Survey of Stem Cell Research and SCI
We touch a flame and we feel heat; we close our fingers around a pen to pick it up. How does our body complete these functions? Every sensation, action and thought revolves around the complicated processes of the central nervous system (CNS), which consists of the brain and the spinal cord. The brain is the central computer of our body interpreting outside information and controlling every action. The spinal cord connects the brain with the rest of the body by sending out millions of electrical signals. When there is injury to the spinal and this connection breaks paralysis occurs. As of now spinal cord damage is irreversible, leaving approximately 250,000 Americans in a devastating position. However, in the past 10 years, as stem cell research has developed, new exciting possibilities have arisen for people suffering from spinal cord injury.
Before understanding the consequences of a damaged spinal cord it is important to understand how the spinal cord functions. Cells called neurons are responsible for receiving and processing every piece of information the brain sends the rest of the body. Neurons are made up of four parts—the cell body which houses the nucleus and most of the cell organelles, dendrites, an axon, and axon terminals. Dendrites are bush like projections that bring information from other neurons to the cell body. The axon, a longer projection, sends information away form the cell body.
One strategy to repair a damaged spinal cord involves stimulation of axon regrowth in order to reestablish the broken connection.
When the dendrites receive information the cell body generates a nerve impulse, which travels along the axon to another “ target” cell. At the target cell—a muscle cell, another nerve cell, or gland cell—the axon divides into a multitude of nerve endings. The tip of each of these endings is called the axon terminal and located very close to the target cell. Here the axon forms a synapse allowing neurotransmitters to travel across a small gap (25 nm wide) and fuse with the receptors of the next cell—this is how electric signals are sent from the brain to other parts of the body. When neurons die, connection between axon terminals and receptors are broken and the CNS can no longer function.
There are over 100 billion neurons in the central nervous system and as many as 10,000 different subtypes of these neurons. The incredible power of the brain to process information exists in the massive amount of neurons and synapses. Neurons aren’t the only cell in the central nervous system—glial cells exist in even greater numbers then neurons. These cells come in different forms with a variety of different functions, all helping the central nervous system to operate. Two glial cells related to the spinal cord are oligodendrocytes and astrocytes. Oligodendrocytes are responsible for producing myelin, a fatty substance that provides electrical insulation on the axons. Myelin allows electric signals to be sent at a rate of 100 meters/sec as opposed to 1 meter/ sec without myelin. Death of oligodendrocytes results in demyelination, halting communication between the brain and the rest of the body. Astrocytes break down and remove harmful proteins, as well as secrete proteins called neurotrophic factors, which help neurons survive and grow. Astrocytes also respond to injury: they clear away debris, an action resulting in formation of glial scarring.
The spinal cord needs more protection then any other organ or system because unlike other organs, the spinal cord cannot repair itself. The complex interactions between the brain and neurons, in combination with the enormous number of individual neurons and synapses, make reconnection of the nerve cells extremely difficult. The spinal column supplies the main defense of the spinal cord, providing a protective barrier against injury. The syrinx, a fluid filled area, offers additional protection by absorbing shock. Unfortunately, both of these defenses cause complications upon injury. Swelling causes additional damage to the spinal cord as pressure builds in the confined space between the cord and vertebrae. The syrinx contributes to scar tissue that builds up around the area of injury. Scar tissue blocks the neurons from reconnecting once the cord has been severed.
Often the cord is not completely severed during injury; even so, swelling cuts off the blood supply to the neurons and glial cells. Without a blood supply these cells die. Additional cell death occurs as cells from the immune system migrate to the injury site. In order for a connection to be reestablished new neurons and glial cells must regenerate to replace the injured ones. Up until about 10 years ago people believed that there was no possibility for neurogenesis of adult nerve cells. Once nerve cells were damaged they were gone, eliminating hope for complete recovery from paralysis. As a result, treatments for spinal cord injury focused on prevention of further damage (secondary damage) and rehabilitation.
While the majority of cells found in the central nervous system are born during the embryonic and early postnatal period, scientists recently discovered that new neurons are continuously added to two specific regions of the adult mammalian brain (Reynolds and Weiss 1992). Neural stem cells were isolated from the dentate gyrus of the hippocampus and the walls of the ventricular system called the ependymal layer. The progeny of these stem cells differentiate in the granule cell layer, meaning neurogenesis continues late into adult rodent life. These stem cells also migrate along the rostral migratory stream to the olfactory bulb, where they differentiate into neurons and glial cells (Luskin, 1993). Nerve cell differentiation has been witnessed in vivo, as well as in vitro when stimulated with an epidermal growth factor (Gage, 1995). The discovery of differentiating stem cells in the brain revolutionized the way scientists think about treating spinal cord injury. Suddenly the chance for partial or possibly full recovery from paralysis seemed like a plausible option. Attention shifted to regenerating the neurons and glial cells as a solution to spinal cord injury.
