Apr 28 2004
In experiments with rodents, Johns Hopkins scientists have used properly directed stem cells to successfully overcome what is thought to be a basic hurdle in restoring function to severely damaged central nervous systems -- getting new motor neurons to migrate through the spinal cord.
In their experiments, the researchers first coaxed embryonic stem cells from mice to begin their transformation into motor neurons. Once these "pre-motor neurons" were implanted into the spinal cords of paralyzed rats, a constant drip of molecules that block the nerve-repelling activity of the spinal cord's myelin sheath gave the cells a chance to break through.
About 80 of an initial 12,000 neurons-in-training implanted into each rat became full-fledged motor neurons and pushed their finger-like extensions, called axons, through the spinal cord, the researchers reported April 26 in the Advance Online section of the Proceedings of the National Academy of Sciences.
"We think that getting new motor neurons to travel properly through the cord is the major hurdle to try to restore muscle control," says Douglas Kerr, M.D., Ph.D., an assistant professor of neurology. "It's significant that axons from these motor neurons make it outside of the cord."
However, he cautions that much remains before stem cells or cells derived from them could be useful for restoring lost or "broken" neurons in people. For example, in their experiments, even though some of the new neurons reached through the myelin coating, they didn't get much farther down the road to the real target -- muscles.
Furthermore, to approach therapy, Kerr says, these and other experiments need to be done with human embryonic stem (ES) cells that may one day be clinically useful. However, the human ES cell lines approved for research under federal grants -- by far the largest source of funding for academic researchers -- have been grown in the presence of mouse cells, which many scientists believe makes them unsuitable for use in people.
"Our work does lay an important and critical early foundation for techniques that we hope will restore function to nervous systems damaged by amyotrophic lateral sclerosis (ALS), spinal motor atrophy (SMA), and other degenerative motor neuron diseases," says Jeffrey Rothstein, M.D., Ph.D., professor of neurology and director of the Packard Center for ALS Research at Johns Hopkins, one source of funding for the work. "But there's a long way to go."
In preliminary test-tube studies, the researchers saw that their ES-derived motor neurons and healthy muscle cells "conversed," each pumping out -- within hours -- agents needed to pave the way for the cells' "hookup."
In the lab dishes, Kerr saw the new motor neurons grow outward toward the muscle cells and form proper connections with proper receptors. The muscle cells that were connected to nerve cells even began to contract in the laboratory dish.
To investigate whether the phenomenon would happen in animals, the team of scientists implanted the pre-motor neurons into the spinal cords of paralyzed adult rats, easing them into areas typically rich in motor neurons. "We transplanted roughly 12,000 cells per animal, and about 4,000 of them 'took,'" says Kerr. "They became true motor neurons and looked gorgeous."
In these first animal studies, however, the axons of these new neurons didn't poke through the spinal cord and out to muscle targets. Kerr reasoned that, because myelin is a potent inhibitor of axon growth, the myelin-coated neurons that ring the spinal cord were probably trapping the new motor neurons inside.
Fortunately, other scientists had recently uncovered molecules that can block myelin's axon-repelling effect. So the team implanted the rats with a pump to drip these myelin "de-squelchers" in the vicinity of the animals' spinal cords. As a result, some motor neurons extended themselves through the cord.
To try to coax the new axons to finish their journey to muscle, the team now is experimenting with several neural growth factors. Kerr also says their laboratory system that shows how ES cell-derived motor neurons and muscle cells normally behave is going to be an important tool in understanding and perhaps combating human motor neuron diseases.
Already, he is culturing muscle cells with ES cells taken from mouse models of ALS and SMA. He hopes to plot what goes wrong and when at the molecular level. "We should be able to see where normal molecular pathways are disrupted in these models of motor neuron disease," he says.
The study was funded by the Families of SMA, Andrew's Buddies/Fight SMA, the Robert Packard Center for ALS Research at Johns Hopkins and the Katie Sandler Fund for Research at Johns Hopkins.
Authors on the paper are Kerr, Rothstein, James Harper, Chitra Krishnan, Deepa Deshpande, Schonze Peck, Irina Shats and Stephanie Backovic, of the Johns Hopkins School of Medicine; and Jessica Darman of the Johns Hopkins Bloomberg School of Public Health, of which Kerr is also a faculty member.
On the Web:http://www.pnas.org