S T A N F O R D M E D I C I N E
Volume 18 Number 1 Winter/Spring 2001
BY KRISTA CONGER
HELEN M. BLAU, PHD, has spent most of her academic career demonstrating that adult cells are more responsive to their environment than many doctors and researchers think. But her lab's recent find- ing that cells from the bone marrow may be able to form new neurons in the brain surprised even Blau, professor and chair of Stanford's molecular pharmacology department and director of gene therapy technology.
Most scientists and physicians, including Blau, have been taught that nerve cells are a precious, irreplaceable commodity. Unlike the cells that make up your skin, intestine and liver, nerve cells are unable to replace their old, worn out relatives. And almost none of the body's mature cells are able to switch roles and become a different kind of cell altogether. While a single fertilized egg can give rise to all the cells of the body, adult cells are believed to have a much narrower range of career choices. Most researchers believe that nearly all adult cells are so highly specialized that they can't become any other type of cell -- they're known as "terminally differentiated."
A few cell types, particularly stem cells found in bone marrow, are less set in their ways. Blau's interest in cell fate and differentiation has led her to study adult bone marrow because of its ability to generate several different types of blood cells. In fact, recent research -- some of which was conducted by members of Blau's lab -- has shown that cells from the bone marrow can, surprisingly, transform themselves into liver or muscle cells. Scientists don't know precisely why the switch occurs or which bone marrow cells are involved.
But in contrast to bone marrow, nerve cells' options are limited. Until recently, scientists believed they couldn't regenerate themselves at all. Now, research has shown that adults can make new neurons when existing cells are damaged. For reasons not understood, though, the new cells aren't able to completely repair the brain after a stroke or following a forceful trauma like that suffered during a car crash or a skiing accident. These brain injuries, as well as neurodegenerative diseases like Alzheimer's and Parkinson's, can often lead to varying degrees of incapacitation with no cure in sight.
Blau's recent research may eventually lead to new therapies for these patients. She and her lab members have shown that adult bone marrow cells can travel to the brain. Even more startling: some of the newcomers began to express neuronal-specific proteins and mimic the shape of their neuronal neighbors.
"We are really excited," says Blau. "You might expect this type of result with fetal cells, but with adult cells it's really amazing."
It's amazing for two reasons: specialization and location. Not only are the cells starting to look and act like "irreplaceable" neurons, but they undertake their trek to the brain without any outside urging.
"These are cells under normal influences, without perturbation," Blau says.
The therapeutic possibilities are tantalizing. If the cells can be coaxed to evolve into functioning neurons -- the researchers are testing this now -- they may be able to pinch-hit for their nonfunctional counterparts. And even if they can't fill in for neurons, it's possible they could be genetically modified to deliver proteins missing in the brains of people with diseases like Parkinson's or Huntington's.
The cells' ability to make their own way to the brain is a key point when planning potential therapies. Bone marrow cells can be harvested from the pelvic bone under local anesthesia, genetically engineered to produce a particular protein and returned to the body with a simple transfusion procedure. If these cells could then be encouraged to migrate to the brain in large numbers and replace damaged neurons, doctors might be able to achieve a perfect immunologic match without having to penetrate the skull.
And if the cells don't need to be genetically modified, it may one day be possible to send an army of bone marrow cells to the rescue by mimicking the signal that calls the cells to the brain, without ever removing them from the body.
PRIOR TO BLAU'S FINDING, the most promising avenue of research for people with neurological damage involved supplementing the remaining neurons in the brain with fetal tissue or embryonic cells. When placed in the right envi-
ronment, the young cells seem to be able to begin forming new neurons. But ethical concerns about the use of aborted fetal tissue or embryos "left over" from in vitro fertilization procedures have made the starting material scarce, and there's always the possibility that the patient's immune system will reject the unfamiliar cells. Additionally, because the fetal cells pretty much stay put, they have to be delivered directly into the brain through the skull to do any good.
"We're not poking holes in the brain. This is far less invasive," Blau says.
Blau has nothing but kudos for her lab members who tracked the bone marrow cells to the brain. Graduate student Timothy Brazelton initiated the research. Later several lab members, including senior scientist Fabio Rossi, MD, PhD, and postdoctoral fellow Gilmor Keshet, PhD, launched a coordinated, determined effort to prove the finding beyond a shadow of a doubt.
"I have tremendous admiration for their work," Blau says. "They worked as a team to prove these cells were in the brain."
Brazelton first became interested in cell flexibility, or plasticity, when he was studying the mechanisms of chronic lung rejection in the laboratory of cardiothoracic surgery professor Randy Morris, MD, director of the laboratory for transplantation immunology at Stanford.
Brazelton noticed that the clumps of cells which clog the airways of the donated lung and contribute to transplant rejection are primarily made of the recipient's own fibroblasts -- cells that, according to traditional wisdom, weren't supposed to be flitting around willy-nilly. The fibroblasts' unusual placement hinted at the existence of a circulating progenitor that was somehow stimulated to settle down in the new tissue.
The migrating cells intrigued Brazelton, who moved to Blau's laboratory because he wanted to know more about this unexpected ability of adult cells to be "plastic" -- to respond to changing environments by switching their locations or functions. Blau was well-known for her work in this area, and the molecular pharmacology department offered a PhD to go with the MD Brazelton had been pursuing.
