A host of preclinical studies have examined the ability of stem cells to improve the condition of laboratory animals that have suffered a spinal cord injury. While these studies vary in their size, design, and quality, there has been little cumulative analysis of the data generated by these studies.
Fortunately, there is a powerful analytical tool that can examine data from many studies and this type of analysis is called a “meta-analysis.” Meta-analyses use sophisticated statistical packages to systematically reassess a compilation of the data contained within these papers. Meta-analyses are exhausting, but potentially very useful. Such a meta-analysis is also very important because it provides researchers with an indication of what problems must be worked out before these treatments advance to human clinical trials and what aspects of the treatment work better than others.
A recent meta-analysis of stem cell therapy on animal models of spinal cord injury has been published by Ana Antonic, MSc, David Howells, Ph.D., and colleagues from the Florey Institute and the University of Melbourne, Australia, along with Malcolm MacLeod and colleagues from the University of Edinburgh, UK in the open access journal PLOS Biology.
The goal of regenerative spinal cord treatments is to use stem cells to replace dead cells within damaged areas of the spinal cord. Such treatments would be given to spinal cord injury patients in the hope of improving the ability to move and to feel below the site of the injury. Many experiments that utilize animal models of spinal cord injury have used stem cells to treat laboratory animals that have suffered spinal cord injury, but, unfortunately, these studies are limited in scale by size (as a result of financial considerations), practical and ethical considerations. Such limitations hamper each individual study’s statistical power to detect the true effects of the stem cell implantation. Also, these studies use different types of stem cells in their treatment scenarios, inject those cells differently induce spinal cord injuries differently, and test their animals for functional recovery differently.
To assess these studies, this new paper examined 156 published studies, all of which tested the effects of stem cell treatments on about 6,000 spinal cord-injured animals.
Overall, they found that stem cell treatment results in an average improvement of about 25 percent over the post-injury performance in both sensory (ability to feel) and motor (ability to move) outcomes. Unfortunately, the variation from one animal to another varied widely.
For sensory outcomes the degree of improvement tended to increase with the number of cells implanted. Such dose-responsive results tend to indicate that the improvements are actually due to the stem cells, and therefore, this stem cell-mediated effect represents a genuine biological effect.
The authors also measured the effects of bias. Simply put, if the experimenters knew which animals were treated and which were untreated, then they might be more likely to report improvements in the stem cell-treated animals. They also examined the way that the stem cells were cultured, the way that the spinal injury was generated and the way that outcomes were measured. In each case, important lessons were learned that should help inform and refine the design of future animal studies.
The meta-analysis also revealed some surprises that should provoke further investigations. For example, there was little evidence that female animals showed any beneficial sensory effects as a result of stem cell treatments. Also, the efficacy of the stem cell treatment seemed to not depend on whether immunosuppressive drugs were administered or not.
The authors conclude, “Extensive recent preclinical literature suggests that stem cell-based therapies may offer promise; however the impact of compromised internal validity and publication bias means that efficacy is likely to be somewhat lower than reported here.”
Even though human clinical trials are in the works, such trials will continue to be informed by preclinical studies on laboratory animals.
After injury to the spinal cord, glial cells and neural stem cells in the spinal cord contribute to the formation of the “glial scar.” This glial scar is rich in molecules known as chondroitin sulfate proteoglycans (CSPGs) that are known to repel growing axons. Therefore, the glial scar is viewed as a major impediment to spinal cord regeneration.
However, new work from the Karolinska Institutet in Solna, Sweden has confirmed that the glial scar actually works to contain the damage within the spinal cord. Far from impairing spinal cord recovery, the stem cell-mediated formation of the glial scar confines the damage to a discrete portion of the spinal cord and prevents it from spreading.
Trauma to the spinal cord can sever those nerve fibers that conduct nerve impulses to from the brain to skeletal muscles below the level of spinal cord injury. Depending on where the spinal cord is injured and the severity of the injury, spinal cord injuries can lead to a various degrees of paralysis. Such paralysis is often permanent, since the severed nerves do not grow back.
The absence of neural regeneration required an explanation, since cultured neurons whose axons are severed can regenerate both in culture and in a living creatures (for an excellent review, see Nishio T. Axonal regeneration and neural network reconstruction in mammalian CNS. J Neurol. 2009 Aug;256 Suppl 3:306-9). Thus, neuroscientists have concluded that the injured spinal contains a variety of molecules that inhibit axonal outgrowth and regeneration.
This hypothesis has been demonstrated since many axon growth inhibitors have been isolated from the injured spinal cord (see Schwab ME (2002) Repairing the injured spinal cord. Science 295:1029–1031). Such molecules include proteins like Nogo, Myelin-Associated Glycoprotein (MAG), and Oligodendrocyte-Myelin Glycoprotein (OMgp). However, as the Nishio review points out, axons from severed nerved have been seen growing throughout the central nervous system. Therefore, most of the blame for a lack of regrowth has been pinned on the glial scar.
