The intestinal epithelium constitutes a system of constant and rapid renewal triggered by proliferation of intestinal stem cells (ISCs), and is an ideal system for studying cell proliferation, migration and differentiation. Primary cell cultures have proved to be promising to unravel the mechanisms involved in the epithelium homeostasis. In 2009, Sato et al. established a long-term primary culture to generate epithelial organoids (enteroids) with crypt- and villus-like epithelial domains representing the complete census of progenitors and differentiated cells. Similarly, isolated ISCs expressing Lgr5 (Leucine-rich repeat-containing G protein-coupled receptor) could generate enteroids. Here, we describe methods to establish gastric, small intestinal, and colonic epithelial organoids (Basic Protocol 1) and the generation of Lgr5+ve single cell-derived epithelial organoids (Basic Protocol 2). We also describe the imaging techniques used to characterize those organoids (Basic Protocol 3). This in vitro model constitutes a powerful tool for studying stem cell biology and intestinal epithelial cell physiology throughout the digestive tract.

Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5 + ) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5 + or Lgr6 + cells from taste tissue can generate continuously expanding 3D structures ("organoids"). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2'-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5 + cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6 + cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5 + or Lgr6 + cells, validating the use of this model for the study of taste cell generation. Lgr5 (leucine-rich repeat-containing G protein-coupled receptor 5), encoded by a Wnt (wingless-type MMTV integration site family) target gene, marks adult stem/progenitor cells in taste tissue in posterior tongue that in vivo give rise to all major types of taste bud cells, as well as perigemmal cells (6, 7). Lgr5 is also known to mark actively cycling stem cells in small intestine, colon, stomach, and hair follicle, as well as quiescent stem cells in liver, pancreas, and cochlea (8). Isolated Lgr5 + adult stem cells from multiple tissues are able to generate so-called organoid structures ex vivo (9-11). For instance, Sato and colleagues (10) developed a 3D culture system to grow crypt-villus organoids from single intestinal stem cells; all differentiated cell types were found in these structures, indicating the multipotent nature of these cells. We hypothesized that Lgr5 + taste stem/progenitor cells in a 3D culture system would be capable of expanding and giving rise to taste receptor cells ex vivo. In the present study, we isolated Lgr5 + stem/progenitor cells from taste tissue and cultured them in a 3D culture system. Single Lgr5 + cells grew into organoid structures ex vivo in defined culture conditions, with the presence of both proliferating cells and differentiated mature taste cells in which taste signaling components are functionally expressed. When organoids were replated onto a 2D sur...

Despite the global prevalence of gastric disease, there are few adequate models to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (PSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/β-catenin signaling in mouse embryos led to conversion of fundic to antral epithelium, while β-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signaling pathways in epithelial morphogenesis and differentiation of fundic cell types, including chief cells and functional parietal cells. While hFGOs are a powerful new model for studying the development of the human fundus and its lineages, they also represent a critical new model system to study the molecular basis of human gastric physiology, pathophysiology, and drug discovery.

Trefoil factor (TFF) peptides, containing a 40-amino acid motif, including six conserved cysteine residues that form intramolecular disulfide bonds, are a family of mucin-associated secretory molecules mediating many physiological roles that maintain and restore gastrointestinal (GI) mucosal homeostasis. TFF peptides play important roles in response to GI mucosal injury and inflammation. In response to acute GI mucosal injury, TFF peptides accelerate cell migration to seal the damaged area from luminal contents, whereas chronic inflammation leads to increased TFF expression to prevent further progression of disease. Although much evidence supports the physiological significance of TFF peptides in mucosal defenses, the molecular and cellular mechanisms of TFF peptides in the GI epithelium remain largely unknown. In this review, we summarize the functional roles of TFF1, 2, and 3 and illustrate their action mechanisms, focusing on defense mechanisms in the GI tract.

A self-organizing organoid model provides a new approach to study the mechanism of human liver organogenesis. Previous animal models documented that simultaneous paracrine signaling and cell-to-cell surface contact regulate hepatocyte differentiation. To dissect the relative contributions of the paracrine effects, we first established a liver organoid using human induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) as previously reported. Time-lapse imaging showed that hepatic-specified endoderm iPSCs (HE-iPSCs) self-assembled into three-dimensional organoids, resulting in hepatic gene induction. Progressive differentiation was demonstrated by hepatic protein production after organoid transplantation. To assess the paracrine contributions, we employed a Transwell system in which HE-iPSCs were separately co-cultured with MSCs and/or HUVECs. Although the three-dimensional structure did not form, their soluble factors induced a hepatocyte-like phenotype in HE-iPSCs, resulting in the expression of bile salt export pump. In conclusion, the mesoderm-derived paracrine signals promote hepatocyte maturation in liver organoids, but organoid self-organization requires cell-to-cell surface contact. Our model demonstrates a novel approach to identify developmental paracrine signals regulating the differentiation of human hepatocytes.