Abstract 2644: In vivo and in vitro stem cell models suggest a role for LGR5 in regulating intestinal epithelial lineage specification.

Abstract The stem cell marker Leucine Rich Repeat Containing G-protein Coupled Receptor 5 (LGR5) is frequently upregulated in human colon cancer. Numerous observations implicate LGR5 in stem cell maintenance during intestinal development, tissue homeostasis and cancer, however the precise role of LGR5 in these processes is still unclear. To explore the role of LGR5, we generated mouse embryonic stem cells (ESCs) in which one or both alleles of Lgr5 were deleted and replaced with either β-galactosidase or green fluorescent protein (gfp) reporter genes, or the human LGR5 gene. These cell lines were used in three complimentary systems to differentiate and/or isolate cell lineages. The first system was an in vivo model in which subcutaneous xenografts of murine ESCs grew and formed tumors (teratomas) containing cellular structures from all three germ cell layers. Immunostaining showed that a subset of E-cadherin-positive epithelial cells also expressed Lgr5 reporter genes. Teratomas derived from Lgr5 null ESCs showed an increase in E-cadherin-positive epithelial structures, and gene expression profiling of Lgr5 null teratomas showed an upregulation of epithelial genes including Cdh1, Epcam, Cldn7, Krt5, and Krt14 compared with control teratomas. In contrast, ESCs constitutively overexpressing a secreted form of LGR5 ectodomain formed teratomas with fewer epithelial structures and reduced expression of epithelial genes compared to control teratomas. The second system used in vitro differentiation of ESCs towards an intestinal epithelial lineage, followed by 3D culturing, to derive intestinal organoids. Lgr5+/+ (WT), Lgr5lacZ/+ (Het), or Lgr5lacZ/gfp (Null) ESCs are being examined for their ability to adopt an intestinal fate and form organoids in vitro. A third system used ex vivo culture of murine intestinal crypts isolated from humanized LGR5 heterozygous mice to form intestinal organoids. In summary, the morphological and gene expression changes observed in teratomas implicate a role of LGR5 in regulating epithelial tissue homeostasis. The ability to grow and manipulate organoid cultures ex vivo/in vitro will allow us to use various soluble reagents and detailed microscopy to further assess the role of the Lgr5 pathway in intestinal cell lineage specification as well as tumorigenesis. Citation Format: Courtney M. Williams, Jessica L. Harper, Hui Huang, Pat Boland, Qing Zhang, George D. Yancopoulos, Zhe Li, O. Gavin Thurston. In vivo and in vitro stem cell models suggest a role for LGR5 in regulating intestinal epithelial lineage specification. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2644. doi:10.1158/1538-7445.AM2013-2644