Behavior Of Brain Stem Cells Controlled By Cerebrospinal Fluid Signals

The choroid plexus is a network of blood vessels in each ventricle of the brain. It is derived from the pia mater and produces the cerebrospinal fluid.  The choroid plexus, unfortunately, has been ignored to some degree when it comes to brain research.  However, CSF turns to be an important regulator of adult neural stem cells, research indicates.

A new study led by Prof. Fiona Doetsch at the Biozentrum of the University of Basel, Switzerland has shown that signals secreted by the choroid plexus dynamically change during aging, and these different signals affect the behavior of aged stem cells.

In the adult brain, neural stem cell populations in various places throughout the central nervous system divide to give rise to neurons and glial cells throughout our lives. These stem cells reside in unique micro-environments (known as niches) that provide key signals that regulate stem cell self-renewal and differentiation. Stem cells in the adult brain contact the ventricles, which are cavities in the brain filled with CSF. CSF bathes and protects the brain and is produced by the cells of the choroid plexus.

Ventricular System of the Brain

Doetsch and her coworkers have shown that the choroid plexus is a key component of the stem cell niche, and that the properties of this stem cell niche change throughout life and affect stem cell behavior.

Doetsch’s group discovered that the choroid plexus secretes a cocktail of important signaling factors into the CSF. These CSF-secreted growth factors are important in stem cell regulation throughout life. As we age, the levels of stem cell division and formation of new neurons decrease. They also showed that although stem cells are still present in the aged brain, and have the capacity to divide, their ability to do so have significantly decreased.

Graphical abstract

“One reason is that signals in the old choroid plexus are different. As a consequence, stem cells receive different messages and are less capable to form new neurons during aging. In other words, compromising the fitness of stem cells in this brain region,” said Violeta Silva Vargas, first author of the paper that appeared in the journal Cell Stem Cell. “But what is really amazing is that when you cultivate old stem cells with signals from young fluid, they can still be stimulated to divide, behaving like the young stem cells.”

In the future, Doetsch and her group plans to tease out the composition of the signaling factors secreted by the choroid plexus.  They would also like to know how the composition of this growth factor cocktail changes as a result of changes in brain states and how these changes affect neural stem cells. This could provide new ways to understand brain function in health and disease.

“We can imagine the choroid plexus as a watering can that provides signals to the stem cells. Our investigations also open a new route for understanding how different physiological states of the body influence stem cells in the brain during health and disease, and opens new ways for thinking about therapy,” said Doetsch.

This work was published here: Violeta Silva-Vargas et al., “Age-Dependent Niche Signals from the Choroid Plexus Regulate Adult Neural Stem Cells,” Cell Stem Cell, 2016; DOI: 10.1016/j.stem.2016.06.013.

Epilepsy Reduces The Formation of New Neurons in the Brain

An ambitious, multidisciplinary project led by Amanda Sierra and Juan Manuel Encinas, Ikerbasque from the Achucarro centre (Achucarro Basque Center for Neuroscience) has discovered that epilepsy in a mouse model system reduces the production of new cells in the brain.

The hippocampus is a region of the brain involved in learning and memory and it is also the site of a robust neural stem cell population that generates new neurons. These hippocampal neural stem cells generate new neurons throughout the adult life of mammals. The cells generated by the hippocampal neural stem cells function in certain types of learning and memory and in responses to anxiety and stress.


This new research by Sierra and Encinas has revealed that in epileptic mice, hippocampal neural stem cells stop generating new neurons and are turn into reactive astrocytes. Reactive astrocytes promote inflammation and alter communication between neurons. Could manipulation of neural stem cells provide new ways to treat epilepsy?

Reactive Astrocyte
Reactive Astrocyte

This work has recently been published in the journal Cell Stem Cell.

The results of this research also confirms previous work by the same group that showed that epilepsy, which causes hyperexcitation of neurons but does not cause convulsions, activates neural stem cells, which leads to their premature exhaustion. Thus the generation of new neurons in the hippocampus ends is chronically reduced.

Juan Manuel Encinas, the leader of this study, highlighted the fact that “this discovery has enabled us to gain a better understanding about how neural stem cells function. We have shown that in addition to generating neurons and astrocytes, neural stem cells in the adult hippocampus can generate reactive astrocytes following an epileptic seizure.”

Encinas and his colleagues carried out this work in experimental animals that were genetically engineered to be epileptic. However, this discovery has clear implications in clinical practice and in the quest to develop new therapies for epilepsy, since the generation of new neurons (neurogenesis) is a process that is negatively affected in epileptic seizures in the hippocampus. Encinas pointed out, “If we can manage to preserve the population of neural stem cells and their capacity to generate new neurons in humans, it may be possible to prevent the development of certain symptoms associated with epilepsy and very likely to mitigate the damage that is caused in the hippocampus.”

In this project, Encinas and his colleagues collaborated with research groups attached to institutions such as the Baylor College of Medicine in Houston (United States), the Université Catholique de Louvain (Belgium), the Achucarro centre itself, and the UPV/EHU’s Genetic Expression Service.

Grafted Stem Cells Display Robust Growth in Spinal Cord Injury Model

University of San Diego neuroscientists have used an animal model of spinal cord injury to test the ability of engrafted stem cells to regenerate damaged nerves. Mark Tuszynski and his team built on earlier work with implanted neural stem cells and embryonic stem cell-derived neural stem cells in rodents that had suffered spinal cord injuries.

In this study, Tuszynski and others used induced pluripotent stem cells that were made from a 86-year-old male. This shows that skin cells, even from human patients who are rather elderly, have the ability to be reprogrammed into embryonic stem cell-like cells. These cells were differentiated into neural stem cells and then implanted into the spinal cords of spinal cord-injured rodents.

The injured spinal cord is a very hostile place for implanted cells. Inflammation in the spinal cord summons white blood cells to devour cell debris. White blood cells are rather messy eaters and they release enzymes and toxic molecules that can kill off nearby cells. Also, regenerating cells run into a barrier made by support cells called glial cells that inhibit regenerating neurons from regenerating. Thus, the injured spinal cord is quite the toxic waste dump.

To get over this, Tuszynski and his coworkers treated their induced pluripotent stem cell-derived neural stem cells with growth factors. In fact, when the cells were implanted into the animal spinal cords, they were embedded in a matrix that contained growth factors. After three months, Tuszynski and his colleagues observed extensive axonal growth projecting from grafted neurons that reached long distances in both directions along the spinal cord from the brain to the tail end of the spinal cord. These sprouted axons appeared to make connections with the existing rat neurons. Importantly, these axons extended from the site of the injury, which is astounding given that the injured area of the spinal cord has characteristics that are inimical to neuronal and axon growth.

Even though Tuszynski and others showed that neural stem cells made from embryonic stem cells can populate the damaged spinal cord, using induced pluripotent stem cell-derived neural stem cells has an inherent advantage since these cells are less likely to be rejected by the patient’s immune system. Furthermore, the induced pluripotent stem cell-derived neural stem cells showed dramatic growth in the damaged spinal cord, but the implanted animals did not regain the use of their forelimbs. The implanted human cells were fairly young when the implanted animals were tested. Therefore, they might need to mature before they could restore function to the implanted animals.