A new study by Jonas Frisén of the Department of Cell and Molecular Biology and his colleagues has shown that the neural stem cell population in the spinal cord are the main contributors to the glial scar. However, when glial scar formation was prevented after spinal cord injury, the injured area in the spinal cord expanded and more nerve fibers were severed. Furthermore, in their mouse model, a great number of nerve cells died in those mice that did not make glial scars when compared to those mice that were able to produce a normal glial scar.
“It turned out that scarring from stem cells was necessary for stabilizing the injury and preventing it from spreading,” said Frisén. “Scar tissue also facilitated the survival of damaged nerve cells. Our results suggest that more rather than less stem cell scarring could limit the consequences of a spinal cord injury.”
According to earlier animal studies, recovery can be improved by transplanting stem cells to the injured spinal cord. These new findings suggest that stimulating the spinal cord’s own stem cells could offer an alternative to cell transplantation therapies.
This paper appeared in the journal Science, 1 November 2013: 637-640, and the first author was Hanna Sabelström. This interesting paper might be leaving one thing out when it comes to spinal cord regeneration. Once the acute phase of spinal cord injury is completed and the chronic phase begins, the glial scar does in fact prevent spinal cord regeneration. This is the main reason Chinese researchers have used chondroitinase enzymes to digest the scar in combination with transplantations on stem cells. By weakening the repulsive effects of the glial scar, these stem cells can form axons that grow through the scar. Also, olfactory ensheathing cells or OECs seem to be able to shepherd axons through the scar, although the degree of regeneration with these cells has been modest, but definitely real. Therefore, negotiating axonal regeneration through the glial scar remains a major challenge of spinal cord injury. Thus, while the glial scar definitely has short-term benefits, for the purposes or long-term regeneration, it is a barrier all the same.
A gaggle of laboratory animal experiments and clinical studies in human patients have established that stem cell injections into the spinal cord after spinal cord injury promote functional recovery (see Beattie, M. S., et al., Exp. Neurol. 148(2):453‐463; 1997; Bennett, D. L., et al., J. Neurosci. 20(1):427‐437; 2000; Kim HK, et al., PLos One 4(3): e4987 2009; Lu, P.; Tuszynski, M. H. Exp. Neurol. 209(2):313‐320; 2008; McTigue, D. M., et al., J. Neurosci. 18(14):5354-5365; 1998; Widenfalk, J.; Lundströmer, K. J. Neurosci. 21(10):3457‐3475; 2001; also see Salazar DL, et al., PLoS ONE, August 2010; Hooshmand M, et al., PLoS ONE, June 2009; Cummings BJ, et al., Neurological Research, July 2006; and Cummings BJ, et al., PNAS, September 19, 2005). Stem Cell, Inc., for example, has conducted several tests with human patients using their HuCNS-SC human neural stem cell line, and transplantation of these stem cells promotes functional recovery in human patients who have suffered spinal cord injury.
However, one factor that has yet to be properly determined is the best site for stem cell injection. Previous work by scientists at the Keio University School of Medicine in Japan has shown that injection of neural stem cells and neural progenitor cells (NS/PCs) into non-injured sites by either intravenous or intrathecal (introduced directly into the space under the arachnoid membrane of the brain or spinal cord) administration failed to produce sufficient engraftment of stem cells at the site of injury.
Instead cells were trapped in the lungs and kidneys, and many mice even developed fatal lung conditions as a result of intravenous administration (see Takahashi Y., et al., Cell Transplant. 2011;20(5):727-39). These data convinced them that intralesional application of the stem cells (injections directly into the damaged site of the spinal cord) might be the most effective and reliable method for NS/PC tranplantations.
A new study by the Keio group has attempted to ascertain the efficacy of the intralesional injections. Mice with spinal cord injuries were injected with NS/PCs that had been derived from mice that expression glowing proteins. This allowed the injected cells to be tracked with bio-luminescence imaging (BLI).
The principal investigator of this research is Masaya Nakamura from the Department of Orthopedic Surgery at the Keio University School of Medicine. Dr. Nakamura and his team gave mice spinal contusions at the level of the tenth thoracic vertebra. Then some mice were given low doses and others high doses of NS/PCs that were derived from fetal mice (for those who are interested, low dose – 250,000 cells per mouse; high dose – 1 million cells per mouse) nine days after spinal cord injury. These mice were further divided into two groups: those injected at the lesion epicenter (E), those injected at sites at the front and back of the lesion (RC for “rostral/caudal”). Thus there were four groups total: High dose E, High dose RC, Low dose E, and Low dose RC.
All four groups showed better functional recovery than the control group, which was injected with phosphate buffered saline. BLI showed that the number of cells that survived in each of the four cell-transplanted groups was about the same across these groups. Thus injecting more cells does not lead to greater numbers of surviving neural stem cells. This makes sense, since the damaged spinal cord in very inhospitable place for transplanted cells.
