Human Enteroids:Establishment Of Ex Vivo Culture System And Expression And Function Of Key Ion Transporters Following Differentiation

Enteroids are organotypic cultures of intestinal epithelium derived from adult intestinal LGR5+ stem cells. Enteroids are comprised of all major types of intestinal epithelial cells and represent the architecture and function remarkably similar to those of intestinal epithelium, faithfully recapitulating intestinal epithelium ex vivo. Enteroids are superior to animal models and traditional cell culture models in many aspects. Enteroids derived from human tissue, in particular, allow the study of human small intestine in the ex vivo setting. Since the first report of mouse enteroids in 2009 and human enteroids in 2011 by Hans Clevers lab, enteroids have soon become a model of interest and are highly desirable as tools for studying intestinal physiology and diseases, drug screening and development, and regenerative medicine.Absorption and secretion of ions are important functions of small intestine. A variety of ion transporters are expressed on the surface of intestinal epithelial cell. These ion transporters mediate various ion transport processes, playing essential roles in maintaining water and electrolyte homeostasis. Cl- secretion and electroneutral NaCl absorption are the most important ion transport processes across intestinal epithelial cell. Dysfunction of the key ion transporters involved in Cl" secretion and electroneutral NaCl absorption is associated with a number of intestinal diseases (eg. diarrhea). Characterization of these ion transporters in human enteroids will improve the understandings in ion transport function of intestinal epithelial cell, and facilitate further studies on intestinal physiology and pathophysiology, development of novel drugs (eg. drugs treating diarrhea/constipation), and regenerative medicine.This project consists of two parts. The first part is establishment of ex vivo culture system of human enteroids, with the aim to build a robust primary culture and long-term expansion system of human enteroids to allow further studies. The second part is expression and function of key ion transporters following differentiation in human enteroids, aiming to determine the physiological function of human enteroids with a focus on ion transporters involved in Cl" secretion and electroneutral NaCl absorption.PartⅠEstablishment of ex vivo culture system of human enteroidsⅠ ObjectiveThis study aimed to build a robust primary culture and long-term expansion system of human enteroids, establish a reliable method of growing human enteroids as mono layers to facilitate functional assays, and validate the differentiation protocol of human enteroids to allow the comparison between non-differentiated and differentiated human enteroids.Ⅱ Methods(1) Adult human duodenal specimens were obtained by endoscopy or surgery. Primary cultures of human enteroids were established and expanded ex vivo using the methods described by Sato et al and Foulke-Abel et al. (2) Human enteroids were cultured in the Transwell system to be grown as monolayers. Localization of cell nuclei, phosphorylated ezrin and Na+-K+-ATPase was determined by immuno fluorescence to reveal cell arrangement and polarity. Transepithellal electric resistance was measured to demonstrate formation of tight junction between cells. (3) Differentiation of human enteroids was induced using the differentiation medium free of WNT3A, and differences between non-differentiated and differentiated enteroids were studied in transepithellal electric resistance, alkaline phosphatase activity, and mRNA expression of markers of LGR5+ stem cell (LGR5,OLFM4), Paneth cell (LYZ) and terminally differentiated intestinal epithelial cell (SI).Ⅲ Results1 A total of five primary cultures of human enteroids derived from adult human duodenal specimens were successfully established in this study. In the ex vivo culture system, human enteroids were continuously passaged and expanded for at least 6 months.2 Human enteroids formed a single layer in the Transwell system, with phosphorylated ezrin localized on the apical side and Na+-K+-ATPase on the baso lateral side, and transepithellal electric resistance elevating and peaking at day 14 after seeding.3 Differentiation caused significant elevations in transepithellal electric resistance and alkaline phosphatase activity, as well as downregulation of LGR5, OLFM4, LYZ and upregulation of SI in mRNA expression in human enteroids.IV ConclusionA robust primary culture and long-term expansion system of human