“There are several important considerations that future studies will address,” Tuszynski said. “These include whether the extensive number of human axons make correct or incorrect connections; whether the new connections contain the appropriate chemical neurotransmitters to form functional connections; whether connections once formed are permanent or transient; and exactly how long it takes human cells to become mature. These considerations will determine how viable a candidate these cells might before use in humans.”

Tuszynski and his group hope to identify the most promising neural stem cell type for repairing spinal cord injuries. Tuszynski emphasized their commitment to a careful, methodical approach:

“Ultimately, we can only translate our animal studies into reliable human treatments by testing different neural stem cell types, carefully analyzing the results, and improving the procedure. We are encouraged, but we continue to work hard to rationally to identify the optimal cell type and procedural methods that can be safely and effectively used for human clinical trials.”

Engineering Stem Cells to Fight Cancer

Advanced brain tumors are typically treated by surgical removal. However, it is difficult in the extreme to extirpate an entire tumor and therefore, tumor relapse is a perennial problem. A special group of small proteins known as ‘cytotoxic proteins” can target and destroy remaining cancer cells, but these proteins have short half-lives in the body and recent clinical trial called the PRECISE trial was not able to demonstrate that administered cytotoxic proteins had any efficacy against glioblastoma multiforme (GBM) brain tumors.

A new study, however, published online from the journal Stem Cells, a research group led by Khakid Shah from the Harvard Stem Cells Institute, have devised a new strategy designed around these engineered cytotoxic proteins has shown that neural stem cells (NSCs) can be genetically engineered to express these proteins and help treat GBM tumors.

So how did Shah and his colleagues design this novel strategy? They engineered NSCs to not only express specific cancer cell-killing toxins, but also have resistance to these toxins. Secondly, they designed cytotoxins that have the ability to enter cancer cells and target proteins known to be over-expressed by GBM tumors. Then these neural stem cells were encapsulated, they were transplanted into the space left after the bulk of the tumor was surgically removed.

In a mouse model of GMB, the implanted engineered stem cells survived and mediated an increase in long-term survival. This therapy was also effective against multiple patient-derived GBM cancer cell lines, which demonstrated their potential clinical relevance and applicability.

Shah and his coworkers want to bring these results to human trials within the next five years. They hope that this strategy can be successfully deployed in combination with surgical removal of the tumor mass. Shah also hopes that this approach can be adapted to treat other tumor types by using tissue-specific stem cells that express tumor-specific cytotoxins.

See Stuckey DW, Hingtgen SD, Karakas N, et al. Engineering toxin-resistant therapeutic stem cells to treat brain tumors. Stem Cells 2014.

Neural Stem Cells Can Enter the Spinal Cord Without Direct Spinal Cord Injection

Several studies in laboratory animals have shown that transplanted neural stem cells have remarkable ability to differentiate into brain and spinal cord cells and replace dead cells. A few clinical trials have also shown that neural stem cells have a great deal of promise to treat neurological diseases.

Unfortunately, getting stem cells into the spinal cord requires injections directly into the spinal cord by highly skilled neurosurgeons using special equipment.  Such a procedure is highly invasive and risky.  It would be much safer and easier if intravenously administered cells could find their way to the spinal cord.

Giacomo Comi and Stefanie Corti from the University of Milan may have found a way to do just that.  With their coworkers, Comi and Corti made neural stem cells from human induced pluripotent stem cells, but they selected their cells in a very unique way.  They screened differentiating induced pluripotent stem cells that expressed high levels of an enzyme called aldehyde dehydrogenase, scattered light in a particular way, and expressed the cell adhesion molecule VLA4.  Previous experiments showed that neural stem cells made from induced pluripotent stem cells that expressed high levels of aldehyde dehydrogenase with low side scattering of light grew well in the spinal cords of rodents with a neurodegenerative disease, differentiated into nerve cells and relieved symptoms (see Corti S., et al. Hum. Mol. Genet. 2006;15:167–187 and Corti S., et al. J. Clin. Invest. 2008;118:3316–3330).  Additional work in other laboratories have shown that cells that express the VLA4 protein on their cell surfaces can enter the central nervous system from the general circulation (see Pluchino S., et al. Nature. 2005;438:266–271; and Winkler E.A., et al. Acta Neuropathol.2013;125:111–120).  Thus, Comi and Corti sought to make neural stem cells from induced pluripotent stem cells that had all the qualities they had previously relied upon, but also expressed the cell adhesion molecule VLA4 to determine if such cells could enter the nervous system from the general circulation.  

After establishing their desired neural stem cell lines in culture, Comi and Corti and their coworkers transplanted these cells into the spinal cords of mice that suffered from an experimental form of Amyotrophic Lateral Sclerosis (ALS).  The implanted cells had previously been labeled with a green-glowing protein, and the presence of green-glowing cells in the spinal cord of the rats was confirmed.  However, another set of ALS rats were given these same cells intravenously, and once again green-glowing cells were found in the spinal cord of the ALS rats.  Donor cells also reached the brain and were detected in the cortical and subcortical areas of the brain.  Even more remarkably, no adverse effects, including tumor formation, abnormal cell growth or inflammation, were detected in any of the recipient animals.

iPSC-derived NSCs migrate and engraft into the spinal cords of SOD1G93A mice after intravenous transplantation. (A) Experimental design: GFP-NSC cells (1 × 106 cells) were delivered by weekly intravenous injection into SOD1G93A mice starting at 90 days of age. (B and C) Donor GFP+ cells were detected in the spinal cord, particularly in the anterior horns. (C) Quantification of GFP-donor cells in the cervical, thoracic and lumbar spinal cord. Error bars indicate the SD. (D) Quantification of the phenotype acquired by the donor cells revealed the presence of cells with an undifferentiated phenotype (nestin), a neuronal (NeuN) phenotype and a glial (GFAP) phenotype. Error bars indicate the SD. (E) Representative images of cells acquiring a neuronal phenotype that are positive for NeuN (red) and GFP (green). Scale bars: (B) 150 µm right, 120 µm left; (E) 50 µm upper panel, 75 µm lower panel.
iPSC-derived NSCs migrate and engraft into the spinal cords of SOD1G93A mice after intravenous transplantation. (A) Experimental design: GFP-NSC cells (1 × 106 cells) were delivered by weekly intravenous injection into SOD1G93A mice starting at 90 days of age. (B and C) Donor GFP+ cells were detected in the spinal cord, particularly in the anterior horns. (C) Quantification of GFP-donor cells in the cervical, thoracic and lumbar spinal cord. Error bars indicate the SD. (D) Quantification of the phenotype acquired by the donor cells revealed the presence of cells with an undifferentiated phenotype (nestin), a neuronal (NeuN) phenotype and a glial (GFAP) phenotype. Error bars indicate the SD. (E) Representative images of cells acquiring a neuronal phenotype that are positive for NeuN (red) and GFP (green). Scale bars: (B) 150 µm right, 120 µm left; (E) 50 µm upper panel, 75 µm lower panel.