However, when the mice were examined for the expression of particular brain-derived neurotropic factors, the expression of such genes was higher in the RC-injected mice than in the E-injected mice. These results seems to explain why the transplanted NS/PCs differentiated more readily into neurons in the RC-injected mice rather than a type of glial cell known as an astrocyte, as was the case in the E-injected mice.
Nakamura and his team interpreted these results to mean that the environments of the E and RC sites can both support the survival of transplanted NS/PCs during the sub-acute phase of spinal cord injury. The authors conclude with a practical note: “Therefore, we conclude that it is optimal to graft a certain threshold number of NS/PCs into the epicenter lesion during the sub-acute phase of SCI, and thereby avoid causing further iatrogenic injury to the intact RC regions of the spinal cord.”
Hopefully Nakamura’s work will be translated into further human clinical trials. One feature of this study is that a particular threshold of stem cells survive when injected into the spinal cord and injecting larger numbers of cells does not increase the number of surviving cells. Injecting more cells might only contribute to the cell debris in the spinal cord. This is certainly a good thing to know when conducting clinical trials with neural stem cells in spinal cord-injured patients.
One of the challenges of stem cell-based therapies is cell survival. Once stem cells are implanted into a foreign site, many of them tend to pack up and die before they can do any good. For this reason, many scientists have examined strategies to improve stem cell survival.
A new technique that improves stem cells survival have been discovered by Yubo Fan and his colleagues at Beihang University School of Biological Science and Medical Engineering. This non-chemical technique, biphasic electrical stimulation (BES) might become important for spinal cord injury patients in the near future.
Spinal cord injury affects approximately 250,000 Americans, with 52% being paraplegic and 47% quadriplegic. There are 11,000 new spinal cord injuries each year and 82% are male.
Stem cell transplantions into the spinal cord to regenerate severed neurons and associated cells provides a potentially powerful treatment. However, once stem cells are implanted into the injured spinal cord, many of them die. Cell death is probably a consequence of several factors such as a local immune response, hypoxia (lack of oxygen), and probably most importantly, limited quantities of growth factors.
Fan said of his work, “We’ve shown for the very first time that BES may provide insight into preventing growth factor deprivation-triggered apoptosis in olfactory bulb precursor cells. These findings suggest that BES may thus be used as a strategy to improve cell survival and prevent cell apoptosis (programmed cell death) in stem cell-based transplantation therapies.”
Since electrical stimulation dramatically accelerates the speed of axonal regeneration and target innervation and positively modulates the functional recovery of injured nerves, Fan decided to test BES. His results showed that BES upregulated all the sorts of responses in stem cells that you would normally see with growth factors. Thus BES can increase stem cell survival without exogenous chemicals or genetic engineering.
Fan and his team examined the effects of BES on olfactory bulb neural precursor cells and they found that 12 hours of BES exposure protected cells from dying after growth factor deprivation. How did BES do this? Fan and other showed that BES stimulated a growth factor pathway called the PI3K/Akt signaling cascade. BES also increase the output of brain-derived neurotrophic factor.
“What was especially surprising and exciting,” said Fan, “was that a non-chemical procedure can prevent apoptosis in stem cell therapy for spinal cord patients.” Fan continued: “How BES precisely regulates the survival of exogenous stem cells is still unknown but will be an extremely novel area of research on spinal cord injury in the future.”
BES can improve the survival of neural precursor cells and will provide the survival of neural precursor cells and will provide the basis or future studies that could lead to novel therapies for patients with spinal cord injury.
Regenerating damaged nerve tissue represents a unique challenge for regenerative medicine. Nevertheless, some experiments have shown that it is possible to regenerate the myelin sheath that surrounds particular nerves.
Myelin is a fatty, insulating sheath that surrounds particular nerves and accelerates the transmission of nerve impulses. The myelin sheath also helps neurons survive, and the myelin sheath is attacked and removed in multiple sclerosis, a genetic disease called Charcot-Marie-Tooth disease, and spinal cord injuries. Being able to regenerate the myelin sheath is an essential goal of regenerative medicine.
Fortunately, a new study from a team of UC Davis (my alma mater) scientists have brought this goal one step closer. Wenbig Deng, principal investigator of this study and associate professor of biochemistry and molecular medicine, said, “Our findings represent an important conceptual advance in stem cell research. We have bioengineered the first generation of myelin-producing cells with superior regenerative capacity.”
The brain contains two main cell types; neurons and glial cells. Neurons make and transmit nerve impulses whereas glial cells support, nourish and protect neurons. One particular subtype of glial cells, oligodendrocytes, make the myelin sheath that surrounds the axons of many neurons. Deng and his group developed a novel protocol to induce embryonic stem cells (ESCs) to differentiate into oligodendrocyte precursor cells or OPCs. Even though other researchers have made oligodenrocytes from ESCs, Deng’s method results in purer populations of OPCs than any other available method.
