Gut Churning

In 2007, Hans Clevers's group at the Hubrecht Institute for Developmental Biology and Stem Cell Research in Utrecht used a combination of inducible gene knock-ins and lineage tracing to identify a gene that serves as an intestinal stem cell marker.³ Cells positive for the gene, leucine-rich, repeat-containing G proteincoupled receptor 5 (Lgr5), localized to the region where CBC cells reside, and generated all the lineages seen in the intestinal and colon epithelium. These Lgr5-positive cells divide once per day—a point of controversy, since many researchers hold that stem cells divide only rarely (on the order of weeks or months), in order to maintain the stability of their genome.

The Nature study, this month's Hot Paper, provided the first genetic marker for intestinal cells with distinct stem cell behavior, and revived Leblond's hypothesis regarding where to find them. "For us, this is old news," says Matthew Bjerknes of the University of Toronto, who, along with Leblond's student Hazel Cheng has continued working on the CBC hypothesis. "But it has opened the debate widely again."

Clevers quickly followed his 2007 Lgr5 paper with several others supporting that Lgr5-positive CBC cells were, in fact, stem cells. In one, a March Nature paper, his group purified and cultured Lgr5- positive cells in the presence of a Wnt factor, and the cultures formed "organoids" with distinct crypt-villus structures.⁴ Culturing gut epithelium has been notoriously challenging, and growing whole segments of gut in culture "is spectacular," says Riccardo Fodde of the Erasmus Medical Center in Rotterdam. "It is unbelievable almost."

Meanwhile, the debate over intestinal stem cells' identity and location is far from over. Last summer, Mario Capecchi at the University of Utah School of Medicine reported that Bmi1, already implicated in self-renewal in neurons and hematopoietic cells, marks intestinal stem cells, and pinpointed them to approximately the +4 position.⁵ More indirect evidence from Linheng Li's lab at the Stowers Institute for Medical Research in Kansas City, Missouri, implicates yet another gene in intestinal stemness that localizes around +4.⁶

According to Potten, those two lines of evidence definitively support his model. Clevers's 2007 study showed a few Lgr5-positive cells in position +4, and Potten says those cells—which would also have been present in the purified cells of the "organoid" study—are the intestinal stem cells. (Lgr5's function is unknown; Potten speculates the labeled cells at the base of the crypt are precursors to another nonstem-cell type.)

Clevers, however, notes Li's marker was never functionally verified, and insists that for stem cells, Lgr5 trumps Bmi1. "On its own," he says, Lgr5 "makes a black and white distinction between a stem cell and a daughter cell. And I don't know any other marker that does that." A recent gene expression study from his lab showed his Lgr5-positive cells also expressed Bmi1. The two labeled populations also have the same kinetics, he adds: just like Lgr5-positive cells, Bmi1-positive cells fill the crypt with lots of cells in 3 or 4 days.⁷ Indeed, says Clevers, he is convinced his group and Capecchi's "are actually tracing the same cells."

Intestine isn't the only tissue to hold putative stem cells carrying Lgr5: Elaine Fuchs at The Rockefeller University notes that the gene has consistently come up in screens of hair follicle stem cells. The biology of hair follicle stem cells is better understood than intestine and other structures, so researchers can use that knowledge to better understand Lgr5's role. "I think what's exciting is that we can now draw on each other's science, and that always propels a field forward," she says.

Clevers agrees that in comparing markers in different systems, "we hope to see a minimum program of stemness." His group is working on teasing out the function of Lgr5 to better understand what precisely it's marking. In addition, they have found Lgr5- positive cells in the stomach, brain, retina, mammary gland, kidney, and pancreas. Except for the mammary gland, he adds, "in all cases, they sit in different places than where people had postulated them to be"—and by extension, mark different populations than past studies have fingered as stem cells.

With the advent of reliable stem cell markers, the debate regarding location may now be irrelevant. "We can ask ourselves, does it really matter anymore?" says Bjerknes. "Now we have tools in addition to simple morphology. We have these markers, let's just push forward and see what these cells can do."

Correction (July 7): A previous version of this article incorrectly stated Charles Philippe Leblond's university affiliation as the University of Toronto. In fact, he was based at McGill University in Montreal. The Scientist regrets the error.