Neural stem cells administered in either manner increased survival in the recipient mice and reduced the loss of neurons and their connections with other cells.  Also, the levels of nerve growth factors were increased in the spinal cords of transplanted animals.

Transplantation of ALDHhiSSCloVLA4+ NSCs improves neuromuscular function, increases survival and reduces motor neuron and axon loss in ALS mice. (A and C) Transplantation of NSCs significantly improved motor performance in SOD1 mice, as demonstrated by the rotarod test both in the intrathecally transplanted group (A) and in systemically injected mice (C) (4 weeks after transplantation, P < 0.001, ANOVA). (B and D) Kaplan–Meier survival curves for mutant SOD1 mice treated intrathecally (B) or systemically (D) with ALDHhiSSCloVLA4+ NSCs or with vehicle. Survival was significantly extended for NSC-transplanted mice compared with vehicle-treated mice for both treatment groups (P < 0.05, log-rank test). (E) The motor neuron count (n = 6 for each group) in the lumbar spinal cord of NSC-transplanted, vehicle-treated SOD1 mice and wild-type mice (data represent the mean ± SD of the number of motor neurons per section) at 140 days of age. The evaluation revealed significantly increased numbers of surviving motor neurons in treated SOD1G93A mice (P < 0.001, ANOVA). (F) Quantification of axons (data represent the mean ± SD) at 140 days of age (n = 6 for each group) demonstrated that transplanted SOD1G93A mice showed a significantly increased number of axons (P < 0.001, ANOVA).
Transplantation of ALDHhiSSCloVLA4+ NSCs improves neuromuscular function, increases survival and reduces motor neuron and axon loss in ALS mice. (A and C) Transplantation of NSCs significantly improved motor performance in SOD1 mice, as demonstrated by the rotarod test both in the intrathecally transplanted group (A) and in systemically injected mice (C) (4 weeks after transplantation, P < 0.001, ANOVA). (B and D) Kaplan–Meier survival curves for mutant SOD1 mice treated intrathecally (B) or systemically (D) with ALDHhiSSCloVLA4+ NSCs or with vehicle. Survival was significantly extended for NSC-transplanted mice compared with vehicle-treated mice for both treatment groups (P < 0.05, log-rank test). (E) The motor neuron count (n = 6 for each group) in the lumbar spinal cord of NSC-transplanted, vehicle-treated SOD1 mice and wild-type mice (data represent the mean ± SD of the number of motor neurons per section) at 140 days of age. The evaluation revealed significantly increased numbers of surviving motor neurons in treated SOD1G93A mice (P < 0.001, ANOVA). (F) Quantification of axons (data represent the mean ± SD) at 140 days of age (n = 6 for each group) demonstrated that transplanted SOD1G93A mice showed a significantly increased number of axons (P < 0.001, ANOVA).

Likewise, transplanted animals did not display the massive proliferation of cells known as astrocytes that is so characteristic of ALS spinal cords.  As it turns out, the administered neural stem cells prevented the astrocyte explosion by activating an astrocyte cell surface protein called TRPV1.  The activation of this cell surface protein prevented the astrocytes from dividing and cluttering up the spinal cord.

These remarkable experiments show, first of all, that neural stem cells can be made that express the VLA4 protein and such cells do not need to be injected into the spinal cord.  Instead they can be given intravenously and they will enter the spinal cord on their own, which is a much safer mode of administration.  Secondly, neural stem cells made from induced pluripotent stem cells are notorious for being able to cause tumors, but these cells, and the screening method used to select them from differentiating induced pluripotent stem cells, produced cells that apparently do not cause readily cause tumors in laboratory animals.  Of course, more intense screening is required to establish the safety of this line, but the initial observations appear hopeful.  Thirdly, this shows that we do not need to rip the spinal cords from 10-week old fetuses to make therapeutically useful neural stem cell lines; induced pluripotent stem cell technology will provide the means to do this.

Neuralstem Treats Final Patient in Phase 2 ALS Stem Cell Trial

NeuralStem, Inc. has announced that the final patient in its Phase 2 clinical trial that assessed the efficacy of its NSI-566 spinal cord-derived neural stem cell line in the treatment of amyotrophic lateral sclerosis (ALS), which is otherwise known as Lou Gehring’s disease.

ALS is a rapidly progressive, invariably fatal neurological disease that attacks the nerve cells responsible for controlling voluntary muscles; that is, muscle action we are able to control, such as those in the arms, legs, and face, etc.  ALS is a member of those disorders known as motor neuron diseases, all of which are characterized by the gradual degeneration and death of motor neurons.

Motor neurons are nerve cells located in the brain, brain stem, and spinal cord that serve as controlling units and vital communication links between the nervous system and the voluntary muscles of the body. Messages from motor neurons in the brain (so-called upper motor neurons) are transmitted to motor neurons in the spinal cord (so-called lower motor neurons) to particular muscles. In ALS, both the upper motor neurons and the lower motor neurons degenerate or die, and stop sending messages to muscles. Unable to function, the muscles gradually weaken, waste away (atrophy), and have very fine twitches (called fasciculations). Eventually, the ability of the brain to start and control voluntary movement is lost.

ALS causes weakness with a wide range of disabilities. Eventually, all muscles under voluntary control are affected, and individuals lose their strength and the ability to move their arms, legs, and body. When muscles in the diaphragm and chest wall fail, people lose the ability to breathe without ventilatory support. Most people with ALS die from respiratory failure, usually within 3 to 5 years from the onset of symptoms. However, about 10 percent of those with ALS survive for 10 or more years.

Although the disease usually does not impair a person’s mind or intelligence, several recent studies suggest that some persons with ALS may have depression or alterations in cognitive functions involving decision-making and memory.

ALS does not affect a person’s ability to see, smell, taste, hear, or recognize touch. Patients usually maintain control of eye muscles and bladder and bowel functions, although in the late stages of the disease most individuals will need help getting to and from the bathroom.

In this multicenter Phase 2 trial, 15 patients who still had the ability to walk were treated in five different dosing cohorts. The first 12 of these patients received injections only in the cervical regions of the spinal cord in increasing doses (5 injections of 200,000 cells per injection to injections of 4000,000 cells each . In the cervical region, these injected stem cells could potentially preserve the nerves that mediate breathing and this is precisely that this part of the trail aims to test.

spinal cord regions

In the final three patients injected in this trial, patients received a total of 40 injections of 400,000 cells each into both cervical and lumbar regions (a total of 16 million cells were injected. This is in contrast to the patients who participated in the Phase 1 study who received 15 injections of 100,000 cells each (total of 1.5 million cells). This trial will continue until six months past the final surgery, after which the data will be analyzed.

“By early next year, we will have six-month follow-up data on the last patients who received what we believe will be the maximum safe tolerated-dose for this therapy,” said Dr. Eva Feldman, principal investigator in this clinical trial, and a member of the ALS Clinic at the University of Michigan. Dr. Feldman also serves as an unpaid consultant to Neuralstem.

