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Lgr5+ intestinal stem cells and their maintenance [electronic resource]

The intestinal epithelium is one of the most rapidly proliferating tissues in the body. A complete turnover of the epithelium occurs every 3-5 days in the mouse, a process that is maintained by a small population of intestinal stem cells (ISCs) that reside in the crypt bases. The signals that regulate the behavior of these ISCs are still poorly understood. However, the recent identification of genes that mark functional stem cells has yielded insights into how ISCs are regulated and maintained. 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. Post-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 versus injury-induced regeneration. How these ISCs maintain their stemness remains unclear. Paneth cells have been suggested to serve as niche cells for the Lgr5+ ISCs, perhaps through the secretion of essential paracrine factors, but recent reports clearly demonstrate that Paneth cells are not required for Lgr5+ ISC maintenance. Recently, the G-protein-coupled receptors Lgr4-6 were reported to associate with Wnt receptors to mediate R-spondin signaling. Given the importance of Wnt/R-spondin signaling in intestinal crypt maintenance, we tested the in vivo function of Lgr5 by adenovirus-mediated overexpression of the soluble ligand-binding Lgr5 extracellular domain. Circulating Lgr5 ectodomain induced the migration of Paneth cells from the crypts, their eventual loss, and a concomitant disappearance of Lgr5+ ISCs. Paneth cell migration was associated with downregulation of Wnt signaling and its target EphB3. The loss of Lgr5+ ISCs did not affect maintenance of the intestinal epithelium, nor did Lgr5+ stem cells disappear from non-intestinal organs. Together, these findings characterize an easily tractable experimental model for the in vivo deletion of Lgr5+ ISCs, and suggest that Lgr receptors function to actively maintain Lgr5+ ISCs in vivo.