Making OPCs from ESCs is one thing, but can these laboratory OPCs do everything native can do? When Deng and his team tested the electrophysiological properties of their laboratory-made OPCs, they discovered that their cells lacked an important component; they did not express sodium channels. When the lab-made OPCs were genetically engineered to express sodium channels, they generated the characteristic electrical spikes that are common to native OPCs. According to Deng, this is the first time anyone has made OPCs in the laboratory with spiking properties. Is this significant?
Deng and his colleagues compared the spiking OPCs to non-spiking OPCs in the laboratory. Not only did the spiking OPCs communicate with neurons, but they also did a better job of maturing into oligodentrocytes.
Transplantation of these two OPC populations into the spinal cord and brains of mice that are genetically unable to produce myelin also showed differences. Both types of OPCs were able to mature into oligodendrocytes and produce myelin sheaths, but only the spiking OPCs had the ability to produce longer and thicker myelin sheaths.
Said Deng, “We actually developed ‘super cells’ with an even greater capacity to spike than natural cells. This appears to give them an edge for maturing into oligodendrocytes and producing better myelin.
Human neural tissue has a poor capacity to regenerate and even though OPCs are present, they do not regenerate tissue effectively when disease or injury damages the myelin sheath. Deng believes that replacing glial cells with the enhanced spiking OPCs to treat injuries and diseases has the potential to be a better strategy than replacing neurons, since neurons are so problematic to work with in the laboratory. Instead providing the proper structure and environment for neurons to live might be the best approach to regenerate healthy neural tissue. Deng also said that many diverse conditions that have not been traditionally considered to be myelin-based diseases (schizophrenia, epilepsy, and amyotrophic lateral sclerosis) are actually now recognized to involve defective myelin.
On that one, I think Deng is dreaming. ALS is caused by the death of motor neurons due to mechanisms that are intrinsic to the neurons themselves. Giving them all the myelin in the world in not going to help them. Also, OPCs made from ESCs will be rejected out of hand by the immune system if they are used to regenerate myelin in the peripheral nervous system. The only hope is to keep them in the central nervous system, but even there, any immune response in the brain will be fatal to the OPCs. This needs to be tested with iPSCs before it can be considered for clinical purposes.
Disclaimer: I am reporting on this experiment because of its significance for people with spinal cord-injuries even though I remain appalled at the manner in which the stem cells were acquired.
An international research team has reported that a single set of injections of human neural stem cells had provided significant neuronal regeneration and improvement of function in rats impaired by acute spinal cord injury.
Dr. Martin Marsala, who is professor of anesthesiology at the University of California, San Diego, with colleagues from academic institutions in Slovakia, the Czech Republic, and the Netherlands, used neural stem cells derived from an aborted human fetus to treat spinal cord-injured rats.
Sprague-Dawley rats received spinal cord injuries at the level of the third lumbar vertebra by means of compression. Such injuries render the rats incapable of using their hind legs. They cannot climb a ladder, walk a catwalk or perform other tasks that require the effective use of their hind legs.
The stem cells that were transplanted into the spinal cords of these rats were NSI-566RSC cells, which were provided by the biotechnology company Neuralstem. These cells were initially isolated from the spinal cord of an eight-week old human fetus whose life was terminated through elective abortion. These cells have been grown in culture and split many times. They are a neural stem cell culture that has the capacity to form neurons and glia.
The rats were broken into six groups, and four of these groups received spinal cord injuries. One of these spinal cord-injured groups received injections of were injured NSI-566RSC cells (12 injections total, about 20,000 cells per microliter of fluid injected), another received injections of only fluid, and the third group received no injections. The final spinal cord-injured group of rats received injections of NSI-566RSC cells that had been genetically engineered to express a green glowing protein. Another group of rats were operated on, but no spinal cord injury was given to these animals, and the final group of rats were never operated on.
All rats that received injections of cells were administered powerful drugs to prevent their immune systems from rejecting the administered human cells before the injections (methylprednisolone acetate for those who are interested at 10 mg / kg), and after the stem cell injections (tacrolimus at 1.5 mg / kg).
The results were significant and exciting. In the words of Marsala, “The primary benefits were improvement in the positioning and control of paws during walking tests and suppression of muscle spasticity.” Spasticity refers to an exaggerated muscle tone or uncontrolled spasms of muscles. Spasticity is a serious and common complication of traumatic injury. It can cause severe cramping and uncontrolled contractions of muscles, which increases the patient’s pain and decreases their control.
First, it is clear from several control experiments that the injection procedure did not affect the spinal cord function of these animals, since the sham injected rats had perfectly normal use of their hind limbs and normal sensory function of their limbs. Thus the injection procedure is innocuous. Also, the use of the drugs to suppress the immune response were also equally unimportant when it came to the spinal cord health of the rats.
