Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers

Background & Aims: Human enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl- and HCO3 - secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation. Methods: Human duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl- and HCO3 -/CO2, to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s). Results: Differentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na+/K+/2Cl- co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na+/HCO3 - co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl- and HCO3 - secretion as th

Human enteroids: Preclinical models of non-inflammatory diarrhea

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

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

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

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

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

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

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

Lipofuscin Formation Catalyzed by the Milk Protein β‑Lactoglobulin: Lysine Residues in Cycloretinal Synthesis

Lipofuscins are toxic autofluorescent byproducts of the visual cycle. The accumulation of lipofuscins such as cycloretinal in the retina is thought to play a role in the progression of age-related macular degeneration (AMD). Intriguingly, the milk protein β-lactoglobulin (BLG) can promote the cyclodimerization of <i>all-trans</i>-retinal to cycloretinal both <i>in vitro</i> and <i>in vivo</i>. Here, site-directed mutagenesis of BLG and mass spectrometric analysis with substrate analogues demonstrate that lysine residues play a key role in catalysis. It is also shown that catalytic activity necessitates the presence of a physical binding site and cannot be mediated by a peptide chain. These studies provide insight into the mechanism of the cyclodimerization process and provide a model system for biocatalysis and biosynthesis of cycloretinal <i>in vivo</i>. In the long term, these studies may pave the way for drug development and inhibitor design as an early treatment regimen for AMD