Neurons Made from Induced Pluripotent Stem Cells Stably Integrate into the Brain

Jens Schwamborn and Kathrin Hemmer from the Luxembourg Centre for Systems Biomedicine (LCSB) of the University of Luxembourg have shown that implanted neurons made from induced pluripotent stem cells show long-term stability in the brain.

Induced pluripotent stem cells (iPSCs) are made from mature adult cells by means of genetic engineering and cell culture techniques. These cells have embryonic stem cell-like capacities and can, potentially differentiate into any adult cell type. Because neurons made from iPSCs have sometimes not shown instability, the ability of neurons derived from iPSCs to stably integrate into brain has been questioned.

Schwamborn and Hemmer showed that six months after implantation, their iPSCs-derived neurons had become fully functionally integrated into the brain. This successful integration of iPSC-derived neurons into lastingly stable implants raises hope for future therapies that will replace sick neurons with healthy ones in the brains of patients with Parkinson’s disease, Alzheimer’s disease and Huntington’s chorea, for example. This work was published in the current issue of Stem Cell Reports.

The LCSB research group hopes to bring cell replacement therapy to maturity as a treatment for neurodegenerative diseases. The replacement of sick and/or dead neurons in the brain could one day cure disorders such as Parkinson’s disease. However, devising a successful therapy in human is a long, arduous process, and for good reasons. “Successes in human therapy are still a long way off, but I am sure successful cell replacement therapies will exist in future. Our research results have taken us a step further in this direction,” declared Schwamborn.

In their latest tests, the LCSB research group, in collaboration with colleagues from the Max Planck Institute and the University Hospital Münster and the University of Bielefeld, made stable neuronal implants in the brain from neurons that were derived from reprogrammed skin cells. They used a newer technique in which the neurons were produced from neural stem cells (NSCs). These NSCs or induced neural stem cells (iNSCs) had, in turn been made from iPSCs that were made from the host animal’s own skin cells, which considerably improves the compatibility of the implanted cells. Mice who received the neuronal implants showed no adverse side effects even six months after implantation. The new neurons were implanted into the hippocampus and cortex regions of the brain. Implanted neurons were fully integrated into the complex network of the brain and they exhibited normal activity and were connected to the original brain cells via newly formed connections known as synapses, which are the contact points between nerve cells.

These tests demonstrate that stem cells researchers are continuing to get a better handle on how to use cells derived from something other than human embryos in order to successfully replace damaged or dead tissue. “Building upon the current insights, we will now be looking specifically at the type of neurons that die off in the brain of Parkinson’s patients – namely the dopamine-producing neurons,” Schwamborn reports.

In future experiments, implanted neurons could provide the neurotransmitter dopamine (which is lacking in patients with Parkinson’s disease) directly into the patient’s brain and transport it to the appropriate sites. Such a result would herald an actual cure for the disease rather than a short-term fix. The first trials in mice are in progress at the LCSB laboratories on the university campus Belval.

Stem Cells Inc. Reports Additional Spinal Cord Injury Patients Transplanted with Neural Stem Cell Line Show Functional Improvements

StemCells, Inc. has developed a proprietary stem cell line called HuCNS-SC.  This stem cell line is a neural stem cell line, and neural stem cells can readily form neurons (the conducting cells of the nervous system), or glial cells (the support cells of the nervous system). In order to determine if these cells can regenerate spinal nerves in patients who have suffered a spinal cord injury, StemCells Inc. has commissioned a clinical trial to test their cells in human spinal cord injured patients.

Early indications showed that the HuCNS-SC cells were safe, but some patients have shows improvements in sensation.  Now StemCells Inc has issued an announcement that these initially reported improvements in only a few patients have also been confirmed in other patients.

According to Armin Curt, M.D., Professor and Chairman of the Spinal Cord Injury Center at Balgrist University Hospital, University of Zurich, and the principal investigator of their Phase I/II trial, the initial improvements that were observed in the first two patients treated with their HuCNS-SC neural stem cells have now been observed in two additional patients who have also been treated with these stem cells. These results come from an interim analysis of recent clinical data.

In a presentation to the Annual Meeting of the American Spinal Injury Association in San Antonio, Texas, Dr. Curt showed data on AIS B subjects who were transplanted with HuCNS-SC neural stem cells in the Phase I/II chronic spinal cord injury trial. This trial is different from the AIS A patients who have no mobility or sensory perception below the point of injury, since AIS B subjects are less severely injured, and are paralyzed but retain sensory perception below the point of injury. Two of the three AIS B patients who are participating in the study showed significant gains in sensory perception. The third patient remained stable.  These interim results confirm the favorable safety profile of these stem cells and the surgical implant procedure used to transplant them into the spinal cords of spinal cord injury patients.

Also included in Dr. Curt’s presentation was data from a total of five new subjects with a minimum six-month follow-up. In total, Stem Cells Inc. has now reported clinical updates on a total of eight of the twelve patients enrolled in its Phase I/II clinical trial that is testing this Company’s proprietary HuCNS-SC (purified human neural stem cells) platform technology for treating chronic thoracic spinal cord injury.

“Thoracic spinal cord injury was chosen as the indication in this first trial primarily to demonstrate safety. This patient population represents a form of spinal cord injury that has historically defied responses to experimental therapies and is associated with a very high hurdle to demonstrate any measurable clinical change. Because of the severity associated with thoracic injury, gains in multiple sensory modalities and segments are unexpected, and changes in motor function are even more unlikely,” said Dr. Curt. “In contrast, the cervical cord, which controls more motor function, may represent a patient population in which motor responses to transplant may be more readily anticipated.”

“We are seeing multi-segmental gains and a return of function in the cord in multiple patients. This indicates something that was not working in the spinal cord, now appears to be working following transplantation. This is even more significant because of the time that has elapsed from the date of injury, which ranges from 4 months to 24 months across the subjects with sensory gains,” said Stephen Huhn, M.D., FACS, FAAP, vice president, CNS clinical research at StemCells, Inc. “These results are exciting with respect to the expansion of this trial into patients with cervical injury because even a gain of one to two segments in cervical spinal cord injury patients can allow for additional function in the upper extremities.”

Genetically Modified As a Potential Treatment of Alzheimer’s Disease

A neurobiology team from UC Irvine (full disclosure, my alma mater) has used genetically engineered neural stem cells to treat mice with a form of Alzheimer’s disease (AD). Such implanted neural stem cells ameliorated some of the symptoms and pathological consequences of this disease in affected mice.

Patients with AD show accumulation of the protein amyloid-beta in their brains. These amyloid-beta clusters form clear plaques in the brain that are also quite toxic to nearby neurons.

Amyloid beta plaques can be cleared with the protein in them is degraded. Fortunately, the enzyme neprilysin can degrade these plaques, but the brains of AD patients show low levels of this enzyme. Neprilysin levels decrease with age and this is probably one of the reasons AD tends to be a disease of the aged.

The UC Irvine group, under the direction of Mathew Blurton-Jones, tried to deliver neprilysin to the brains of afflicted mice and used neural stem cells to do it. The goal of this work was to determine if increased degradation of the amyloid plaques abated the pathological effects of AD.

