Isolating Mammary Gland Stem Cells


Female mammary glands are home to a remarkable population of stem cells that grow in culture as small balls of cells called “mammospheres.” Clayton and others were able to identify these stem cells in 2004 (Clayton, Titley, and Vivanco, Exp Cell Res 297 (2004): 444-60), and Max Wicha’s laboratory at the University of Michigan showed that a signaling molecule called Sonic Hedgehog and a Polycomb nuclear factor called Bmi-1 are necessary for the self-renewal of normal and cancerous mammary gland stem cells (Lui, et al., Cancer Res June 15, 2006 66; 606). The biggest problem with mammary gland stem cells is isolating them from the rest of the mammary tissue.

Mammary gland stem cells or MaSCs are very important for mammary gland development and during the induction of breast cancer. Getting cultures of MsSCs is really tough because the MaSCs share cell surface markers with normal cells and they are also quite few in number.

Gregory Hannon and his co-workers at Cold Spring Harbor Laboratory used a mouse model to identify a novel cell surface protein specific to MaSCs. By exploiting this unusual marker, Hannon and his team were able to isolate MaSCs from mouse mammary glands of rather high purity.

Camila Do Santos, the paper’s first author, said that “We are describing a marker called Cd1d.” Cd1d is also found on the surfaces of cells of the immune system, but is specific to MaSCs in mammary tissue. Additionally, MaSCs divide slower than the surrounding cells. Do Santos and her colleagues used this feature to visually isolate MaSCs from cultured mammary cells.

They used a mouse strain that expresses a green glowing protein in its cells and then made primary mammary cultures from these green glowing mice. After shutting of the expression of the green glowing protein with doxycycline, the cultured cells divided, and diluted the quantity of green glow protein in the cells. This caused them to glow less intensely. However, the slow-growing MaSCs divided much more slowly and glowed much more intensely. Selecting out the most intensely glowing cells allowed Dos Santos and her colleagues to enrich the culture for MaSCs.

“The beauty of this is that by stopping GFP expression, you can directly measure the number of cell divisions that have happened since the GFP was turned off,” said Dos Santos. She continued: “The cells that divide the least will carry GFP the longest and are the ones we characterized.”

Using this strategy, Dos Santos and others selected stem cells from the mammary glands in order to examine their gene expression signature. They also confirmed that by exploiting Cd1d expression in the MaSCS, in combination with other techniques, they could enhance the purity of the cultures several fold.

Hannon added, “With this advancement, we are now able to profile normal and cancer stem cells at a very high degree of purity, and perhaps point out which genes should be investigated as the next breast cancer drug targets.”

Will we be able to use these cell for therapeutic purposes some day?  Possibly, but at this time, more must be known about them and MaSCs must be better characterized.

Taste Stem Cells Identified


Researchers at the Monell Center in Philadelphia, PA have successfully identified the location and markers for taste stem cells on the tongue. These findings will almost certainly allow scientists to grow and manipulate taste cells for clinical and research purposes.

Neurobiologist Robert Margolskee with the Monell Center who was also one of the authors of this study said: “Cancer patients who have taste loss following radiation to the head and neck and elderly individuals with diminished taste function are just two populations who could benefit from the ability to activate adult taste stem cells.”

Taste cells are located in rosette-like clusters known as taste buds in bumpy structures called papillae on the upper surface of the tongue. Two types of taste cells contain chemical receptors that initiate the perception of sweet, bitter, unami salty, and sour taste qualities. A third type of taste cell appears to serve as a support cell for these taste cells.

Gustatory_receptor_cell

A truly remarkable characteristic of these sensory cells is that they regularly regenerate, and all three taste cells undergo frequent turnover. The average lifespan of these cells is 10-16 days, which means that constant regeneration must occur in order for these cells to replace the cells that constantly die.

For decades, scientists who study taste have tried to identify the stem cell population that form these different taste cells. Scientists were also completely uncertain as to the location of these taste cell progenitors. Where they in the taste buds, near the taste buds, or someplace entirely different?

Monell scientists drew upon the strong association between oral taste cells and endocrine cells in the intestine. They reasoned that the cell-surface markers for stem cells in the intestine might also serve as markers for stem cells in the tongue. By using antibodies to a surface protein called Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5), the Monrell team observed two strong expression patterns for this marker in the tongue. One signal was underneath taste papillae at the back of the tongue and the second signal was an even weaker signal underneath taste buds in those papillae.

The Monell group hypothesized that the two levels of expression could indicate two different populations of cells that expressed Lgr5 at different levels. The stronger-expressing cells are probably the actual stem cells and those that more weakly express Lgr5 are those progeny of these stem cells that are beginning to differentiate. Therefore, the expression of the stem cell marker in these cells is fading.

Additional work showed that Lgr5-expressing cells were capable of differentiating into any of the three different types of taste cells.

Peihua Jiang. who is also a neurobiologist at the Monell Center, said: “THis is just the tip of the iceberg. Identification of these cells opens up a whole new area for studying taste cell renewal, and contributes to stem cell biology in general.”

In the future, the Monell group plans to program the Lgr5-expressing cells to differentiate into the different taste cell types, and explore how to grow these cells in culture. This will create a renewable source of taste receptor cells for research and perhaps even clinical use.

See Karen Yee, et al., “Lgr5-EGFP Marks Taste Bud Stem/Progenitor Cells in Posterior Tongue.” Stem Cells 2013 DOI: 10.1002/stem.1338.