Why do you think your paper is highly cited?

The intestine is a popular choice for scientists wanting to study the regulation of tissue renewal because it is the most rapidly self-renewing organ in the body, with the epithelium being completely regenerated every 5-7 days in mice and humans.

It has been known for many decades that this regeneration process is driven by a small population of stem cells residing in pockets (termed crypts) within the gut wall. However, their precise identity has proved elusive due to a lack of specific markers that distinguish them from other cell types present in the crypts.

In the Nature article we report our discovery of the first unique marker, Lgr5 (Leucine-rich G Protein-Coupled Receptor 5) for these intestinal stem cells. We describe the generation of a new mouse model which allows us to perform in-vivo lineage tracing from Lgr5+ve cell populations, providing solid functional evidence of stem cell identity. This paper was also the first to challenge the current dogma that adult stem cells should be quiescent—we showed the Lgr5+ve stem cells to be proliferating every 24 hours, which we believe makes more sense considering the large number of new cells that need to be generated in the crypts each day to sustain epithelial renewal.

Finally, we describe preliminary data that Lgr5 is likely to be a specific marker for stem cell populations in other rapidly regenerating organs such as the stomach, skin, and mammary gland. Ongoing lineage tracing experiments confirm this, thereby establishing Lgr5 as a broad marker for adult stem cells.

 Does it describe a new discovery, methodology, or synthesis of knowledge?

The paper largely details a new discovery—namely the identification of Lgr5 as the first marker of cycling stem cells in the small intestine and colon.

 Would you summarize the significance of your paper in layman’s terms?

The intestine is basically a long tube, whose inner lining (the epithelium) is largely responsible for efficiently absorbing nutrients and water from our diet and compacting the left-over material into feces for subsequent "disposal". Within this hostile environment, the epithelium is constantly being subjected to mechanical shearing and nasty bug attack as digested food/water passes through, which means it rapidly wears out and needs replacing.

We’ve known for a long time that special cells called stem cells are responsible for ensuring this constant renewal of the epithelium, but until now we had no way of identifying them. We recently solved this problem when we found a gene called Lgr5 to be expressed only on these stem cells in the epithelium. This now allows us to intensively study the stem cells and improve our understanding of how tissue renewal is controlled in the intestine.

Armed with this knowledge we expect to be able to better understand why this renewal process breaks down in human diseases such as Crohn's disease, inflammatory bowel disease, and cancer. Ultimately, we hope to purify the stem cells from the intestine using markers such as Lgr5 for helping to repair damaged intestines, or even other adult tissues.

 How did you become involved in this research and were any particular problems encountered along the way?

We have been working on the role of the Wnt signaling pathway in intestinal renewal and cancer for the past decade. The Wnt pathway exerts its effects by controlling the expression of a select set of target genes influencing various biological processes ranging from cell proliferation and migration to cell differentiation and death.

In 2002, we found that Lgr5 is one such Wnt target gene which has a unique expression pattern in the intestinal crypts. These Lgr5 cells turned out to be identical to a population of cells termed crypt base columnar cells (CBC) by Charles Philippe Leblond, Matthew Bjerknes, and Hazel Cheng in the early 1980s—they proposed that these CBC cells were good candidates for the elusive stem cells, but had no direct way of proving this. We therefore set out to test this hypothesis using Lgr5 a specific marker for the CBC cells.

The lack of commercial antibodies against this cell-surface receptor together with the lack of in-vitro assays for assaying intestinal stem cells dictated that we had to pursue an in-vivo lineage tracing approach in order to assess the stem cell capacity of these Lgr5 cells. This meant embarking on a lengthy project to generate a mouse model (Lgr5-EGFP-ires-CreERT2) that would allow us to perform this in-vivo lineage tracing when combined with an inducible reporter mouse strain. Ultimately, this proved very successful and has also allowed us to determine that Lgr5 is marking stem cell populations in other tissues including the stomach, skin, and mammary gland.

 Where do you see your research leading in the future?

The mouse model described in the Nature article not only allows us to perform in-vivo lineage tracing, but also allows us to isolate and purify the stem cells from the intestine (and other Lgr5+ve organs) by virtue of the fact they express a fluorescent marker GFP (Green Fluorescent Protein). This has allowed us to set up an in-vitro culture system that allows us to grow new pieces of intestine from single Lgr5 stem cells (Sato, T. et al., Nature 459:262, 2009).

Although this is currently limited to the generation of very small pieces of epithelium in the laboratory, future advances in the technique may facilitate production of intestinal pieces large enough to have therapeutic value (Crohn's disease patients and cancer patients for example). We hope to set up similar culture systems using Lgr5 stem cells from other adult tissues to explore similar therapeutic opportunities in a range of human organs.

We are also currently investigating the function of the Lgr5 gene on the stem cells using knockout mice and are actively pursuing the ligand for this orphan GPCR. Having identified the stem cells, we can now investigate their role in cancer. We have already shown that mutated intestinal stem cells are the likely source of colon cancer (Barker, N. et al., Nature 457: 608, 2009). Future studies will further explore the contribution of Lgr5 stem cells to epithelial cancers.

 Do you foresee any social or political implications for your research?

The ability to isolate various adult stem cell populations using Lgr5 as a specific marker will hopefully allow us (and others) to exploit their capacity for effecting tissue renewal for therapeutic use. Assessing the role of the Lgr5 stem cells in various cancers will also improve our understanding of cancer development and may ultimately translate into the generation of novel therapies.

Dr. Nick Barker
Hubrecht Institute
Utrecht, The Netherlands

KEYWORDS: PROTEIN-COUPLED RECEPTOR; MOUSE SMALL-INTESTINE; ADULT-MOUSE; EPITHELIAL PROGENITORS; CANCER; OVEREXPRESSION; EXPRESSION; MUSASHI-1; RENEWAL; GPR49.