Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology

Abstract

Identification of Lgr5 as the intestinal stem cell marker as well as the growth factors necessary to replicate adult intestinal stem cell division has led to the establishment of the methods to generate "indefinite" ex vivo primary intestinal epithelial cultures, termed "mini-intestines." Primary cultures developed from isolated intestinal crypts or stem cells (termed enteroids/colonoids) and from inducible pluripotent stem cells (termed intestinal organoids) are being applied to study human intestinal physiology and pathophysiology with great expectations for translational applications, including regenerative medicine. Here we discuss the physiologic properties of these cultures, their current use in understanding diarrhea-causing host-pathogen interactions, and potential future applications.

Keywords: differentiation; enteroid; induced pluripotent stem cell (iPS cell) (iPSC); intestinal epithelium; lgr5; organoid; stem cells; transport.

FIGURE 1.

Intestinal stem cells (i.e. CBC) located at the base of the crypts can be cultured ex vivo to generate indefinitely propagating enteroid cultures. The enteroid epithelium is composed of the same intestinal cell types that exist in vivo.

FIGURE 2.

Human enteroids as a model to study NHE3 activity. Human duodenal enteroid was loaded with the pH-sensitive dye, SNARF-4F (left; pseudocolor image), and imaged using a two-photo microscope (Olympus). NHE3 activity, which is inhibited by ethylisopropyl amiloride or S3226, is defined as Na⁺-dependent intracellular alkalinization following an acid load in the presence of 50 μm HOE-694 (inhibits all other plasma membrane NHE isoforms) (61). To measure NHE3 activity (right panel), enteroids were acid-loaded by pulsing NH₄Cl followed by Na⁺-free, tetramethylammonium (TMA) solution, resulting in intracellular acidification. After the addition of Na⁺-containing solution, brush-border NHE3 exchanged extracellular Na⁺ for intracellular H⁺. Intracellular pH of enteroids was calibrated after final exposure to K⁺ clamp solution containing nigericin at various pH values (e.g. 6.25, 6.70, 7.50). Measurements were calculated from regions of interest (multi-colored traces in left pseudocolor image) using MetaMorph Image Analysis software.