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.
GO TO