enteroids, as well as a method of growing human enteroids as mono layers are demonstrated in this study. The differentiation medium free of WNT3A is effective in inducing differentiation in human enteroids. Non-differentiated and differentiated enteroids, respectively, represent features of cell types located in crypts and villi of intestinal epithelium.Part IIExpression and function of key ion transporters following differentiation in human enteroidsI ObjectiveThis study aimed to determine the expression and function of the key ion transporters involved in Cl" secretion and electroneutral NaCl absorption in non-differentiated and differentiated human enteroids.II MethodsIon transporters including apical chloride channels CFTR, ANO1, AN06, ANO10 and basolateral ion transporters NKCC1, KCC, KCNQ1/KCNE3, KCNN4 that participate in Cl- secretion, as well as apical Na+/H+exchangers NHE3, NHE2 and anion exchangers DRA, PAT1 that mediate electroneutral NaCl absorption were studied. (1) Expression of mRNA and protein of these ion transporters in non-differentiated and differentiated human enteroids were determined by quantitative real-time PCR and immunoblotting, respectively. (2) Localization of CFTR and NHE3 in human enteroids was determined by immunofluorescence. (3) Forskolin-induced Cl" secretion was studied in non-differentiated and differentiated human enteroids by the Ussing chamber/current clamp technique. Roles of the ion transporters in forskolin-induced Cl" secretion were determined by specific inhibitors. (4) Na+/H+exchange activity was compared between non-differentiated and differentiated human enteoids by the PTI fluorometer using the intracellular pH sensitive dye BCECF-AM.III Results1 Expression and function of ion transporters involved in Cl" secretion1.1 There were no significant changes in protein expression of the apical chloride channels following differentiation in human enteroids. mRNA and protein expression of the basolateral ion transporters, however, were greatly reduced in differentiated human enteroids. Immunofluoresence showed that CFTR was localized on the apical side in both non-differentiated and differentiated human enteroids with similar signal intensity.1.2 Forskolin induced elevations in transepithelial voltage and short-circuit current in both non-differentiated and differentiated human enteroids. The forskolin effects were completely inhibited by the CFTR inhibitor CFTRinh-172 and partially inhibited by the cAMP-activated K+channel inhibitor chromanol 293B.1.3 The NKCC1 inhibitor bumetanide was able to completely inhibit the forksolin-induced elevations in transepithelial voltage and short-circuit current in non-differentiated but not in differentiated human enteroids.1.4 The KCC inhibitor DIOA was able to completely inhibit the forksolin-induced elevations in transepithelial voltage and short-circuit current in differentiated human enteroids that were not inhibited by bumetanide.2 Expression and function of ion transporters involved in electroneutral NaCl absorption2.1 Following differentiation, protein expression of NHE3 and NHE2 was not significantly changed, but mRNA expression of DRA and PAT1 was significantly upregulated. Immunofluorescence demonstrated that NHE3 was localized on the apical side in both non-differentiated and differentiated human enteroids with similar signal intensity.2.2 Non-differentiated and differentiatd human enteroids exhibited comparable Na+/H+exchange activity.IV ConclusionThis study demonstrates that human enteroids have both Cl" secretory and electroneutral NaCl absorptive function. (1) Following differentiation, protein expression of the apical chloride channels is not changed, but mRNA and protein expression of the basolateral ion transporters involved in Cl" secretion are greatly reduced. Forskolin is able to stimulate Cl" secretion in human enteroids, which involves CFTR and basolateral cAMP-activated K+channels and is dependent on NKCC1 and KCC as the basolateral Cl" transporter in non-differentiated and differentiated human enteroids, respectively. These findings support the hypothesis that epithelial cells located in intestinal villi also have Cl" secretory function, and highlight the critical role of KCC in C-" secretion in intestinal epithelial cells located in villi. (2) Non-differentiated and differentiatd human enteroids are similar in protein expression of Na+/H+exchangers and Na+/H+ exchange activity. These results are in favor of the hypothesis that epithelial cells located in intestinal crypts also have electroneutral NaCl absorptive function.