Two months after the stem cell injections, the rats were subjected to the “catwalk test,” in which the animals walked a narrow path and their paw position was assessed. As you can see in the figure below, the stem cell-injected rats have a paw position that is far more similar to the normal rats than to the spinal cord injured rats.
Secondly, when muscle spasticity was measured, the stem cell-injected rats showed definite decreases in muscle spasticity. The spinal cord-injured rats that received no stem cell injections showed no such changes.
Sensory assessments also showed improvements in the stem cell-treated rats, but the improvements were not stellar. Nevertheless, the stem cell-treated rats progressively improved in their sensory sensitivity whereas the non-treated spinal cord-injured rats consistently showed no such improvement.
What were the implanted cells doing? To answer this question, Marsala and his co-workers examined tissue sections of spinal cords from the rats implanted with the glowing green stem cells. According to Marsala, the implanted neural stem cells are stimulating host neuron regeneration and partially replacing the function of lost neurons.
Marsala explained: “Grafted spinal stem cells are a rich source of different growth factors which can have a neuroprotective effect and can promote sprouting of nerve fibers of host neurons. We have demonstrated that grafted neurons can develop contacts with the host neurons and, to some extent, restore the connectivity between centers, above and below the injury, which are involved in motor and sensory processing.”
The implanted neural stem cells definitely showed extensive integration with the spinal nerves of the host rats. Again Marsala, “In all cell-grafted animals, there was a robust engraftment and neuronal maturation of grafted human neurons was noted.” Marsala continued: “Importantly cysts or cavities were not present in any cell-treated animal. The injury-caused cavity was completely filled by grafted cells.”
Marsala’s goal is to used a neuronal stem cell line derived from a patient-specific induced pluripotent stem cell line in a clinical trial. For now, the UC San Diego Institutional Review Board or IRB is reviewing a small phase 1 clinical trial to test the safety and efficacy of this neural stem cell line in patients with spinal cord injuries who have no feeling or motor function below the level of the spinal cord injury.
Spinal cord injuries represent one of the most intractable problems for regenerative medicine. When the spinal cord is injured, a tissue that is normally isolated from the bloodstream, now comes into contact with a variety of inflammatory factors and cells that increase the destruction of the original lesion. The spinal responds with a glial scar that plugs the lesion and prevents further exposure of the spinal cord to damaging inflammation, but the scar is also filled with molecules that repel neuronal axon growth cones. This spells curtains for neuronal regeneration, and finding a cell type that can negotiate around the glial scar and find the original muscle is a genuine tour de force.
Given this to be the case, there have been many experiments in rodents to examine the efficacy of various stem cell populations to as treatments for spinal cord injuries. A recent paper in Stem Cell Research and Therapy (van Gorp et al., 2013, 4:57) has examined human fetal spinal cord-derived neural stem cells (HSSCs) and their ability to restore motor function in rats with spinal cord injuries to the lower back. Because this group examined movement and spinal cord tissue samples, this paper contributes something significant to our knowledge of HSSC-mediate healing of spinal cord injuries.
The HSSC line used in this paper is neural stem cell line NSI566RSC, which was extracted from the spinal cord of an 8-week old “fetus.” I have placed fetus in quotes because at eight weeks, the fetus is actually a very old embryo, since the end of the eighth week is end of embryonic development. I realize that these types of age calculations have room for error, and therefore, the baby might very well have been at the early fetal stage. However, the baby’s mother terminated her pregnancy (yes it was an abortion and no I am not cool with that) and donated the dead baby’s tissue to UC San Diego for research purposes.
Sprague-Daley rats were subjected to spinal cord injuries at the level of the third lumbar vertebra. Three days later, half of the rats were given saline injections into their spinal cord and the other half were given HSSC injections into their spinal cords. The animals were evaluated for two months after the treatments on a daily basis. After two months, the rats were sacrificed (put down) and the spinal cord tissue was extensively analyzed.
Of the 35 animals employed in this study, 3 were excluded because of paw injuries or drug toxicity. Eight weeks after the cells were implanted, the rats were tested with a CatWalk apparatus to determine their gait. The rats injected with HSSCs showed a much more normal gait than those injected with saline. To give you some idea of the improvement, the rats that were not injured had a RCHPP or rostro-caudal hind paw positioning score of 0+/- 1.7mm, and the saline injected animals had an average RCHPP of -18 +/- 3.1 mm, and those injected with HSSCs had an RCHPP of -9.0 +/- 1.9 mm.
Despite these improvements, there were no significant differences in ladder climbing, stride length, overall coordination, or single-frame motion.
