In experiments with fruit flies, Johns Hopkins scientists have restored the insect's sperm-making stem cells by triggering cells on the way to becoming sperm to reverse course. The unexpected findings are described in the May 13 issue of Science.
Like all stem cells, the fruit fly's sperm-making stem cells can renew themselves or can develop into more specialized cells -- eventually sperm in this case. While a few types of fairly specialized cells can naturally revert to their stem cell origins at times -- think regrowth of salamanders' lost limbs -- the researchers' experiments document what is thought to be one the first clear examples of an artificially triggered reversal of cell fate in an adult creature.
"With a few exceptions, it is thought that once cells start down the path toward specialization, they can't go back," says Erika Matunis, Ph.D., assistant professor of cell biology in Hopkins' Institute for Basic Biomedical Sciences. "But we've clearly shown in fruit flies that lost sperm-making stem cells can be replaced, not by replication of remaining stem cells, but by reversal of more specialized cells."
The Hopkins team studied fruit flies whose "don't-specialize" signal for stem cells can be turned on or off by changing the temperature around them. In experiments to examine what happens when the signal is turned off and then turned back on, second-year graduate student Crista Brawley discovered that cells that are two steps -- but not more -- away from their stem cell origins can revert to the more primitive state.
Understanding how and until what point specialized cells can reverse course might help scientists figure out how to use stem cells to regenerate lost or injured tissue, or how to trigger remaining tissue to better heal itself. There is no immediate application for people, however, because little is known about the corresponding process in humans.
"In fruit flies, we literally can count each stem cell in the testis because we can detect proteins that distinguish them, and we know exactly where the stem cells are supposed to be," says Matunis. "In fruit flies, we also know the signal that keeps the stem cells in their primitive state, and we can turn it off and on. We don't have any of this information for people, or even for mice."
In the fruit flies, sperm are made by a system that consists of a raspberry-like cluster of sperm-making stem cells centered around a "hub."
The hub emits a chemical signal that tells only the closest cells to maintain their stem cell status. When one of these stem cells divides in two, the "daughter" cell nearest the hub remains a stem cell while the one slightly farther away gets one step closer to being sperm. These more-distant daughter cells, called gonialblasts, then become cells called spermatogonia, each of which divides a number of times before becoming spermatocytes, precursors to sperm.
To see whether lost stem cells could come back, Brawley put the temperature-sensitive fruit flies in warmer climes of 29 degrees Celsius (a balmy 84 degrees Fahrenheit) for two days, and discovered that only 22 percent of testes still contained sperm-making stem cells. After four days, almost no stem cells remained at all.
Brawley then used a special microscope to examine whether stem cells returned in flies allowed to "recover" at the cooler temperature of 18 degrees Celsius (about 64 degrees Fahrenheit) for two days after spending two days at the signal-squelching warmer temperature.
"We were very happy to see that stem cells did return," says Matunis, who discovered the hubs' signal in 2001 with Natasha Tulina.
But much to their surprise, while only 22 percent of testes had remaining stem cells going into the recovery period, 76 percent of them had stem cells after two days at 18 degrees.
"We had expected that remaining stem cells replenished the supply, but even in testes with none left, stem cells reappeared once the signal was turned back on," says Matunis. "So, the stem cells had to be coming from some other cell type within the testis."
These testes looked different, too, she says. Ones that had regained stem cells now lacked spermatogonia, and the raspberry-like cluster of stem cells essentially touched the spermatocytes in the researchers' microscopic pictures.
However, after four days of recovery, spermatogonia repopulated the real estate between the stem cells and the spermatocytes, indicating the new stem cells worked. In flies that had only spermatocytes left after life at 29 degrees, no stem cells returned, the researchers report.
Because these observations suggested that spermatogonia might be returning to their stem cell roots, Brawley labeled some of them with fluorescent markers and exposed the flies to the warmer temperature for four days. Sure enough, only those cells that retained spermatogonia regained stem cells, and some of the new stem cells were fluorescent, she says.
Now the scientists will examine whether this reversal, called "dedifferentiation," happens naturally in the flies, and whether spermatogonia retrace the path taken by stem cells -- a true reversal -- or whether they use different signals, proteins and processes to revert to stem cells.
The studies were funded by the National Institutes of Health. Authors on the paper are Matunis and Brawley, a student in Hopkins' Biochemistry, Cellular and Molecular Biology graduate program.
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