In this work, two different AD model systems were used. Thy1-APP and 3xTg-AD mice both exhibit many of the pathological effects of AD, and both were used in this study. Neural stem cells were transfected in express 25 times more neprilysin that normal. Then these genetically modified neural stem cells were transplanted into two areas of the brain known to be affected by AD: the hippocampus and the subiculum, which lies just below the hippocampus. Other AD mice were transplanted with neural stem cells that had not been transformed with neprilysin.

Post-mortem examination of both groups of mice even up to three months after transfection of the neural stem cells showed that those mice that received injections of neprilysin-expressing neural stem cells had significant reductions in amyloid-beta plaques within their brains compared to control mice. The neprilysin-expressing cells even seemed to promote the growth of neurons and the establishment of connections between them.

A truly remarkable finding of this work was that numbers of amyloid-beta plaques were also reduced in area of the brain that were some distance from the areas where the stem cells were injected. This suggests that the injected stem cells migrates across the brain, reducing plaque formation as they went.

Future experiments will seek to see if the reduction in amyloid-beta plaques also leads to improvements in cognition. Also, before this protocol can make its transition from animal models of human trials, the UC Irvine group will need to determine if the neprilysin also degrades soluble forms of amyloid-beta.

Every AD mouse model varies as to the types of pathologies observed in the brains of the affected mice. For this reason, this group tested their treatment strategy in two distinct AD mouse models, and in both cases, the neprilysin-expressing neural stem cells reduced the incidence of amyloid beta plaques. This strengthens the conclusion and neprilysin-expressing neural stem cells can indeed degrade amyloid-beta plaques.

More work needs to be done before this work can be used to support a human trial, but this is certainly an encouraging start to something great.

Long-Term Survival of Transplanted Human Neural Stem Cells in Primate Brains

A Korean research consortium has transplanted human neural stem cells (hNSCs) into the brains of nonhuman primates and ascertained the fate of these cells after being inside the brains of these animals for 22 and 24 months. They discovered that the implanted hNSCs had not only survived, but differentiated into neurons and never caused any tumors.

This important study is slated to be published in the journal Cell Transplantation.

To properly label the hNSCs so that they were detectable inside the brains of the animals, Lee and others loaded them with magnetic nanoparticles to enable them to be followed by magnetic resonance imaging (MRI). Also, they did not use immunosuppressants when they transplanted their hNSCs into the animals. This study is the first to examine the long-term survival and differentiation of hNSCs without the need for immunosuppression.

“Stroke is the fourth major cause of death in the US behind heart failure, cancer, and lower respiratory disease,” said study co-author Dr. Seung U. Kim of University of British Columbia Hospital’s department of neurology in Canada. “While tissue plasminogen activator (tPA) treatment within three hours after a stroke has shown good outcomes, stem cell therapy has the potential to address the treatment needs of those stroke patients for whom tPA treatment was unavailable or did not help.”

Based on the ability of hNSCs to differentiate into a variety of types of nerves cells, Lee and his colleagues thought that these cells have remarkable potential to treat damaged brain tissue and replace what was lost after a stroke, head injury or other type of brain trauma. Cell regeneration therapy can potentially treat brain-specific diseases like Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), spinal cord injury and stroke.

Dr. Kim and colleagues in Korea grafted magnetic particle-labeled hNSCs into the brains of laboratory primates and evaluated their performance to assess their survival and differentiation over 24 months. Of particular interest was determining their ability to differentiate into neurons and to determine whether the cells caused tumors.

“We injected hNSCs into the frontal lobe and the putamen of the monkey brain because they are included in the middle cerebral artery (MCA) territory, which is the main target in the development of the ischemic lesion in animal stroke models,” commented Dr. Kim. “Thus, research on survival and differentiation of hNSCs in the MCA territory should provide more meaningful information to cell transplantation in the MCA occlusion stroke model.”

Lee’s team said that they chose NSCs for transplantation because the existence of multipotent NSCs “has been known in developing rodents and in the human brain with the properties of indefinite growth and multipotent potential to differentiate” into the three major CNS cell types – neurons, astrocytes and oligodendrocytes.

“The results of this study serve as a proof-of-principle and provide evidence that hNSCs transplanted into the non-human primate brain in the absence of immunosuppressants can survive and differentiate into neurons,” wrote the researchers. “The study also serves as a preliminary study in our planned preclinical studies of hNSC transplantation in non-human primate stroke models.”

“The absence of tumors and differentiation of the transplanted cells into neurons in the absence of immunosuppression after transplantation into non-human primates provides hope that such a therapy could be applicable for use in humans.” said Dr. Cesar V. Borlongan, Prof. of Neurosurgery and Director of the Center of Excellence for Aging & Brain Repair at the University of South Florida. “This is an encouraging study towards the use of NSCs to treat neurodegenerative disorders”.

Teaching Old Neural Stem Cells New Tricks

In our brains, cells called neurons produce nerve impulses and are responsible for thinking, learning memory, reasoning, and so on. Neurons do not exist in isolation, but in combination with cells called glial cells that support the neurons, nourish them, and protects them from stress damage. Neurons and glial cells are replenished by brain-specific neural stem cell populations in the brain.

Unfortunately, the neural stem cell population in our brains tends to produce far fewer neurons as they age. This deficit of new neurons can play a role in the onset of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Also, our own “senior moments” when we forget where we placed our iPod or car keys comes from a loss of neurons as we age.

Fortunately, some recent research might change this trend. A team from Japan’s Keio University, and the Riken national research institute, has reported the discovery of a small RNA molecule (micro-RNA) that controls neuron production in young mice. When this micro-RNA was manipulated in older mice, their neural stem cells started to make neurons again. The Japanese team also has reasons to believe that the same mechanism is at work in human brains as well. This research was reported in the journal Proceedings of the National Academies of Science. The mechanism is believed to exist in humans as well.

Senior Author Hideyuki Okano said, “We observed the neurogenic-to-gliogenic switching in developing NSCs.” Translation: Okano and his team examined embryonic mouse brains and their neural stem cell (NSC) populations. They found what many other groups have previously observed: that the developing embryonic brain NSCs create neurons first, then switch over to making glial cells later. Okano’s team also discovered the microRNA-17/106-p38 axis that is responsible for this initial neuron-to-glial cell switch during embryonic development.

When they manipulated this embryonic microRNA-17/106-p38 pathway in older, post-natal NSCs in culture, these older post-natal NSCs switched from making glial cells to producing neurons.

In culture, NSCs are difficult to control, since getting large supplies of neurons from cell cultures that various research groups call NSCs is very difficult.

Nevertheless, “there is general agreement that neurogenesis (make neurons) largely precedes gliogenesis (making glial cells) during CNS development in vertebrates,” Okano explained. And adult NSCs, according to Okano, clearly can produce neurons in the body, “whereas they exhibit strong gliogenic characteristics under culture conditions in vitro (that is, in the laboratory).”

Adult NSCs in two regions of the brain—the subventricular zone and hippocampus—also “make neurons, even though transplant studies have shown us that the adult CNS is a gliogenic environment.”