Next, Marsala and colleagues showed that the muscle spasms associated with spinal cord injury were slightly decreased by the implantation of HSSCs and not by injection of saline. To measure spasticity, the ankle or front paw is rotated and the electromyograph of the muscle is measured. The electromyograph or EMG measures the electrical activity of the muscle showed modest improvements in the HSSC-injected animals
Sensory sensitivity was improved in the HSSC-injected animals, and this improvement was progressive. When the rats were prodded below the level of the injury, where they should have no feeling, the HSSC-injected rats showed better response to the stimulation. This was the case with mechanical stimulation and thermal stimulation.
Post-mortem analysis also showed something interesting. When the fluid-filled cavity of the damaged spine was examined, the HSSC-injected animals had a significantly small cavity. Because the injected cells had been labeled with green fluorescent protein, they glowed under UV light and any neuronal cells derived from the injected HSSCs glowed green too. The lesioned areas in the HSSC-injected mice were repopulated with cells. Motorneurons, interneurons and glial cells were detected.
What to make of this study? The repopulation of the spinal cord and the growth of spinal nerve elements within the fluid-filled cavity is remarkable, but the lack of better motor function is disappointing. The recovery of sensory ability is significant, especially, since it is pretty clearly not due to spinal hypersensitivity.
There are two possibilities for the low motor recovery. First, there is a possibility that the these experiments were not conducted for as long a time period as they needed to be. Since the sensory ability improvement was progressive, maybe the motor recovery was too, perhaps? Secondly, maybe the grow and connection of motor neurons had trouble with the glial scar. Why the sensory nerves did not have such a problem and the motor neurons would is inexplicable at this time. However, another possibility is that the muscular targets of motor neurons are not as obvious in adult animals as they are in a developing animal. Finding ways to “paint” the muscles might be a way to increase motor neuron innervation in the future.
Thus, this cell line, NSI-566 RSC is certainly a potential treatment for spinal cord patients. A phase I trial is in the works.
Spinal cord injuries are a very difficult challenge for regenerative medicine. The damaged spinal cord is a toxic waste dump encased in a barrier that prevents regenerating cells from entering or exiting. However, a group of stem cells that have been used in clinical trials have produced extremely encouraging results for spinal cord injury patients.
Collaboration between the Cambridge University’s Veterinary School and Medical Research Council’s Regenerative Medicine Centre, has given scientists a chance to test a particular type of stem cell found inside your nose to regenerate the damaged part of the spinal cord of dogs. The researchers are cautiously optimistic that their work could play an important role in the future treatment of human patients who suffer from similar injuries, but only if they are used in combination with other treatments.
For over a decade, scientists have known about olfactory ensheathing cells (OECs) and their potential usefulness for treating damaged spinal cords. OECs have unique properties, since they have the ability to support nerve fiber growth. This is the role OECs play in the nose; they maintain a pathway for the growth and extension of nerve fibers between the nose and the brain.
Previous research in laboratory animals has shown that OECs can aid regeneration of those extensions of nerve cells that transmit signals (axons). The growth and re-extension of damaged neurons helps them form a kind of bridge between damaged and undamaged spinal cord tissue. A Phase 1 trial in human patients with spinal cord injuries (SCI) established that transplantation of OECs into the spinal cords of SCI patients is safe.
This present study was published in the latest issue of the neurology journal Brain, and it is the first double-blinded randomized controlled trial to test the effectiveness of OEC transplants to improve function in ‘real-life’ spinal cord injury. The trial was performed on animals that had spontaneous and accidental injury rather than in the controlled environment of a laboratory. Unlike other experiments, these test subjects had suffered their spinal cord injuries at least one year ago. Thus, this experiment more closely resembles the way in which this procedure might be used in human patients.
The 34 pet dogs had all suffered severe spinal cord injuries. Twelve months or more after the injury, they were unable to use their back legs to walk and unable to feel pain in their hindquarters. Not surprisingly, many of the dogs were dachshunds, which are particularly prone to this type of injury because of their long body and short legs. Additionally, dogs are more likely to suffer from SCIs because the spinal cord may be damaged as a result of what in humans is the relatively minor condition of a slipped disc. Thus while four-leggedness has its advantages, it also has its drawbacks.
In this study, which was funded by the MRC, one group of dogs had OECs from the lining of their own nose injected into the injury site. The other group of dogs was injected only with the liquid in which the cells were transplanted. Neither the researchers nor the owners (nor the dogs!) knew which injection they were receiving.
The dogs were observed for adverse reactions for 24 hours before being returned to their owners. After this time, the animals were tested at one month intervals for neurological function and to have their gait analyzed on a treadmill while being supported in a harness. In particular, researchers were interested in the ability of the dogs to coordinate the movement of their front and back limbs.
The group of dogs that had received the OEC injection showed considerable improvement that was not seen in the other group. Just view this video here to see the amazing improvement in the OEC transplanted animals. These animals moved previously paralyzed hind limbs and coordinated all their movement with their front legs (again, see this video). This means that in these dogs neuronal messages were being conducted across the previously damaged part of the spinal cord. However, the researchers established that the new nerve connections accounting for this recovery were occurring over short distances within the spinal cord and not over the longer distances required to connect the brain with the spinal cord.
