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.

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.

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.

NHE3 is the brush-border (BB) Na⁺/H⁺exchanger of small intestine, colon, and renal proximal tubule which is involved in large amounts of neutral Na⁺absorption. NHE3 is a highly regulated transporter, being both stimulated and inhibited by signaling that mimics the postprandial state. It also undergoes downregulation in diarrheal diseases as well as changes in renal disorders. For this regulation, NHE3 exists in large, multiprotein complexes in which it associates with at least nine other proteins. This review deals with short-term regulation of NHE3 and the identity and function of its recognized interacting partners and the multiprotein complexes in which NHE3 functions.