Stem Cells Marked by the R-Spondin Receptor LGR5 Bon-Kyoung Koo, Hans Clevers  Gastroenterology  Volume 147, Issue 2, Pages 289-302 (August 2014) DOI: 10.1053/j.gastro.2014.05.007 Copyright © 2014 AGA Institute Terms and Conditions

Figure 1 (A) Structure of the small intestinal epithelium. The inner surface epithelium of the small intestine is folded into villi, which resemble digit-like protrusions, and crypts corresponding to pocket-like structures. The LGR5+ crypt base columnar stem cells, located at the bottom of the crypt, are interspersed between Paneth cells, which constitute the niche cells. Through daily proliferation, the stem cells give rise to progeny called transit-amplifying cells. During their upward migration, the progeny differentiate into nutrient-absorbing enterocytes as well as other secretory cells that produce mucins (goblet cells) and hormones (enteroendocrine cells). M cells and tuft cells are rarer cell types present in the small intestine. (B) Lineage specification. Six differentiated cell types populate the small intestine. Enteroendocrine, goblet, and Paneth cells belong to the secretory lineage, whereas enterocytes originate from the absorptive lineage. Enterocytes and goblet cells are most abundant, whereas enteroendocrine, M, and tuft cells are less common. LRC, label-retaining cell; CBC cell, crypt base columnar cell; M cell, microfold cell. Gastroenterology 2014 147, 289-302DOI: (10.1053/j.gastro.2014.05.007) Copyright © 2014 AGA Institute Terms and Conditions

Figure 2 (A) Principle of lineage tracing. To perform lineage tracing, a reporter (eg, a fluorescent protein or lacZ) is inserted in the locus of a ubiquitously expressed gene (eg, Rosa26). Initially, expression of the reporter is prevented by an upstream “stopper” cassette. To initiate expression of the reporter, tamoxifen is added. Consequently, the earlier expressed Cre recombinase fused to a mutant estrogen ligand-binding domain is able to enter the nucleus, where it excises the “stopper” cassette, allowing the reporter to be expressed. By having the expression of CreERT2 under the control of a cell type–specific promoter, the reporter can be selectively induced in specific cell types or lineages. This method can be used to label stem cells and their daughter cells, because the genetic modification is irreversible and inherited by all progeny of the initially labelled stem cell. UTR, untranslated region; FP, fluorescent protein; IRES, internal ribosome entry site; CreERT2, Cre recombinase fused to mutant estrogen ligand-binding domain; polyA, polyadenylation sequence; SA, splice acceptor. (B) Lineage tracing at different time periods. The initial tracing event, approximately 24 hours after the administration of tamoxifen, reveals a stem cell–specific expression of the reporter. Five days later, a blue ribbon, corresponding to the progeny of the initially labelled stem cell, can be observed. Finally, 60 days after initiation, a full gland tracing can be seen. (C) Schematic drawing of an icosidodecahedron. The black pentagons represent Paneth cells, and the green triangles represent LGR5+ stem cells. The icosidodecahedral structure maximizes the shared membrane between LGR5+ stem cells and Paneth cells while minimizing homologous interactions. (D) Confocal image of a section of the crypt. LGR5+ stem cells are expressing GFP (shown in green), and the black area corresponds to Paneth cells. Gastroenterology 2014 147, 289-302DOI: (10.1053/j.gastro.2014.05.007) Copyright © 2014 AGA Institute Terms and Conditions

Figure 3 Schematic drawing summarizing the current knowledge of plasticity in the intestinal crypt. (A) During homeostasis, LGR5+ stem cells represent the source of self-renewal and give rise to different lineages of the small intestine. (B) On γ-irradiation treatment, fast proliferating cells are killed and secretory progenitor cells marked by DLL1 can revert back to become LGR5+ stem cells and give rise to full gland tracings. (C) Label-retaining cells are distinguished by their label-retaining ability and can generate full gland tracings on γ-irradiation treatment like DLL1+ cells. (D and E) HOPX+ and BMI1+ progenitors are also shown to have a similar capacity. Gastroenterology 2014 147, 289-302DOI: (10.1053/j.gastro.2014.05.007) Copyright © 2014 AGA Institute Terms and Conditions

Figure 4 (A) In the absence of Wnt, β-catenin is phosphorylated by the destruction complex consisting of APC, AXIN, GSK3, and CK1. It is subsequently recognized by β-TrCP, leading to its ubiquitination and degradation by the ubiquitin-proteasome system. (B) In the presence of Wnt, which simultaneously binds to Frizzled and LRP5/6, LRP5/6 is phosphorylated by CK1 and GSK3 and disheveled (DVL) is recruited to the plasma membrane to interact with Frizzled and other DVL molecules. The interaction between AXIN and phosphorylated LRP5/6 and DVL polymer inactivates the destruction complex. Consequently, β-catenin is stabilized, leading to its cytosolic accumulation and translocation into the nucleus, where it binds to LEF/TCF and mediates the transcription of Wnt responsive genes. RNF43 removes the Frizzled/LRP receptors from the cell surface, resulting in their degradation, while LGR5-RSPO inhibits the action of RNF43 by forming a tripartite complex sequestering RNF43 from Frizzled/LRP receptors. Gastroenterology 2014 147, 289-302DOI: (10.1053/j.gastro.2014.05.007) Copyright © 2014 AGA Institute Terms and Conditions

Figure 5 (A) Schematic drawing of a “mini-gut,” also called an organoid. This 3-dimensional self-organizing structure can be derived from a single LGR5+ stem cell or a crypt part of the gastrointestinal tract. Organoids have an architecture and physiology that closely resemble that of the in vivo situation. Stem cells, progenitors, and Paneth cells can be found in the budding “crypt domain” structures, while the differentiated cell types (eg, enterocytes, goblet cells, and enteroendocrine cells) can be identified in the “villus domain.” Progenitors are pushed up and away from the crypt bottom; when reaching the “villus domain,” they have differentiated and are shed into the lumen (depicted by gray cells). (B) Light microscope image of a mouse small intestinal organoid (scale bar = 200 μm). (C) Media composition for different types of mouse and human organoids. Gastroenterology 2014 147, 289-302DOI: (10.1053/j.gastro.2014.05.007) Copyright © 2014 AGA Institute Terms and Conditions

Figure 6 (A) Genetic tools available for organoids. Functional genetics can be performed in organoids derived from endodermal organs (eg, small intestine, colon, stomach, pancreas, or liver) by using retroviral transduction, BAC-mediated transgenesis, or CRISPR/Cas9 genome engineering. (B) A table listing the advantages and disadvantages of the genetic tools that can be applied to organoids. Gastroenterology 2014 147, 289-302DOI: (10.1053/j.gastro.2014.05.007) Copyright © 2014 AGA Institute Terms and Conditions