Professor Robin Franklin, a co-author of the study from the Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, said: “Our findings are extremely exciting because they show for the first time that transplanting these types of cell into a severely damaged spinal cord can bring about significant improvement. We’re confident that the technique might be able to restore at least a small amount of movement in human patients with spinal cord injuries but that’s a long way from saying they might be able to regain all lost function. It’s more likely that this procedure might one day be used as part of a combination of treatments, alongside drug and physical therapies, for example.”
The head of Regenerative Medicine at the Medical Research Council, Dr. Rob Buckle, commented: “This proof of concept study on pet dogs with the type of injury sustained by human spinal patients is tremendously important and an excellent basis for further research in an area where options for treatment are extremely limited. It’s a great example of collaboration between veterinary and regenerative medicine researchers that has had an excellent outcome for the pet participants and potentially for human patients.”
The researchers stress that human patients with a spinal injury rate a return in sexual function and continence far higher than improved mobility. Some of the dogs in the study did regain bowel and bladder control but the number of these was not statistically significant.
Mrs. May Hay, the owner of a dog named Jasper, who took part in the trial (and can be seen in the video), said: “Before the trial, Jasper was unable to walk at all. When we took him out we used a sling for his back legs so that he could exercise the front ones. It was heartbreaking. But now we can’t stop him whizzing round the house and he can even keep up with the two other dogs we own. It’s utterly magic.”
Human patients have already been treated with their own OECs. Jacki Rabon traveled to Brazil to receive an OEC transplantation with her own OECs. Today, even though she has been severely spinal cord injured, she can walk with braces. Read her story here. Another patient, Laura Dominguez, was also treated by the same Brazilian physician. See her video here. It certainly seems as though we have hit upon a sound strategy upon which to build. Let’s pursue this rather than killing human embryos.
Spinal cord injuries to the upper spinal cord (around T6 or even T10) can damage the nerve fibers that connect the brainstem and the spinal cord. Damage to the spinal cord at this level can make the patient susceptible to a potentially life-threatening condition known as autonomic dysreflexia.
Autonomic dysreflexia occurs if a patient has an upper spinal cord injury and receives some sort of strong stimulus to the lower part of the body, often the bladder or bowel. The strong sensory impulse travels up the spinal cord and evokes a response from the sympathetic nervous system. The sympathetic nervous system is a branch of the autonomic nervous system, which controls all those things that occur automatically (beating of your heart, breathing moment-to-moment adjustments of the blood pressure, etc.). The sympathetic branch of the autonomic nervous system is associated with those elements of the “fight or flight” response. If you are scared or surprised, your heart beats faster, face flushes, skeletal muscle become ready for action and so on. A strong sympathetic response will increase the heart rate and cause other blood vessels to constrict to redirect blood flow to the organs that are vital for survival under extreme conditions. In short, the blood pressure rises dramatically, but only transiently.
This widespread and strong sympathetic response causes widespread constriction of blood vessels, particularly in the muscles below the diaphragm. This causes hypertension (high blood pressure). The brain detects this by means of detectors in the arteries known as baroceptors in the arteries. The brain tries to defuse the situation by sending inhibitory impulses down the spinal cord, but these inhibitory signals never reach the constricted blood vessels because of the spinal cord injury. The brain has a back-up plan in which it tries to decrease peripheral blood pressure by slowing the heart rate. This, however, only works a bit and the hypertension continues.
This sudden increase in blood pressure and a decrease in heart rate is the hallmark of autonomic dysreflexia. Patients also tend to lose control of their bladder and bowel. If this all sounds unpleasant, it is.
Fortunately, a stem cell treatment for autonomic dysreflexia might be in the works. Armin Blesch, PhD, of the Spinal Cord Injury Center at Heidelberg University Hospital, in collaboration with colleagues from the University of California, San Diego, tested a treatment protocol for autonomic dysreflexia. Blesch and colleagues isolated neural stem cells from the brainstem of fetal rats. The goal was not the completely heal the spinal cord injury, but to provide some healing so that the compensatory impulses from the brain could find their way down to the abdominal area to relieve the hypertension.
Blesch tested spinal cord-injured rats that had received transplantations of the fetal brainstem stem cells with spinal cord-injured rats that had received no such treatment. Then Blesch and his colleagues compared the response of these animals when the lower portion of the body was heavily stimulated eight weeks after the stem cell transplantations.
According to Blesch: “Only the animals that received transplants of fetal cells derived from the brainstem had a resting heart rate and blood pressure that was similar to normal values,” Blesch said. “When we looked at their spinal cords, we found evidence that the transplanted cells may be serving as nerve relays to reconnect the brain with centers of control for cardiovascular function.”
