Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology

Abstract

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 demonstrate their functional relevance to ion transport physiology and pathophysiology. METHODS Human intestinal tissue specimens were obtained from 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 qRT-PCR, 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 withdrawl of WNT3A, yielding decreased crypt markers and increased villus-like characteristics. NHE3 activity was similar in undifferentiated and differentiated enteroids, and was affected by known inhibitors, second messengers, and bacterial enterotoxins. Forskolin-induced swelling (FIS) was completely dependent on CFTR and partially dependent upon NHE3 and NKCC1 inhibition in undifferentiated and differentiated enteroids. Increases in cAMP with forskolin caused enteroid intracellular acidification in HCO3−- free buffer. cAMP-induced enteroid pHi acidification as part of duodenal HCO3− secretion appears to require CFTR and NBCE1. 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 revealed that NBCE1 might be a target in the intestinal mucosa for treatment of secretory diarrheas.

Guanylate cyclase 2C agonism corrects CFTR mutants

“…Importantly, these structures represent key features of the in vivo structure and function of the source tissue. This closed structure of an enterosphere encompassing a central lumen allows investigators to study fluid dynamics (i.e., secretion and absorption) caused by physiological alterations in the epithelia that can result in either swelling or shrinkage of the enterospheres (22). It turned out that enterospheres are extremely useful models to study CFTR function in which forskolin, a cAMP agonist, induces rapid swelling of the enteropsheres, with normal CFTR caused by fluid filling the central lumen as CFTR becomes active (23)(24)(25).…”

Using primary murine intestinal enteroids to study dietary TAG absorption, lipoprotein synthesis, and the role of apoC-III in the intestine

“…A limitation of this organoid system, as for other reported tissue organoids (23,38,39), is that our enteroid models cannot exactly recapitulate the in vivo tissue. As we have demonstrated, although the cellular lineage composition, polarity around a central lumen, and stimulation by FFA mimic in vivo tissue physiology, there are several key aspects of the in vivo environment that are lacking.…”

“…1C). Whereas Caco-2 cells grow in a 2D monolayer composed solely of epithelial cells, mature enteroids are composed of multiple cell types, including stem cells, goblet cells, and enterocytes (17,23). Compared with freshly isolated intestine ( Fig.…”

“…Previous studies in enteroids have focused on stem cell biology, lineage tracing, and Na + /H + exchange (23,(26)(27)(28). We asked whether enteroids were able to accomplish TAG absorption, lipoprotein synthesis, and lipoprotein secretion, which are major functions of the intestine.…”

Intestinal Enteroids Model Guanylate Cyclase C-Dependent Secretion Induced by Heat-Stable Enterotoxins

“…While prevention and therapy of ST-induced secretory diarrhea remain important priorities, available biological models limit development of effective agents (16)(17)(18). In vitro models (e.g., human colon cancer cells) may not recapitulate normal tissue architecture, biology, or molecular signaling events (13,16,17,19), while in vivo models, such as suckling mice (20) and ileal loops (21), are labor-intensive and not suited to high-throughput screening.…”