Lgr5 stem cells in self-renewal and cancer
Session type: Plenary lectures
The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using an inducible Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were performed in adult mice. The Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that it represents the stem cell of the small intestine and colon. Similar obserations were made in hair follicles and stomach epithelium.
Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids in 3D culture. Tracing experiments indicate that the Lgr5+ve stem cell hierarchy is maintained in these organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche. The same technology has now been developed for the Lgr5+ve stomach stem cells.
Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in stem cells, but not in other crypt cells results in progressively growing neoplasia, identifying the stem cell as the cell-of-origin of adenomas. Moreover, a stem cell/progenitor cell hierarchy is maintained in early stem cell-derived adenomas, lending support to the "cancer stem cell"-concept.
Fate mapping of individual crypt stem cells using a multicolor Cre-reporter revealed that, as a population, Lgr5 stem cells persist life-long, yet crypts drift toward clonality within a period of 1-6 months. Lgr5 cell divisions occur symmetrically. The cellular dynamics are consistent with a model in which the resident stem cells double their numbers each day and stochastically adopt stem or TA fates after cell division. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products. We find that Paneth cells are CD24+ and express EGF, TGF-a, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitantloss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.