This is a preclinical study, but it is an important proof-of-principle study that shows that complete healing of the spinal cord in not necessary to repair particular functions that can prevent a life-threatening condition such as autonomic dysreflexia.
Hopefully in humans, the neural stem cell line made by StemCells Inc, could work in patients with upper spinal cord injuries who are at risk for autonomic dysreflexia. Perhaps human clinical trials that address this very issue might be in the works.
Umbilical cord stem cells (UCSCs) have been differentiated into clinically significant cell types that might, potentially, lead to new treatment options for spinal cord injuries, multiple sclerosis, and other nervous system diseases.
James Hickman, a University of Central Florida bioengineer and leader of the research group that accomplished this work, said, “This is the first time this has been done with non-embryonic stem cells. . . . We’re very excited about where this could lead because it overcomes many of the obstacles present with embryonic stem cells.” Hickman’s work and that of his colleagues was published in the Jan. 18 issue of the journal ACS Chemical Neuroscience.
UCSCs do not pose the ethical dilemma represented by embryonic stem cells (ESCs). ESC lines are made from 5-day old human embryos and in order to derive them, the inner cell mass cells are extracted from the embryo by means of destroying the embryo. Destruction of a human embryo ends the life of a very young human person. UCSCs, however, come from a source that would otherwise be discarded, and the acquisition of UCSCs do not compromise the life of a human person. Another major benefit is that umbilical cells generally are not rejected by the immune system, and this simplifies their potential use in medical treatments.
The Menlo, California-based pharmaceutical company, Geron, developed a treatment protocol for spinal cord repair that utilized oligodendrocyte precursor cells that were derived from embryonic stem cells. However, it took Geron scientists 18 months to secure approval from the Food & Drug Administration (FDA) for human clinical trials. This is due, largely, to the ethical and public concerns attached to human ESCs. These concerns, in addition to anxieties over ESC-caused tumors, led the company to shut down its ESC division. This highlights the need for other stem cell alternatives.
One of the greatest challenges in working with any kind of stem cell is determining the precise chemical or biological cues that trigger them to differentiate into the desired cell type. The lead author on this paper, Hedvika Davis, a postdoctoral researcher in Hickman’s lab, transformed UCSCs into oligodendrocytes (those structural cells that surround and insulate nerves in the brain and spinal cord). Davis learned from research done by other groups that surface proteins on the surfaces of oligodendrocytes bind the hormone/neurotransmitter norepinephrine. This suggests that cells normally interact with this chemical and that it might be one of the factors that stimulates oligodendrocyte production. Therefore Davis decided to treat USCSs with epinephrine as a starting point.
In early tests, Davis found that norepinephrine, plus several other stem cell growth promoters, caused the UCSCs to differentiate into oligodendrocytes. However, that conversion was incomplete, since the cells grew but stopped short of becoming completely mature oligodendrocytes. Clearly something else was needed to push UCSCs completely into mature oligodendrocytes.
Many stem cells differentiate into particular cell types only if the appropriate environment is offered to them. For example, mesenchymal stem cells can form cartilage, but cartilage formation is extremely sensitive to environmental factors like cell density, and the matrix in which cells are embedded. Thus, Davis decided that, in addition to chemistry, the physical environment might be critical. In order to more closely approximate the physical restrictions cells face in the body, Davis constructed a more confined, three-dimensional environment. She grew the cells on a microscope slide, covered by a glass cover slip. Once the UCSCs had the proper confined environment and norepinephrine plus the appropriate growth factors, they differentiated into completely mature oligodendrocytes. Davis noted, “We realized that the stem cells are very sensitive to environmental conditions.”
The use of these differentiated oligodendrocytes is exciting. There are two main options for the use of these cells. First, the cells could be injected into the body at the point of a spinal cord injury to promote repair. Another intriguing possibility for the Hickman team’s work relates to multiple sclerosis and similar conditions. Hickman explained, “Multiple sclerosis is one of the holy grails for this kind of research.” Hickman’s research group is collaborating with Stephen Lambert at UCF’s medical school, another of the paper’s authors, to explore biomedical possibilities.
Oligodendrocytes produce a protein called myelin, which insulates nerve cells. Myelin sheaths make is possible for neurons in the central nervous system to conduct those nerve impulses that guide movement and other functions. Myelin loss is responsible for conditions like multiple sclerosis, and is also observed in other related conditions such as diabetic neuropathy.
The injection of new, healthy oligodendrocytes might improve the condition of patients suffering from such neurological diseases. These research teams are also hoping to develop the techniques needed to grow oligodendrocytes in the lab and use them a model system to better understand the loss and restoration of myelin, and for testing potential new treatments. Hickman enthusiastically said, “We want to do both. We want to use a model system to understand what’s going on and also to look for possible therapies to repair some of the damage, and we think there is great potential in both directions.”