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Along with pluripotent stem cells progenitor cells, a more restricted type of stem cells, are found in the hippocampus and ependymal layer. These cells are immature cells that are predetermined to differentiate into neurons, oligodendrocytes, and astrocytes. In 1995 Frissen observed that the presence of nestin increases in response to spinal cord injury. Nestin is a protein expressed by stem cells: presence of it indicates neural stem cells are much more active then previously believed. Our brain naturally increases the production of stem cells to aid an injured CNS. If the brain responds in this way, why doesn’t the spinal cord repair itself? In 1999, Johansson and Momma observed that the only active progenitor cells were differentiating into astrocytes. They labeled ependymal cells with a Dil injection so migration could be followed. After making lesions in the spinal cord they waited four weeks and then observed the progress of the ependymal cells. They tested the cells found in the scar tissue around the site of injury and found that all DIL marked cells were astrocytes. This indicates that the progeny from ependymal cells had only differentiated to astrocytes. Stem cells do respond to spinal cord injury, just not for the purpose of reestablishing connection between neurons.
This realization sparked scientist’s interest in understanding what triggers these progenitor cells to proliferate. The active progenitor cells may be ineffective in maintaining a functional CNS after injury, but if scientists could learn what signals triggered differentiation, perhaps they could induce differentiation of neurons and oligodendrocytes. Scientists began to focus on neurotrophic factors that triggered this differentiation, specifically the presence of brain derived neurotrophic factors (BDNF) and neurotrophin 3 and 4 (NT-3 and NT-4). In the early 90’s these trophic factors were targeted as what triggered axon growth during early development. NT-3 also is expressed in greater amounts in response to spinal cord injury. In 1994 Schwab reported dramatic increase in function, and regrowth of a partially severed cord of rats after treatment with NT-3. In 1997 Grill, Gage, and colleagues published a paper examining the effects of transplanted NT-3 on motor skills and morphology after induced spinal injury in mice. They focused on the corticospinal tract, the pathway in charge of making voluntary movements. NT-3 has been previously observed to promote regrowth of corticospinal axons, and preserves degenerating motor neurons.
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Grill and colleagues induced lesions in the dorsal hemisection of adult rat’s spinal cord, resulting in severely limited motor ability. Next grafts of syngenic fibroblasts, genetically altered to produce NT-3, were transplanted into the lesion cavity of the experimental group. These rats were kept alive for three months and put though a series of tests to monitor motor improvement. These tests examined coordination, ability to walk on inclined surfaces and precision of foot placement. After three months these rats were killed for the purpose of a quantitative cell count.
Recipients of the NT-3 secreting grafts showed significant improvement in motor skills over the control group, although they did not recover to the full ability they had before injury. After three months recipients of the NT-3 grafts demonstrated growth of corticospinal axons up to 8 mm from where the transplant had taken place. Only the injured axons at the lesion site showed any sign of regrowth. Uninjured axons showed no effort to reestablish connections across the site of injury. This suggests that NT-3 only responds when corticospinal axons are injured. If scientists could pinpoint signals triggering this response there is potential to manipulate the process in a manner causing neural cells to differentiate.
Triggering neurotrophic factors in hopes of inducing progenitors to proliferate is one of two major areas of study in spinal cord regeneration. Scientists also can derive undifferentiated embryonic stem cells (ES cells) from fetal spinal cord tissue and then mature them into cells that are suitable to implant into the damaged spinal cord. When using ES cells, researchers have two options: they can treat ES cells, allowing them to mature into CNS cells in vitro before transplantation, or they can directly implant differentiated cells and depend on signals from the brain mature the cells. This technique became possible when Reynolds and Weiss found that stem cells taken from the brain could be propagated in vitro. This allowed labs to duplicate what occurs naturally in the brain, and attempt to use the product to regrow the damaged cells.
In December of 1999 McDonald and colleagues from Washington University School of medicine successfully implanted ES cells in laboratory rats. McDonald induced thoracic spinal cord injury in rats using a metal rod 2.5 mm in diameter resulting in paralysis. Nine days after the injury McDonald and colleagues transplanted roughly 1 million ES embryoid bodies pretreated with retinoic acid into the syrinx that had formed around the contusion. During the nine days that passed between injury and transplantation, all the standard events following a spinal cord injury occurred. At the time of injury some cells died immediately, followed by a second wave of apoptosis within the first 24 hours. The center of the bruised spine filled with fluid becoming a cyst referred to as syrinx. McDonald injected the ES cells into this cavity.
Two weeks after the transplantation ES stem cells filled the area normally occupied by glial scarring. After five weeks the stem cells had migrated further away from the implantation site. Although a number of them had died, there was still enough for the rats to have a growing supply of neurons and glial cells. Most of the surviving cells were oligodendrocytes and astrocytes, but some neurons were found in the middle of the cord. The rats regained limited use of their legs. Paralysis had been cured!!
McDonalds work in 1999 represented new successes in stem cell technology but there are still many years of work ahead of us before any of this technology can be tested in humans. A major obstacle remains: although scientists are achieving results, they don’t understand the factors responsible for what occurs. In McDonalds study, the regaining of functions could result from the few differentiated neurons. Another possibility could be that the high differentiation of oligodendrocytes remyelinted enough axons to reestablish communication. Or perhaps functions regained due to ES cells producing growth factors—more research will have to be done before these options are narrowed down. Additional to unclear understanding of the process, other complications exist. Any introduction of foreign cells into the body triggers the immune system. ES cells would not simply be accepted into the host CNS. McDonald used cyclosporine to prevent rejection in the rats, but things get more complicated when testing begins on humans. The brain and spinal cord are complex, mysterious realms of the body—until science can predict the exact affect of evolving technologies, no testing on humans can occur.