Subventricular Zone

So it seems clear that old NSCs can make neurons, at least under certain conditions. However, it is very difficult to determine the age at which NSCs begin making substantially more glial cells than neurons. According to Okano, “It is difficult to clearly explain the association between total glial cell number and changes in NSC abilities. Moreover, there is less evidence about gliogenic ability of aged NSCs because most of studies about NSCs have mainly focused on the neurogenic ability. “

Still, Okano says: “There are some reports about decline of neurogenesis ability of NSCs with age. These reports indicate that reduction in paracrine Wnt3 factors, and increase of (chemokine) CCL11 concentration in blood, impaired adult neurogenesis in the hippocampus, for example.”

Could the group’s microRNA approach improve memory in humans? Okano believes so, but says more work needs to be done.

“We observed the neurogenic effect by overexpression of miR-17 in primary cultured neurospheres” – spheres of a variety of cells, including NSCs—“derived from the SVZ at postnatal day 30. Similar phenomenon by overexpression of miR-106b-25 cluster has been reported by another group.”

Okano also warns that his approach has only been attempted in cultured cells. He cautioned, “There is no evidence using knock-out mice. Therefore, the functions of them in adult neurogenesis and learning/memory functions are still unclear.”

Next, Okano’s group will develop “a useful method for precise manipulation of cytogenesis from NSCs. “

However, he says, “we think that further understanding of basic molecular mechanisms underlying the neural development is also an important issue.” He will study the ways in which his microRNA system interacts with other glia-producing genes. He wants to fully understand the mechanisms underlying “the end of neurogenic competence and acquisition of gliogenic competence.”

Finally, the group will “examine the significance of miR-17/p38 pathway in various somatic stem cells other than NSCs,” he says.

Engineered Neural Stem Cells Deliver Anti-Cancer Drug to the Brain

Irinotecan is an anticancer drug that was approved for use in 1996. It is a modified version of the plant alkaloid camptothecin, and even though it shows significant activity against brain tumors in culture, but in a living body, this drug poorly penetrates the blood-brain barrier. Therefore irinotecan usually does not accumulate to appreciable levels in the brain and is typically not used to treat brain tumors.

That could change, however, if a new strategy published in paper by Marianne Metz and her colleagues from the laboratory of Karen Aboody at the Beckman Research Institute at the City of Hope in Duarte, California, in collaboration with colleagues from several other laboratories.

In this paper, Metz and her co-workers genetically engineered neural stem cells to express enzymes called “carboxylesterases.” These carboxyesterase enzymes convert irinotecan, which is an inactive metabolite, to the active form, which is known as “SN-38.” The efficient conversion of irinotecan to SN-38 in the brain greatly accelerates the therapeutic activity of this drug in the brain. Also, the constant conversion of irinotecan to another molecule accelerates the transport of irinotecan past the blood brain barrier.

To test this strategy. Metz and others grew the engineered neural stem cells in culture and measured their ability to make carboxylesterases in culture, and their ability to convert irinotecan into SN-38 in culture.  In both cases, the engineered neural stem cells made a boat-load of carboxylesterase and converted irinotecan into SN-38 in spades.  More importantly, the genetically engineered neural stem cells behaved exactly as they did before, which shows that the genetic manipulation of these cells did not change their properties.

Next, Metz others tested the ability of the engineered neural stem cells to kill human brain tumor cells in culture in the presence of irinotecan.  Once again, the genetically engineered neural stem cells effectively killed human brain tumor cells in culture in a irinotecan-concentration-dependent manner.  When these genetically engineered neural stem cells were injected into the brains of mice with brain tumors, intravenous administration of irinotecan produced high levels of SN-38 in the brain.  This shows that these cells have the capacity to increase the production of SN-38 in the brain.

This strategy is similar to other strategies that been used in various clinical trials, but because neural stem cells have a tendency to move into brain tumor tissue and surround it, they represent an efficient and effective way to deliver anticancer drugs to brain tumors.  Also, since the particular neural stem cell line used in this experiment (HB1.F3.CD) does not cause tumors and is also not recognized as foreign by the immune system, it is a particularly attractive stem cell line for such an anti-tumor strategy.

Human Neural Stem Cells Heal Damaged Limbs

The term “ischemia” refers to conditions under which a part of your body, organ, or tissue is deprived of oxygen. Without life-giving cells begin to die. Therefore, ischemia is usually a very bad thing.

Critical limb ischemia or CLI results when blood vessels to the legs, feet or arms are severely obstructed. The results of CLI are never pretty, and CLI remains a medical condition that presents few treatment options.

A study from a research team and the University of Bristol’s School of Clinical Sciences has used stem cells in a trial that uses laboratory mice to treat CLI. The success of this study provides a new direction and new hope for procedures that relieve symptoms and prolong the life of the limb.

Autologous stem cells treatments, or those stem treatments that utilize a patient’s own stem cells care subject to clear limitations. After collection from bone marrow, fat, or other source, the stem cells must be expanded in culture after stimulation with chemicals called cytokines. After growth in culture, the cells typically contain a collection of different types of stem cells of variable quality and potency. Also, if the patients has had a heart attack or has diabetes, then the quality and potency of their own stem cells are seriously compromised.

To circumvent this problem, Paulo Madeddu and his team at the Bristol Heart Institute have used an immortalized human neural stem cell line called CTX to treat animals who suffered from diabetes mellitus and CLI.

The CTX cell line comes from a biotechnology company called ReNeuron. This company is using this cell line in a clinical trial for stoke patients, and wants to use the CTX cell line in a clinical trial for CLI patients in the future.

When CTX cells are injected into the muscle of diabetic mice with CLI, the cells promote recovery from CLI. The CTX cells do so by promoting the growth of new blood vessels.

Madeddu said, “There are not effective drug interventions to treat CLI. The consequences are a very poor quality of life, possible major amputation and a life expectancy of less than one year from diagnosis in 50 percent of all CLI patients.”

Dr. Madeddu continued: “Our findings have shown a remarkable advancement towards more effective treatments for CLI and we have also demonstrated the importance of collaborations between universities and industry that can have a social and medical impact.”

Skin Tissue as a Treatment for Multiple Sclerosis

Italian researchers have derived stem cells from skin cells that can reduce the damage to the nervous system cause by a mouse version of multiple sclerosis. This experiment provides further evidence that stem cells from patients might be a feasible source of material to treat their own maladies.

The principal investigators in this work, Cecilia Laterza and Gianvito Martino, are from the San Raffaele Scientific Institute, Milan and the University of Milan, respectively.

Because multiple sclerosis results from the immune system attacking the myelin sheath that surrounds nerves, most treatments for this disease consist of agents that suppress the immune response against the patient’s own nerves. Unfortunately, these treatments have pronounced side effects, and are not effective in the progressive phases of the disease when damage to the myelin sheath might be widespread.

