and tight junctions (white arrow, control). Enteroids incubated with serine protease EspP, an important virulence factor from EHEC bacteria that may facilitate systemic transepithelial Shiga toxin delivery in human disease, caused actin remodeling of the brush border and basolateral membranes. Yellow arrows indicate actin rearrangement in basolateral membranes; orange arrows denote clumps of microvilli with prominent rootlets and terminal webs; red arrows mark macropinosomes containing internalized horseradish peroxidase (darkened areas) and there are also wide-opened lateral intercellular spaces. (A color version of this figure is available in the online journal.)

Figure 3

(a) Human jejunal enteroids plated on a fibronectin-based peptide-coated Transwell insert formed flat sheets of cells that merged to yield a monolayer with TER ~1000 Ωcm⁻². Immunostaining with apical (villin), basolateral (Na⁺/K⁺-ATPase), and nuclear markers (Hoescht 33342) verified the polarized character of the single cell layer. (b) A similar differentiated monolayer of human duodenal enteroids expresses apical NHE3 and CFTR. (A color version of this figure is available in the online journal.)

Figure 4

Human jejunal enteroids exposed to lentivirus stably express vector-encoded eGFP over a period of at least four weeks, suggesting transduction of Lgr5⁺ stem cells. Lentivirus transduction was carried out by spinoculation with mechanically dissociated enteroids. The panels show a single enteroid viewed by fluorescence (left panel) and bright field microscopy (right panel) obtained after four weekly passages. (A color version of this figure is available in the online journal.)

Figure 5

Assessment of cell viability in enteroids following exposure to rotavirus. Human jejunal enteroids were mock treated (a) or treated with rotavirus (b) for 24 h. Single cell suspensions were incubated with propidium iodide (PI) and 50,000 events examined for the presence of PI fluorescence using an LSRII flow cytometer. (a) and (b) Representative histograms showing percentage of cells that take up PI (non-viable cells) after each treatment. (c) Quantitation of viable cells after each treatment (n =3 ± SD). *P <0.05 using Mann–Whitney test