Even when an organ is believed to harbour stem cells, the cells themselves can be elusive. The standard method used to hunt for mammary stem cells involves injecting cells into the mammary fat pad of mice and seeing if the cells regenerate mammary tissue. Unfortunately, this approach is time consuming, highly variable and allows no way to count the cells. In this month's Nature Medicine, Connie Eaves and her team at the British Columbia Cancer Agency, Canada, report a new technique which has allowed them, for the first time, to characterize and quantify mammary gland stem cells in humans1.

The team first suspended individual human mammary gland cells in small collagen gels along with support cells called fibroblasts that had been rendered incapable of dividing. They then implanted the gels under the kidney capsule in immunodeficient mice, where dense capillaries would provide sufficient nutrients for the cells to grow. Excitingly, the transplanted cells proliferated and self-organized into human mammary structures inside the gels. They even produced milk proteins when mice reached the late stages of pregnancy.

But Eaves wasn't satisfied. “The collagen gels were opaque. They had to be fixed so we could look at the histology, and this couldn't give us any quantitative information.”

To get around this, the authors used enzymes to digest the four-week-old gels to release the mammary gland cells. Then they used the cells for an in vitro assay to look for single progenitor cells capable of creating colonies of more mature cells, in this case, the epithelial lineages associated with the mammary gland. “In addition to the colonies we found in vitro, we were also able to show that the regenerated gland also contained daughter mammary stem cells that could generate new structures in vivo [in secondary transplants],” says Eaves. Together, this information proved that regenerative cells do indeed exist in the population. By seeding collagen gels with different numbers of cells in the initial transplantation, the authors were able to work out that about 1 in 5,000 cells were stem cells. The researchers could also show that many of the progenitor cells able to make colonies in vitro were different from the cells that produced new mammary glands in vivo.

The method also allowed the group to determine cell-surface markers of the repopulating cells, including an important anchoring protein on the cell surface.

Jane Visvader, an expert in mammary gland development at The Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia, praises the technique for “bypassing inherent challenges” associated with the mammary fat pad implantation. “The work is particularly important because they have developed an efficient assay that allows quantitation of mammary stem cells.”

This could have important implications in our understanding of breast cancer. “It will be important to see how expression of this marker changes during tumorigenesis. Time will tell whether this is a useful marker of breast cancer stem cells,” says Visvader. But in the meantime, she added, “the assay offers an effective means of addressing the sensitivity of stem cells to growth factors, drugs, etc., in terms of studying their effect on proliferation and maturation.”