The symptoms of loss of the myelin sheath might one or more of the following: problems with touch or other such things, muscle cramping and muscle spasms, bladder, bowel, and sexual dysfunction, difficulty saying words because of problems with the muscles that help you talk (dysarthria), lack of voluntary coordination of muscle movements (ataxia), and shaking (tremors), facial weakness or irregular twitching of the facial muscles, double vision, heat intolerance, fatigue and dizziness; exertional exhaustion due to disability, pain, or poor attention span, concentration, memory, and judgment.

Clinically, multiple sclerosis is divided into the following categories on the basis of the frequency of clinical relapses, time to disease progression, and size of the lesions observed on MRI.  These classifications are:

A)         Relapsing-remitting MS (RRMS): Approximately 85% of cases and there are two types – Clinically isolated syndrome (CIS): A single episode of neurologic symptoms, and Benign MS or MS with almost complete remission between relapses and little if any accumulation of physical disability over time.

B)         Secondary progressive MS (SPMS)

C)         Primary progressive MS (PPMS)

D)        Progressive-relapsing MS (PRMS)

The treatment of MS has 2 aspects: immunomodulatory therapy (IMT) for the underlying immune disorder and therapies to relieve or modify symptoms.

To treat acute relapses:

A)    Methylprednisolone (Solu-Medrol) can hasten recovery from an acute exacerbation of MS.

B)    Plasma exchange (plasmapheresis) for severe attacks if steroids are contraindicated or ineffective (short-term only).

C)    Dexamethasone is commonly used for acute transverse myelitis and acute disseminated encephalitis.

For relapsing forms of MS, the US Food and Drug Administration (FDA) include the following:

A)    Interferon beta-1a (Avonex, Rebif)

B)    Interferon beta-1b (Betaseron, Extavia)

C)    Glatiramer acetate (Copaxone)

D)    Natalizumab (Tysabri)

E)    Mitoxantrone

F)    Fingolimod (Gilenya)

G)    Teriflunomide (Aubagio)

H)    Dimethyl fumarate (Tecfidera)

For aggressive MS:

A)    High-dose cyclophosphamide (Cytoxan).

B)    Mitoxantrone

In order to treat multiple sclerosis, restoring the damaged myelin sheath is essential for returning patients to their former wholeness.

In this study, this research team reprogrammed mouse skin cells into induced pluripotent skin cells (iPSCs), and then differentiated them into neural stem cells. Neural stem cells can differentiate into any cell type in the central nervous system.

(a) Bright field image of miPSC-NPCs obtained from miPSC Sox2βgeo. Scale bar, 50 μm. (b) GFP expression on miPSC-NPCs upon LV infection. Scale bar, 50 μm. (c–h) Immunostaining for Nestin (c), Vimentin (d), Olig2 (e), Mash1 (f), Sox2 (g) and GLAST (h). Scale bar, 50 μm. (i) Growth curve of miPSC-NPC. (j–l) Differentiation of miPSC-NPCs in three neural-derived cell populations, neurons (j), astrocytes (k) and oligodendrocytes (l). Scale bar, 50 μm. (m–o) Expression of the adhesion molecule CD44 (m), the chemokine receptor CXCR4 (n) and the integrin VLA-4 (o) on in vitro cultured miPSC-NPCs analysed by flow-activated cell sorting (FACS). Grey line represents the isotype control, whereas red line indicates the stained cells.
(a) Bright field image of miPSC-NPCs obtained from miPSC Sox2βgeo. Scale bar, 50 μm. (b) GFP expression on miPSC-NPCs upon LV infection. Scale bar, 50 μm. (c–h) Immunostaining for Nestin (c), Vimentin (d), Olig2 (e), Mash1 (f), Sox2 (g) and GLAST (h). Scale bar, 50 μm. (i) Growth curve of miPSC-NPC. (j–l) Differentiation of miPSC-NPCs in three neural-derived cell populations, neurons (j), astrocytes (k) and oligodendrocytes (l). Scale bar, 50 μm. (m–o) Expression of the adhesion molecule CD44 (m), the chemokine receptor CXCR4 (n) and the integrin VLA-4 (o) on in vitro cultured miPSC-NPCs analysed by flow-activated cell sorting (FACS). Grey line represents the isotype control, whereas red line indicates the stained cells.

Next, Laterza and her colleagues administered these neural stem/progenitor cells “intrathecally,” which simply means that they were injected into the spinal cord underneath the meninges that cover the brain and spinal cord to mice that had a rodent version of multiples sclerosis called EAE or experimental autoimmune encephalomyelitis.

EAE mice are made by injecting them with an extract of myelin sheath. The mouse immune system mounts and immune response against this injected material and attacks the myelin sheath that surrounds the nerves. EAE does not exactly mirror multiple sclerosis in humans, but it comes pretty close. While multiple sclerosis does not usually kill its patients, EAE either kills animals or leaves them with permanent disabilities. Animals with EAE also suffer severe nerve inflammation, which is distinct from multiple sclerosis in humans in which some nerves suffer inflammation and others do not. Finally, the time course of EAE is entirely different from multiple sclerosis. However, both conditions are caused by an immune response against the myelin sheath that strips the myelin sheath from the nerves.

The transplanted neural stem cells reduced the inflammation within the central nervous system. Also, they promoted healing and the production of new myelin. However, most of the new myelin was not made by the injected stem cells. Instead the injected stem cells secreted a compound called “leukemia inhibitory factor” that promotes the survival, differentiation and the remyelination capacity of both internal oligodendrocyte precursors and mature oligodendrocytes (these are the cells that make the myelin sheath). The early preservation of tissue integrity in the spinal cord limited the damage to the blood–brain barrier damage. Damage to the blood-brain barrier allows immune cells to infiltrate the central nervous system and destroy nerves. By preserving the integrity of the blood-brain barrier, the injected neural stem cells prevented infiltration of blood-borne of the white blood cells that are ultimately responsible for demyelination and axonal damage.

(a) EAE clinical score of miPSC-NPC- (black dots) and sham-treated mice (white dots). Each point represents the mean disease score of at least 10 mice per group (±s.e.m.); two-way ANOVA; *P<0.05; **P<0.01. (b) Quantification of spinal cord demyelination and axonal damage at 80 dpi in miPSC-NPC- (black bars) versus sham-treated (white bars) EAE mice (N=6 per group). Data (mean values ±s.e.m.) represent the percentage of damage. Student’s t-test; *P<0.05; **P<0.01. (c,d) Representative images of spinal cord sections stained with Luxol Fast Blue (c) and Bielschowsky (d). Red dotted lines represent areas of damage. Scale bar, 200 μm. (e) Quantitative analysis of the localization of miPSC-NPCs upon transplantation into EAE mice (N=3). (f–k) Immunostaining for CD45, MBP, Nestin, Ki67, Olig2, GFAP, βIII tubulin and GFP shows accumulation of transplanted cells (arrowheads) within perivascular spinal cord damaged areas. Dotted lines represent vessels. Nuclei are visualized with DAPI. Scale bars, 50 μm. (l) Percentage (±s.e.m.) of the miPSC-NPCs expressing the different neural differentiation markers upon transplantation into EAE mice; the majority of transplanted miPSC-NPCs remained undifferentiated. (N=3 mice per group).
(a) EAE clinical score of miPSC-NPC- (black dots) and sham-treated mice (white dots). Each point represents the mean disease score of at least 10 mice per group (±s.e.m.); two-way ANOVA; *P<0.05; **P

“Our discovery opens new therapeutic possibilities for multiple sclerosis patients because it might target the damage to myelin and nerves itself,” said Martino.