A major motivation behind spinal cord research has been Christopher Reeve. Injured in a horseback riding incident, Christopher Reeve suffered a cervical spinal cord injury that left him quadriplegic. Christopher Reeve began the Christopher Reeve Paralysis Foundation (CRPF). CPRF funds research to treat or cure paralysis resulting from spinal cord injury or other CNS disorders. CPRF supports a Research Consortium, which collaborates the work of nine laboratories, as well as funds an international individual grants program. Several of the labs involved in the Research consortium focus on stem cells, making a lot of progress. The Salk Institute, run by Dr. Fred Gage examines the progenitor cells differentiating into glial cells. Someday they hope to manipulate these progenitor cells, inducing differentiation into neural cells.
Celebrity support towards a cause is extremely beneficial: in addition to putting a tremendous amount of money into research, Reeve shows support for continuing stem cell research despite moral concerns. There are a lot of people who find stem cell research extremely unethical. Scientists have found the most success with ES cells taken from embryoid spinal cords: although the ES cells are taken from embryos consisting at most of 64 cells, they still have potential to develop into a human being. People who believe life begins at conception remain morally against stem cell research. Justification is that the stem cells are derived from embryos discarded from fertility clinics. These embryos would be wasted if not used for stem cell research. Christopher Reeve published a position paper in response to the moral concerns and President Bush's decisions on stem cell researching. CPRF supports responsible stem cell research, recognizing the fine ethical boundaries existing in this technology.
Spinal cord injury research represents a new and rising field—more progress has been made in the last five years then in the previous fifty. This sudden success resulted from the new understanding of stem cell technology. The concept of stem cells has gotten a lot of press lately—from Time Magazine to the television show South Park. The realization that stem cells have potential to differentiate into neural cells opens new doors, destroying the accepted idea that adult neurogenesis is not an option. With these new possibilities, stem cell research has evolved into an exciting new field. There is a lot of room to grow—who knows what new discoveries the future will bring.
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Grill, R., Gage, F.H., Murai, K., Blesch, A. & Tuszynski, M.H. Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury. J. Neuroscience 17: 5560-5572 (1997)
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Temple. S. The development of neural stem cell. Nature (London) 414 112-117, 1 Nov. 2001
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McDonald, J.W., Xiao-Zhong, L., Qu, Y. Su, L., Mickey, S.K., Turestsky, D. Gottlieb. D.I. & Choi, D. Transplanted embryonic stem cells survive, differentiate and promote recvery in the injured rat spinal cord. Nature Medicine 5, no 12, 1410-1412 Dec. 1999
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Johansson, C.B., Momma, S., Clarke, D.L., Risling, M., Lendahl, U. & Frisen, J. Identification of a neural stem cell in the adult mammalian central nervous system. Cell, vol 96, 25-34, Jan 8, 1999.
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Gage, Fred H.; Kempermann, Gerd; Palmer, Theo D.; Peterson, Daniel A.; Ray, Jasodhara. Multipotent progenitor cells in the adult dentate gyrus. In: Journal of Neurobiology Aug., 1998. 36 (2): 249-266
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Gage, Fred H.; Coates, Penelope W.; Palmer, Theo D.; Kuhn, H. Georg; Fisher, Lisa J.; Suhonen, Jaana O.; Peterson, Daniel A.; Suhr, Steve T.; Ray, Jasodhara. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. In: Proceedings of the National Academy of Sciences of the United States of America 1995. 92 (25): 11879-11883.
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Nakamura, Masaya; Bregman, Barbara S.. Differences in neurotrophic factor gene expression profiles between neonate and adult rat spinal cord after injury. In: Experimental Neurology June, 2001. 169 (2): 407-415.
Websites
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Cristopher Reeve Paralysis Foundation: http://www.christopherreeve.org/research/researchmain.cfm
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Rebuilding The Nervous System with Stem cells (off the National Institutes of Health Website) http://www.nih.gov/news/stemcell/chapter8.pdf
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Human Neuronal Progenitor Cells: http://www.neuroguide.com/hnpcs.html
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SC 4. Lateral Corticospinal Tract intro: http://www.medsch.wisc.edu/anatomy/sc97/text/p4/intro.htm
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Brain Briefings- astrocytes: http://web.sfn.org/content/Publications/BrainBriefings/astrocytes.html
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Glial Cells: http://www.vet.ed.ac.uk/pvs/glial.htm
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How Do Nerve Cells Communicate messages? - http://apu.sfn.org/content/Publications/BrainBackgrounders/communication.htm
Editor's note: This paper was written by Noelle Huskey, whose brother suffers from schizophrenia. In August, 2001, the voices tormenting him commanded him to jump from the roof of his house -- now he is paraplegic and slowly recovering. Noelle also wrote a paper last year on the impact of her brother's mental |