"Our first hypothesis was that functional recovery came from human cells reconstituting the nerve circuits destroyed by the paralysis-inducing virus we gave the rats," says first author Douglas Kerr, M.D., Ph.D., assistant professor of neurology at the Johns Hopkins School of Medicine. "Some of the tens of thousands of implanted primitive human stem cells did become nerve cells or the like, but not enough to account for the physical improvements.
"Instead, these human embryonic germ cells create an environment that protects and helps existing rat neurons -- teetering on the brink of death -- to survive," he says.
It turns out that the implanted human cells spew out two important molecules that help protect rats' existing nerve circuits. One of the molecules helps promote nerve cells' survival, and the other encourages nerve cells to stay connected to their neighbors, says Kerr.
"The rats that got human stem cells were still far from normal, but even the improvements we saw could be important clinically," says Kerr, who emphasizes that any clinical application is still many years away.
In their experiments, spearheaded and majorly funded by the private organization Project ALS, the scientists first infected rats with a virus (Sindbis) they developed that selectively destroys nerve cells that control muscles in the hind limbs. Lou Gehrig's disease, also known as ALS or amyotrophic lateral sclerosis, is similarly marked by a gradual loss of the nerves that control muscles, although its cause is unknown.
One-third of the animals then received transplants of human embryonic germ cells, which are capable of becoming any cell type, into their spinal fluid. The other rats served as controls and received either hamster kidney cells or human cells that don't have stem cell properties.
Twelve weeks later, the 15 paralyzed rats that got human stem cells partially recovered control of their hind limbs. Moreover, their hind limbs were 40 percent stronger than control animals'. By 24 weeks, 11 of the 15 turned over at least three seconds faster when placed on their backs than before getting the human cells. Control rats did not improve, on average, over the 24 weeks of the study.
In paralyzed rats, Kerr and his team found that most of the implanted human cells migrated into the spinal cord, and many became cells of the nervous system -- astrocytes, neurons and even motor neurons -- while in uninjured animals the transplanted cells just sat on the spinal cord's outer surface. However, even in injured animals, only about four human cells per rat became motor neurons that actually extended out of the spinal cord and into muscle, potentially creating a circuit that could control movement.
"We saw some physical recovery, and we saw human stem cells that had become motor neurons, but it turns out that the two observations weren't related," says Kerr. "We saw functional recovery that wasn't due to new neurons, and we had no idea how that could be possible."
Kerr then discovered that the rats' own neurons were healthier in animals that received human stem cells. In subsequent laboratory experiments, Kerr found that the human stem cells produced copious amounts of two key growth signals. These were transforming growth factor-alpha (TGF-alpha), which promotes neurons' survival, and brain derived neurotrophic factor (BDNF), which strengthens their connections to other neurons. When the scientists blocked these two signals in the laboratory, the stem cells' beneficial effects disappeared.
"Even before motor neurons die, connecting neurons peel back as if they sense a sinking ship," says Kerr. "Simply keeping a neuron alive can't improve physical abilities if it's not connected to other neurons. It must be part of a circuit.
"In some ways our results reduce stem cells to the non-glamorous role of protein factories, but the cells still do some amazing, glamorous things we can't explain," he adds. "For example, the white matter that surrounds the spinal cord was thought to be an impenetrable barrier to axon growth, but some of the transplanted cells not only migrated into the spinal cord, but also sent axons back out. It is just incredible."
"These are important first steps as we begin to analyze the potential of various types of stem cells in disorders of motor neurons," adds Jeffrey Rothstein, M.D., Ph.D., director of the Robert Packard Center for ALS Research at Johns Hopkins and a participant in the research team. "The unexpected role of non-neuronal cells in the recovery of motor function may have important therapeutic implications someday."
Human embryonic germ cells, derived from fetal tissue, were first isolated in the laboratory of co-author John Gearhart at Johns Hopkins. They are one of two types of human cells collectively referred to as pluripotent stem cells. The experiments were funded by Project ALS, Families of SMA and Andrew's Buddies/FightSMA. Authors on the paper are Kerr, Jeronia Llado, Michael Shamblott, Nicholas Maragakis, David Irani, Thomas Crawford, Chitra Krishnan, Sonny Dike, John Gearhart and Rothstein, all of The Johns Hopkins University School of Medicine.
Under a licensing agreement between Geron Corporation and The Johns Hopkins University, Gearhart and Shamblott are entitled to a share of royalty received by the University on sales of products described in this article. Gearhart, Shamblott, and the University own Geron Corporation stock, which is subject to certain restrictions under University policy. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with its conflict of interest policies.
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