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The newspaper of The Johns Hopkins University June 26, 2006 | Vol. 35 No. 38
Embryonic Stem Cells Awaken Latent Nerve Repair

JHU scientists engineer working motor neuroncircuits in paralyzed rats

By Marjorie Centofanti
Johns Hopkins Medicine

In a dramatic display of stem cells' potential for healing, a team of Johns Hopkins scientists reports that it has engineered new, completed, fully working motor neuron circuits — neurons stretching from spinal cord to target muscles — in paralyzed adult animals.

The research, in which mouse embryonic stem cells were injected into rats whose virus-damaged spinal cords model nerve disease, shows that such cells can be made to retrace complex pathways of nerve development long shut off in adult mammals, the researchers say.

"This is proof of the principle that we can recapture what happens in early stages of motor neuron development and use that to repair damaged nervous systems," said Douglas Kerr, a neurologist who led the Johns Hopkins team.

Elias A. Zerhouni, director of the National Institutes of Health, which provided a grant for the study, said, "It's a remarkable advance that can help us understand how stem cells can begin to fulfill their great promise. Demonstrating restoration of function is an important step forward, though we still have a great distance to go."

The researchers created what amounts to a cookbook recipe to restore lost nerve function, Kerr said. The approach could one day repair damage from such diseases as ALS (Lou Gehrig's disease), multiple sclerosis and transverse myelitis, or from traumatic spinal cord injury, the researchers say. "With small adjustments keyed to differences in nervous system targets," Kerr said, "the approach may also apply to patients with Parkinson's or Huntington's disease."

In a report on the study, released online today, June 26, in the Annals of Neurology, the Johns Hopkins team says that 11 of the 15 treated rats gained significant, though partial, recovery from paralysis after losing motor neurons to an aggressive infection with Sindbis virus — one that, in rodents, specifically targets motor neurons and kills them. The animals recovered enough muscle strength to bear weight and step with the previously paralyzed hind leg.

Kerr likens the approach to electrical repair. "Paralysis is like turning on a light switch and the light doesn't go on," he said. "The connectivity is messed up, but you don't know where. We've asked stem cells to go where needed to fix the circuit."

For a brief period after a nerve dies, it leaves behind what's essentially an empty shell, with some scaffolding and non-nerve substances remaining. "But with [embryonic stem cell] injections at the right time and place, and by adding the right cues, we've learned to restore the biological 'memory' for growing neurons, which is clearly still in place," he said.

The motor circuit engineering combines recent discoveries on stem cell differentiation, a growing understanding of early development of the nervous system and insights into behavior of the nervous system in traumatic injury, Kerr noted.

"As adults, our cells no longer respond to early developmental cues because those cues are usually gone," Kerr said. "That's why we don't recover well from severe injuries. But that's what we believe we have changed. We asked what was there when motor neurons were born, and specifically what let motor neurons extend outward. Then we tried to bring that environment back, in the presence of adaptable, receptive stem cells."

In the study, Kerr's team pretreated cultures of mouse embryonic stem cells with growth factors that both increase survival and prompt specialization into motor neurons. Adding retinoic acid and sonic hedgehog protein — agents that direct cells in the first weeks of life to assume the proper places in the spinal cord — readied the conditioned embryonic stem cells for the motor neuron circuit that starts in the spinal cord. Then, stem cells were fed into the paralyzed rats' spinal cords.

Extending new motor neurons in an adult nervous system, however, meant overcoming hurdles. One involved myelin, the fatty material that insulates mature motor neurons. Like the coating on electrical wire, myelin prevents weakening of the traveling electrical impulse and lets it continue long distances. In humans, the myelinated sciatic nerve, for example, exits the spinal cord and extends to the leg muscles it activates, carrying impulses several feet.

Once laid down, however, myelin inhibits further nerve growth — nature's way of discouraging excessive wiring in the nervous system.

"We had to overcome inhibition from myelin lingering in the dead nerve pathways," Kerr explained. Two recently developed agents, rolipram and dbcAMP, enabled that.

The assorted treatments let the new motor neurons survive, grow through the spinal cord and extend slightly into the outlying nervous system. A second hurdle remained in getting the neurons to skeletal muscle targets.

As suggested by earlier work by team member Ahmet Hoke on repair in the outlying, peripheral nervous system, the researchers applied GDNF, a powerful stimulator of neuron growth, to the remains of the newly dead sciatic nerve at a point near its former leg muscle contacts. GDNF attracted the extending motor neurons, "luring" them to the muscles.

To ensure a continuous supply of GDNF, the researchers relied on injected fetal mouse neural stem cells, a known source of the molecule.

Of some 4,100 new motor neurons created in the spinal cord, roughly 200 exited the cord and 120 reached skeletal muscle, forming typical nerve-muscle junctions, with appropriate, typical chemical markers.

Microscopically, the neurons and their muscle associations appear identical to natural ones in healthy animals.

Fifty of the new neurons were found to carry electrical impulses. (Because such testing is time and labor intensive, only a small area of leg muscle was assayed. The improved ability of treated rats, however, suggests more functional neurons are likely.) The rats gained weight and were more mobile in their cages, and measures of muscle strength increased.

Animals treated without even one component of the "cocktail" experienced no such recovery. Novel ways of tracing the neurons back to their source assured the scientists that they indeed had come from the injected stem cells not from lingering host neurons.

Research begins this summer to see how well the technique applies to human nerve recovery, using federally approved human embryonic stem cells in larger mammals like pigs, Kerr said. Each of six academic institutions in a new collaboration will tackle a different major question of safety and effectiveness. Questions of tumor formation, often a concern with embryonic stem cells; of the safety of surgery; and of the embryonic stem cells' ability to form healthy motor circuits are major questions to answer. Several years of testing and thorough data evaluation would occur before applying to the FDA to approve human clinical trials.

The study was supported by Families of SMA, Andrew's Buddies/Fight SMA, the ALS Association and the Robert Packard Center for ALS Research at Johns Hopkins, the Muscular Dystrophy Association, Wings Over Wall Street and a grant from the NIH.

Kerr is a grantee of the Packard Center for ALS Research at Johns Hopkins. He also directs Project RESTORE, a Johns Hopkins-based undertaking to advance therapies for transverse myelitis and multiple sclerosis.

Others on the research team from Johns Hopkins are Jeffrey Rothstein, Hoke, Nicholas Maragakis, Yun Sook Kim, Sonny Dike, Deepa Deshpande, Chitra Krishnan and Jennifer Drummond, all of the Department of Neurology at the School of Medicine; and Jessica Carmen, Tara Martinez and Irina Shats, all of the Department of Molecular Microbiology and Immunology at the Bloomberg School of Public Health. Jeremy Shefner, of the Department of Neurology at the State University of New York Upstate Medical University, also contributed to the study.


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