The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1 + ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1 + ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5 + ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.R-spondin | Dickkopf-1 | intestinal regeneration T he G protein-coupled receptor Lgr5 and the Polycomb group protein Bmi1 are two recently described molecular markers of self-renewing and multipotent adult stem cell populations residing in the crypt of the small intestine, capable of supporting regeneration of the intestinal epithelium (1, 2). Despite their similar ability to functionally repopulate the intestinal epithelium as demonstrated by independent in vivo lineage tracing experiments in reporter mice, the intestinal stem cells (ISCs) identified by these two molecular markers are spatially distinct. Whereas Lgr5 + ISCs are crypt base columnar (CBC) cells (1, 3) interspersed between Paneth cells and expressed throughout the intestine, Bmi1 + ISCs are mostly restricted to the "+4" cell position abutting the uppermost Paneth cell in proximal small intestine crypts (2). Lgr5 + ISCs are actively cycling (1), equipotent, and contribute to intestinal homeostasis by neutral drift competition (4-6). By comparison, Bmi1 + ISCs are less well characterized, and because of the lack of direct evidence, their cell cycle status is variably ascribed to be rapidly (7) vs. slowly cycling (8). It has been suggested that Bmi1 and Lgr5 mark an overlapping and possibly identical or redundant population of ISCs (5, 7, 9); however, no direct exploration of their functional similarities and differences has been performed. Further, it is unknown how Bmi1 + and Lgr5 + ISCs relate to a proposed model in which the intestine differentially uses an actively cycling ISC population during homeostasis and a distinct quiesce...

The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the γ-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein–coupled receptor-5–positive (Lgr5+) and B lymphoma moloney murine leukemia virus insertion region homolog-1–positive (Bmi1+) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5+ cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.

SUMMARY The canonical Wnt/β-catenin signaling pathway governs diverse developmental, homeostatic and pathologic processes. Palmitoylated Wnt ligands engage cell surface Frizzled (Fzd) receptors and Lrp5/6 co-receptors enabling β-catenin nuclear translocation and Tcf/Lef-dependent gene transactivation1–3. Mutations in Wnt downstream signaling components have revealed diverse functions presumptively attributed to Wnt ligands themselves, although direct attribution remains elusive, as complicated by redundancy between 19 mammalian Wnts and 10 Fzds1 and Wnt hydrophobicity2,3. For example, individual Wnt ligand mutations have not revealed homeostatic phenotypes in the intestinal epithelium4, an archetypal canonical Wnt pathway-dependent rapidly self-renewing tissue whose regeneration is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs)5–9. R-spondin ligands (Rspo1–4) engage distinct Lgr4-6 and Rnf43/Znrf3 receptor classes10–13, markedly potentiate canonical Wnt/β-catenin signaling and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo8,14–17. However, the interchangeability, functional cooperation and relative contributions of Wnt versus Rspo ligands to in vivo canonical Wnt signaling and ISC biology remain unknown. Here, we deconstructed functional roles of Wnt versus Rspo ligands in the intestinal crypt stem cell niche. We demonstrate that the default fate of Lgr5+ ISCs is lineage commitment, escape from which requires both Rspo and Wnt ligands. However, gain-of-function studies using Rspo versus a novel non-lipidated Wnt analog reveal qualitatively distinct, non-interchangeable roles for these ligands in ISCs. Wnts are insufficient to induce Lgr5+ ISC self-renewal, but rather confer a basal competency by maintaining Rspo receptor expression that enables Rspo to actively drive and specify the extent of stem cell expansion. This functionally non-equivalent yet cooperative interplay between Wnt and Rspo ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precision control of tissue regeneration.

The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here, a single air-liquid interface culture method was used without modification to engineer oncogenic mutations into primary epithelial/mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia upon KrasG12D expression and/or p53 loss, and readily generated adenocarcinoma upon in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), and versus more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the Insulin-like growth factor-2 (IGF2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

This study aimed to evaluate the correlation between symptoms and endoscopic findings in reflux esophagitis. Subjects, 8031 persons without medication for gastrointestinal disease, were briefly asked about the presence of heartburn, dysphagia, odynophagia, and acid regurgitation by associated medical staff before endoscopy for assessment of esophagitis utilizing the Los Angeles Classification. Endoscopically, 1199 (14.9%) were classified as positive reflux esophagitis, and 2223 (27.7%) had heartburn, 1522 (19.0%) had dysphagia, 493 (6.1%) had odynophagia, and 1466 (18.3%) had acid regurgitation. Multivariate analysis indicated that the symptom most related to esophagitis was heartburn (odds ratio: 2.46), although approximately 40% of subjects with grade C or D did not complain of heartburn. Regarding the other symptoms, less than 30% subjects with severe esophagitis complained of the symptoms and the odds ratio was approximately 1. These results indicate that endoscopic esophagitis was not equivalent to any reflux symptoms from which subjects suffered in their daily lives.