Some markers scientists rely on to identify stem cells are proving to be flawed

Magic marker myths

Bookmarks can help you quickly find just the right page among hundreds or even thousands. But we know better than to confuse a bookmark for the page it points to, and we all know that bookmarks can get misplaced. So it is with stem cells. Cell-surface proteins and other markers may seem a convenient way of identifying stem cells, but they're no substitute for assessing what the cells can really do.

Sean Morrison, director of the University of Michigan's Center for Stem Cell Biology, thinks misplaced trust in stem-cell markers is one of the field's biggest problems. "A high percentage of discoveries that don't hold up fail to hold up because people weren't careful enough about the markers they used to identify the stem cells," he says. That's because many tissues lack good functional assays, or markers, or both. So when researchers hear of a stem-cell marker from another system that just might apply to their own, they tend to latch on.

Morrison recently undermined the case for one cherished 'magic marker' for stem cells that equated 'stemness' with cells' retention of the nucleotide-analogue label bromodeoxyuridine (BrdU). The longest-running rationale for this marker holds that stem cells divide more slowly than the progenitor cells they generate. So, after the label is withdrawn from the growth medium, the relatively quiescent stem cells hoard the stash of label already deposited in their genomes, whereas label incorporated into progenitor cells' DNA is steadily diluted as those cells proliferate.

But there's at least one fly in that ointment. If a progenitor undergoing a final division to become two long-lived, differentiated cells happens to get labelled, those cells can retain the label for years, confounding the assay.

An alternative notion posits that BrdU is an even more specific marker of stemness. The 'immortal strand' hypothesis proposes that when a stem cell divides into another stem cell plus a differentiation-committed daughter cell, the former retains its original DNA strands with the newly synthesized (and, presumably, imperfectly copied) strands shunted off to the latter1. Thus, you would expect stem-cells' labeled strands to be always retained in those stem cells.
The trouble is that the label-retention hypothesis has not been validated, says Morrison. "A lot of people take it as an article of faith that DNA-label-retaining cells are highly enriched for stem cells," even in tissues where stem cells cannot be clearly defined by phenotype or function.

Morrison found himself thinking: "If we're going to build a whole field based on this proposition, then somebody should test it." So he did, in the most extensively characterized system available: haematopoietic stem cells (HSCs).2 His team pulsed mice with BrdU and examined their blood at various times to see where the labeled DNA turned up.

Labeled DNA gradually disappeared from HCSs, Morrison says, and more than 99% of those cells that did retain BrdU were not stem cells. Although at least one well-designed study has shown evidence supporting BrdU retention in another tissue, the hair follicle3, Morrison believes label retention is one of the worst markers that has ever been used in published papers. "It's your textbook example of a terrible stem-cell marker."

It's also ubiquitous. "Until half a year, or a year, ago I never heard anybody damn or doubt label retention as a stem-cell marker," says Hans Clevers, director of the Netherlands Institute for Developmental Biology's Hubrecht Laboratory in Utrecht. Gut epithelium is renewed from cells located in the crypts at the base of intestinal villi. In a recent study4, Clevers' group found that the well-known Paneth cells in the crypts had been falsely identified as the gut epithelial stem cells. That widespread assumption had been based largely on observations that the cells retained BrdU.

Clevers's team made transgenic mice in which the gene lacZ, which encodes an enzyme that can yield an intense blue stain in tissue sections, was tethered to the promoter for Lgr5, a gene expressed only, and rarely, in the crypts, making it a possible marker for stem cells. The cells that stained blue were not the Paneth cells, however, but small, sparse cells called crypt-based columnar cells (CBCs).

http://www.nature.com/stemcells/2008/0801/080131/full/stemcells.2008.26.html