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St. Louis, Nov. 29, 1999 - Scientists have developed a treatment that improves the locomotion of rats when given more than a week after spinal cord injury. They turned embryonic stem cells into precursors of nerve cells and transplanted the precursors into the injury site. Inside the animals, some of the cells survived and developed into the three major types of cells needed for spinal cord repair. In the future, the researchers hope to use this approach to design repair strategies for humans.
"Establishing regenerative therapies that promote substantial improvements in locomotion when instituted after the injury process is complete has been a difficult obstacle," says John W. McDonald, M.D., Ph.D. "This is a key threshold that has not before been crossed."
McDonald and colleagues report the advance in the December issue of Nature Medicine. McDonald is an assistant professor of neurology at Washington University School of Medicine in St. Louis and director of the Spinal Cord Injury Unit at the medical school and Barnes-Jewish Hospital.
"The biggest problem in treating spinal cord damage is helping people with existing injuries regain function," says Dennis W. Choi, M.D., Ph.D., the Andrew B. and Gretchen P. Jones Professor and head of neurology. "This paper is the first to report a delayed treatment that promotes recovery."
Between 250,000 and 500,000 Americans have spinal cord injuries. Because many of them are young, they will spend decades in a wheelchair unless new therapies can be developed. "You might need to repair only a small fraction of the cord's damaged connections to convey valuable benefits to patients," says Choi, who also is neurologist-in-chief at Barnes Jewish Hospital.
The neurons in the spinal cord have arms called axons that can extend up to several feet in humans. Serving as telephone wires, the axons carry messages between the brain and the rest of the body. But traumatic injury to the spinal cord can kill neurons or sever their axons, interrupting this flow of information. "One of the biggest limitations to recovery of function is that the mammalian central nervous system isn't capable of generating a sufficient number of cells to replace those that are lost through injury," McDonald says. "So transplantation seems like a possible solution."
Unlike hearts or kidneys, pieces of adult spinal cord don't survive transplantation. So researchers have tried transplanting embryonic neural tissue, which survives but doesn't divide and therefore can provide only limited numbers of replacement cells. Building on this initial progress, some investigators are exploring the transplantation of nerve cell precursors isolated from the adult nervous system.
The Washington University researchers took another promising extension to this approach, using undifferentiated cells called embryonic stem (ES) cells, which were derived from mouse embryos. During development, ES cells give rise to all of the different cell types in the body, though any particular cell's fate depends on the chemical signals it receives. In 1994, David I. Gottlieb, Ph.D., a professor of neurobiology, discovered that a precisely timed treatment with retinoic acid, a chemical used by the developing nervous system, prompts cultured embryonic stem cells to become nerve cell precursors. "These precursors are very exciting cells," Gottlieb says, "because in gene expression, morphology and physiology they are indistinguishable from normal neurons."
The research team treated rats nine days after a thoracic-level spinal cord injury that affected the animals' hind legs. They transplanted about 1 million nerve cell precursors - derived from mouse embryonic stem cells - into a fluid-filled cavity that had developed at the injury site. To prevent rejection, they also gave the animals the immunosuppressant drug cyclosporine, which is used for organ transplantation in humans.
Two weeks to five weeks later, the researchers looked for the transplanted cells, which they had labeled in various ways, including with genetic markers. They also used specialized techniques to identify any axons that had sprouted from the transplanted cells.
Some of the cells survived. By two weeks, transplanted cells had filled the cavity, and some had migrated up to 1 cm in both directions, a distance that spans several segments of a rat's spinal cord. By five weeks, the cells were not as dense, but the injured region contained mouse axons.
By following the injured cord's chemical cues, the precursors had differentiated into some of the appropriate cells for repair - neurons, which transmit information; oligodendrocytes, which wrap the axons of neurons in the fatty sheath needed for efficient conductance; and astrocytes, which maintain an optimal environment for nerve cell function. None of the cells had divided into tumors.
Using an open field locomotor test to assess voluntary locomotion, the researchers compared the performance of the transplanted rats with the performance of injured rats that had undergone sham operations. One month after the surgery, the hind limbs of the control rats could move but not in a coordinated fashion. They also were completely unable to support the weight of the body. But the hind limbs of the transplanted rats had partly regained some coordinated movement. They also were able to partly support the body's weight.
"Their walking certainly wasn't normal," McDonald says. "But this functional recovery was especially encouraging because the precursor cells were transplanted nine days after the spinal cord injury - a time period that has not been explored before. Moreover, only a small percentage of the transplanted cells survived. If cell survival could be enhanced, it might be possible to restore bowel and bladder control or even walking."
To approach this goal, the researchers plan to generate designer ES cells through genetic manipulation. For example, it should be possible to inactivate genes that make cells commit suicide, preserving a larger proportion of the transplant. Yet other genetic modifications might persuade all of the transplanted cells to develop into, say, oligodendrocytes, if re-insulation of intact axons was a priority. The researchers also could turn ES cells into factories that manufacture growth factors known to promote nerve cell survival. "Embryonic stem cells have an added advantage of being genetically flexibile," McDonald says. "So we feel they may be a good source of cells to replenish those lost from the injured spinal cord."
Although the investigators currently are using ES cells from rodents, they would like to experiment with human ES cells. In 1998, a group at the University of Wisconsin in Madison and a group at Johns Hopkins School of Medicine in Baltimore isolated stem cells from human embryos and made them grow into cell lines that potentially could divide forever. Such human ES cells are not yet available for federally funded research, however, because a U.S. law forbids human embryo studies. Whether research with cultured ES cells should fall under this law is currently under debate. "Our stem cell research is encouraging," McDonald says, "because right now there is no regenerative therapy for humans with spinal cord injury."
Grants from the National Institute of Neurological Disorders and Stroke, the National Center for Research Resources, the Alan A. and Edith L. Wolff Charitable Trust, the Christopher Reeve Paralysis Foundation and the Keck Foundation supported this research.
McDonald JW, Liu X-Z, Qu Y, Liu S, Mickey SK, Turetsky D, Gottlieb DI, Choi DW. Transplanted embryonic stem cells survive, differentiate, and promote recovery in injured rat spinal cord. Nature Medicine, December 1999.
See also: McDonald JW and the Research Consortium of the Christopher Reeve Paralysis Foundation. Repairing the damaged spinal cord. Scientific American, vol. 281, pp. 64-73, September 1999.
Journal
Nature Medicine