In Blau's lab, Brazelton switched from studying lung rejection to investigating bone marrow. The plan was to track down what organs might be harboring the changeling cells that originated in the bone marrow to better understand why and how often such cellular switches occur.
Brazelton used bone marrow cells that had been engineered to express a protein that glows green (green fluorescent protein, or GFP) to track the cells' movement. He injected the cells into the tail veins of normal mice whose bone marrow had been destroyed with radiation -- a typical bone marrow transplant procedure.
"The idea was that we would evaluate every organ in the body," says Brazelton. Green, nonblood cells would indicate cells that had originally been bone marrow and had changed their function. The researchers included brain on their list of organs to be investigated, although they didn't really expect the cells to be able to bypass the notorious blood-brain barrier.
When the brains of the mice were analyzed two to six months after the transplant, Brazelton was surprised. He saw labeled cells that looked a lot like neurons sprinkled throughout the central nervous system.
Most of the donor cells were found in the olfactory bulb, which undergoes a high rate of regeneration in rodents, perhaps due to their reliance on their sense of smell. Cells expressing GFP were also found in the hippocampus, cortical areas and cerebellum -- areas responsible for a variety of functions in humans, including learning and memory, conscious thought and emotion.
Brazelton and Blau were both initially skeptical about the finding but Rossi, who had previously studied the plasticity of hematopoietic cells at the European Molecular Biology Laboratories, decided to examine whether specific antibodies could identify surface markers on the GFP-expressing cells. When they used the antibodies to look at the cells more closely, the researchers found that about 5 percent of the cells expressing GFP in the brain no longer expressed surface markers indicative of bone marrow cells, suggesting they had begun to assume a new role.
To be certain that the GFP-expressing cells were actually mimicking neurons, Brazelton examined many hundreds of individual cells using a confocal laser scanning microscope, a laborious and time-consuming process. To avoid interfering with other scientists' research, Brazelton worked from 8 p.m. to 8 a.m. for several months.
"He essentially changed his circadian rhythms to complete the project," says Blau. "I would bring him bagels in the night and we would talk about the research."
BUT THE LONG NIGHTS WERE WORTH IT. When Brazelton microscopically examined individual donor cells in the olfac-tory bulb, he was able to tell that the cells expressed multiple proteins specific to neuronal cells.
"This is the first time that these bone marrow-derived cells have been found in the brain," says Blau.
To confirm their results and determine what further experiments were needed, the Blau lab collaborated with neurobiologist Sue McConnell, PhD. McConnell, a biological sciences professor, reviewed their data at monthly intervals.
"When Sue said she was convinced, I was too," Blau says.
It's not yet known what calls these cells so far from home, Blau says. And the relative number of migrating cells is low: About 0.2 to 0.3 percent of the cells in the olfactory bulb expressed GFP. But the ability of the cells to migrate from the bone marrow to these areas and express neural proteins has exciting therapeutic possibilities.
The fact that the cells were found mostly in the olfactory bulb in the mice doesn't bother Blau. Mice depend on their noses to a much greater extent than do humans, who devote a larger portion of their brains to cognitive functions.
"It makes biological sense," says Blau. "In humans you would expect to see something quite different. We hope these cells will be concentrated in the area of regeneration and memory."
Although Blau cautions that this research hasn't shown the bone marrow-derived cells actually function as neurons, one experiment by Keshet indicated the new cells are able to activate a common transcription pathway in concert with their neighbors. The result suggests the cells may be able to respond appropriately in their new environment.
Blau's team is working to increase the number of cells migrating to the brain in order to test their function and maximize therapeutic potential.
"Probably this migration is going on at a low rate all the time," says Blau. "But it's not high enough to help fight degeneration from disease, or injury from stroke or trauma. We need to learn to enlist this ability." The team would like to investigate whether the migration rate increases in response to damage. If so, they might one day be able to use this to their advantage.
"If we can show that injury and damage increase proliferation and differentiation, it may help us develop potential treatments," Keshet says.
To do that, it's necessary to understand the signals that beckon the bone marrow cells to the brain and tell them to express neuronal-specific proteins. Blau and her lab members would also like to determine whether only a specific subset of bone marrow cells are capable of responding to the call or if any bone marrow cell can assume neuronlike characteristics under the proper conditions.
"We don't know if it's one of the original stem cells, or one of the progeny," says Rossi, who is using a fluorescence-activated cell sorting machine to try to determine which population of bone marrow cells is responsible. Rossi is also continuing to characterize the neuronlike cells to determine how far the cells have traveled on the path to a complete career change.
Additionally, senior scientist Jim Weimann, PhD, is probing whether the conversion from bone marrow can occur in the culture dish. If so, it may be possible to identify biological signals that encourage the change to occur. Weimann, previously from McConnell's lab, is also poised to test whether the new cells can transmit action potentials.
"This discovery has opened up a lot of interesting research areas," Blau says. "What's badly needed in this field are ways to characterize these cells. Does damage attract them? Or do they respond to certain growth factors? Clearly, the potential to regenerate at will all sorts of adult tissues would have tremendously exciting clinical applications.
"There are so very many questions that just fascinate me. I feel like a kid in a candy store," Blau says. SM