Dec. 12, 2003 Researchers at the Howard Hughes Medical Institute at The Rockefeller University have discovered a new method to track and isolate elusive stem cells. The new animal model they developed was successfully tested by isolating and characterizing skin stem cells, but may also be valuable in searching for stem cells that produce the cells of the heart, pancreas or other specific body tissues.
The finding, to appear in the journal Science and reported online in the Dec. 12 issue of Science Express, offers promise for what could be the first broadly adaptable way to find such master cells, which can create tissue as needed and are seen as the foundation for regenerative medicine. Before this finding, the only stem cells that had been isolated and characterized were from blood, nerve cells and embryonic tissue.
In order to avoid accumulating damaging mutations, long-lived stem cells in their natural home (niche) are often "slow cycling," dividing only infrequently. However, upon injury or normal "wear and tear," stem cells are mobilized to leave their niche, divide and replenish the damaged tissue. The study provides a list of more than 150 genetic factors that distinguish the long-lived, "slow cycling" stem cells of the skin from their short-lived, rapidly dividing "daughter cells."
This information will help scientists understand how these mysterious cells are able to replenish both skin epidermis and hair, and what nutrients may help stem cells produce more stem cells in the laboratory. In addition, some of these new genes are likely to serve as markers for these stem cells, making them easier to identify and isolate in the future.
"We now have a much better picture of the traits of mouse skin stem cells, and we can now use these genetic data to examine whether human skin stem cells possess similar traits to the mouse stem cells," says the study's lead investigator, Elaine Fuchs, Ph.D., professor and head of the Laboratory of Mammalian Cell Biology and Development at Rockefeller and an investigator at HHMI.
Fuchs suggests that the skin stem cell model system could hold promise for a variety of medical applications, such as improved regeneration of skin epidermis for burn victims and new understanding of what prompts skin stem cells to grow new hair.
"And this powerful tool may now help us to identify stem cells in other tissues that undergo extensive rejuvenation, like those in the gut and in cornea. We may even be able to identify and isolate mutated stem cells that lead to certain kinds of cancer," Fuchs says.
The system, which took several years to develop, pairs two kinds of genetically altered mice -- one of which carries a fluorescent protein and the other a gene that activates it -- to create mice offspring that will bathe the skin's slow cycling cells in a green glow.
"Now anyone can use the mouse model to search for the stem cells of their choice," says first author Tudorita Tumbar, Ph.D., a postdoctoral associate in the Fuchs lab, who led the effort to create the mouse model.
Because they are so powerful, and so few in number, stem cells are used sparingly by the body, and are tucked away in protected places. In the skin, researchers have long suspected that the "slow cycling" cells are actually stem cells. These slow cycling cells have been found in a "niche," a tiny bulge halfway up the side of a hair follicle shaft.
Similar niches have been found in the eye's cornea and in intestinal cells, and are suspected to be present in several different tissue systems.
Researchers discovered several years ago that skin niche cells (so called label-retaining cells) in mice that had been tagged with a chemical marker could travel out of the niche, and move down to the bulb of the hair follicle to form new hair or move up to create new skin epidermis. But scientists were unable to isolate these label-retaining cells, and hence had only a scant view of the properties of these very special skin cells.
Reasoning that some of these label-retaining cells were skin stem cells, the Fuchs team developed a system to identify them, isolate them and compare their genetic profile with known stem cells.
The system, dubbed "pulse and chase," required development of two different genetically altered mice. In one mouse, a fusion gene (histone H2B-GFP) that can express a green fluorescent protein when cells divide was introduced into a mouse embryo. But in order for the mouse to express this green fusion protein, it needed to be turned on by an "activator" protein. That is where the second mouse came in. The Fuchs lab mated their mouse to one that possessed a second fusion gene (keratin 5-tetracycline-off-activator) that only produces the "activator" protein in the skin epithelial cells. In the final transgenic mice, the glowing histone-H2B-GFP protein was made in all the skin epithelial cells, where it entered the nucleus and bound to DNA. That was the "pulse" part of the formula.
To turn the fluorescent protein off and watch what happened in the "chase," the researchers simply added tetracycline to the mouse food, and no more fluorescent protein was made in the skin. Those cells that rapidly divide soon diluted out their glow and became dim, whereas cells that slowly cycled - the putative stem cells - were the only ones left that glowed brightly. Some cells, the slow-cycling cells, glowed for weeks after others had stopped, says Tumbar.
"After a month the remaining glowing cells, which we predicted to be stem cells, were in the bulge," Tumbar says.
The researchers took those cells out of the bulge, isolated their RNA, and used microarray technology to obtain their "transcriptional profile." (The DNA in genes produces RNA, which codes for protein production). A number of the 150-plus sequences code for proteins that are found on the surface of the slow-cycling stem cells or are secreted by them. Those proteins are new markers that may help find stem cells in human tissue, the researchers say.
The researchers then compared genes found in the skin stem cell niche with those found in blood, neuronal and embryonic stem cells. They found that 40 percent of the genes expressed in slow-cycling skin cells, compared with the genes found in their daughter cells, were also expressed in the other stem cell types. And 80 to 90 percent of the genes expressed in the skin cells were expressed in at least one of the three other stem cell populations.
"This comparison sets the ground for future research regarding similarities between slow-cycling cells and other stem cells," says Tumbar.
Another ongoing comparison between the transcriptional profile of the skin slow-cycling cells (compared to their progeny) and blood stem cells (compared to their progeny) shows at least a 10 percent match, so far, Tumbar says. "This suggests possible commonalities in how different adult stem cell types distinguish themselves from their own progeny," Fuchs explains.
"Previously we knew of just a handful of proteins that distinguish skin stem cells from their progeny, and now we know of more than 100," says Fuchs. "The laboratory is now working to understand how these proteins define the characteristics of these stem cells, such as their slow-cycling properties, their ability to be mobilized to repair skin epidermis in response to wound injuries, and their ability to grow new hair in the normal course of rejuvenation. We are just at the tip of the iceberg."
Finally, the pulse-and-chase system described in the Science paper can be used to hunt for stem cells in other tissue types. Researchers can use the histone H2B-GFP fusion gene mouse as it is, with no modifications, and just mate it with a mouse they expressing the activator gene in their favorite cells or tissue, the investigators say.
Also participating in the research were, from the Fuchs laboratory, were Geraldine Guasch, Ph.D., Valentina Greco, Ph.D., Cedric Blanpain, Ph.D., William E. Lowry, Ph.D., and Michael Rendl, Ph.D.
This research was supported by the Howard Hughes Medical Institute, National Institutes of Health, Human Frontier Science Program, European Molecular Biology Organization, NATO, BAEF, Life Sciences Research and Shroedinger foundations.
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