Human enteroids: preclinical models of non-inflammatory diarrhea

Abstract

Researchers need an available and easy-to-use model of the human intestine to better understand human intestinal physiology and pathophysiology of diseases, and to offer an enhanced platform for developing drug therapy. Our work employs human enteroids derived from each of the major intestinal sections to advance understanding of several diarrheal diseases, including those caused by cholera, rotavirus and enterohemorrhagic Escherichia coli. An enteroid bank is being established to facilitate comparison of segmental, developmental, and regulatory differences in transport proteins that can influence therapy efficacy. Basic characterization of major ion transport protein expression, localization and function in the human enteroid model sets the stage to study the effects of enteric infection at the transport level, as well as to monitor potential responses to pharmacological intervention.

Figure 1

Cells within human enteroids reflect morphology of native intestinal tissue. (a) Human enteroids are well polarized, expressing apical villin (green) and basolateral Na⁺/K⁺-ATPase (red). Nuclei are indicated by Hoescht 33342 (blue). (b) Transmission electron microscopy demonstrates presence of intact tight junctions. n = 3 for each experiment.

Figure 2

Human enteroids recapitulate two major functions of the small intestine: Na⁺ absorption and Cl^- secretion. (a) Enteroids exhibit NHE3 activity, which can be inhibited by forskolin (FSK) treatment before or after initiation of transport measurements. Differentiated enteroids were loaded with the pH-sensitive dye SNARF-4F and prepulsed with 50 mM NH₄Cl to acidify the cytosol. Na⁺-dependent alkalinization in the presence of 50 μM HOE694 is due to NHE3 activity (no alkalinization in the presence of the NHE3 inhibitor S3226, added after HOE694). n = 6 for each condition. CTL, control; STD, pH standards; TMA, tetramethylammonium chloride. (b) Duodenal enteroid lumens (L) significantly dilate in response to elevated cAMP levels via FSK treatment. After FSK treatment, the size of enteroid lumens increased 203 ± 16% over controls. The same optical section of enteroids loaded with SNARF-4F was compared before (red) and after (green) 30 minutes of FSK treatment. Arrows indicate change in position of epithelial layer due to luminal dilation. n = 3 for each experiment.

Figure 3

Human jejunal enteroids support rotavirus growth. (a) Nonstructural viral proteins, NSP4 (green) and NSP5 (red), were detected in enteroids by confocal microscopy. Basolateral membrane marked by E-cadherin (purple) and nuclei (blue) by 4',6-diamidino-2-phenylindole (DAPI) staining. (b) GAPDH-normalized rotavirus VP7 RNA levels are shown as fold increases relative to 0 hours post infection. Values are means of triplicates and standard deviation for three amplifications per sample.