Timothy Coetzee, chief research officer of the National Multiple Sclerosis Society, said of this work: “This is an important step for stem cell therapeutics. The hope is that skin or other cells from individuals with MS could one day be used as a source for reparative stem cells, which could then be transplanted back into the patient without the complications of graft rejection.”

Obviously, more work is needed, but this type of research demonstrates the safety and feasibility of regenerative treatments that might help restore lost function.

Martino added, “There is still a long way to go before reaching clinical applications but we are getting there. We hope that our work will contribute to widen the therapeutic opportunities stem cells can offer to patients with multiple sclerosis.”

See Cecilia Laterza, et al. iPSC-derived neural precursors exert a neuroprotective role in immune-mediated demyelination via the secretion of LIF. NATURE COMMUNICATIONS 4, 2597: doi:10.1038/ncomms3597.

Stem Cell-Mediated Scarring of the Spinal Cord Aids in Recovery

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.

Ependymal cell incorporation of 5-ethynyl-2′-deoxyuridine is reduced in the absence of Ras genes in intact spinal cord (A and B) and 7 days after injury (C to E). Arrowheads and arrows point to proliferating recombined (A and C) and unrecombined (C and D) ependymal cells, respectively. Injury-induced migration is blocked in rasless ependymal cells (F). Sagittal view of the lesion site 14 weeks after injury in a FoxJ1 control mouse (G) and FoxJ1-rasless mice (H to J). Recombined ependymal cells express YFP in (A) to (D), and cell nuclei are labeled with 4′,6-diamidino-2-phenylindole (DAPI) and appear blue. *P < 0.05, **P < 0.01; Student’s t test. Error bars show SEM. Scale bars represent 10 μm in (A) to (D) and 200 μm in (G) to (J). GFAP, glial fibrillary acidic protein.
Ependymal cell incorporation of 5-ethynyl-2′-deoxyuridine is reduced in the absence of Ras genes in intact spinal cord (A and B) and 7 days after injury (C to E). Arrowheads and arrows point to proliferating recombined (A and C) and unrecombined (C and D) ependymal cells, respectively. Injury-induced migration is blocked in rasless ependymal cells (F). Sagittal view of the lesion site 14 weeks after injury in a FoxJ1 control mouse (G) and FoxJ1-rasless mice (H to J). Recombined ependymal cells express YFP in (A) to (D), and cell nuclei are labeled with 4′,6-diamidino-2-phenylindole (DAPI) and appear blue. *P < 0.05, **P < 0.01; Student’s t test. Error bars show SEM. Scale bars represent 10 μm in (A) to (D) and 200 μm in (G) to (J). GFAP, glial fibrillary acidic protein.

“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.

Special Brain Cell Helps New Neurons Survive

A specialized type of brain cell that down-regulates stem cell activity seems to encourage the survival of stem cell progeny, according to new research from the laboratory of Hongjun Song, professor of neurology and director of Johns Hopkins Medicine’s Institute for Cell Engineering’s Stem Cell Program.

Uncovering the precise mechanism by which these cells regulate the life and death of neurons is a central to understanding neurodegenerative diseases, aging, and Alzheimer’s disease, since the activity of these cells is linked to these conditions.

“We’ve identified a critical mechanism for keeping newborn neurons alive,” said Song.  “Not only can this help us understand the underlying causes of some diseases, it may also be a step toward overcoming barriers to therapeutic cell transplantation.”

Song collaborated with Guo-li Ming and the members of his research group. Ming is a professor of neurology at the Institute for Cell Engineering.  Song’s team first reported last year that special brain cells called “parvalbumin-expressing interneurons” signal to nearby stem cells not to divide.  They means by which the parvalbumin-expressing interneurons (PEIs) signal to nearby stem cells is by releasing a neurotransmitter called “gamma-aminobutyric acid” (GABA).  In this present study, Ming and Song examined how GABA from surrounding PEIs affects nearby neurons produced by stem cells.

arvalbumin-expressing interneurons
parvalbumin-expressing interneurons

Many of these newborn neurons naturally die soon after they are born.  According to Song, if the new cells survive, these neurons will migrate to a permanent home in the brain and forge connections called synapses with other cells.

To determine whether GABA is a factor in the survival of newborn neurons and their behavior, Song’s team tagged neurons in mouse brains with a fluorescent protein and watched their response to GABA.

“We didn’t expect these immature neurons to form synapses, so we were surprised to see that they had built synapses from surrounding interneurons and that GABA was getting to them that way,” Song said.

In an earlier study, this research team had found that GABA was getting to the synapse-less stem cells by a less direct route – it was drifting across the spaces between cells.

To confirm the finding, the team engineered the interneurons to be stimulated or suppressed by light.  When stimulated by light, the cells activated nearby neurons.  Then they used this light stimulation procedure in live mice, they found that when the specialized neurons were stimulated and gave off more GABA, the newborn neurons survived in greater numbers than otherwise.  This was the opposite of the response of the neural stem cells, which become dormant when given GABA.

Song interpreted these data in the following manner: “This appears to be a very efficient system for tuning the brain’s response to its environment.  When you have a high level of brain activity, you need more newborn neurons, and when you don’t have high activity, you don’t need newborn neurons, but you need to prepare yourself by keeping the stem cells active.  It’s all regulated by the same signal.”

According to Song, the PEIs behave abnormally in neurodegenerative diseases such as Alzheimer’s disease and mental illnesses such as schizophrenia.

“Now we want to see what the role of these interneurons is in the newborn neurons’ next steps” migrating to the right place and integrating into the existing circuitry.  That may be the key to their role in disease,” said Song.  His team is also interested in using the GABA signal to keep transplanted cells alive without affecting other brain processes as a side effect.

See Song J, Sun J, Moss J, Wen Z, Sun GJ, Hsu D, Zhong C, Davoudi H, Christian KM, Toni N, Ming GL, Song H. Parvalbumin interneurons mediate neuronal circuitry-neurogenesis coupling in the adult hippocampus. Nat Neurosci. 2013 Dec;16(12):1728-30. doi: 10.1038/nn.3572. Epub 2013 Nov 10.

Finding the Optimal Spot for Stem Cell Injections In Spinal Cord Injured Patients

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.

Arachnoid space

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.

Human Astrocytes
Human Astrocytes

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.

How Neural Stem Cells Become Neurons and Glia

How do neural stem cells differentiate into neurons or glia? A new paper from researchers at the University of California, Los Angeles (UCLA) seeks to explain this very phenomenon.

Neurons serve as the conductive cells of the nervous system. They transmit electrochemical signals from one neuron to another and provide signals to muscles, glands, and so on. They are responsible for consciousness, thought, learning and memory, and personality.

Despite their immense utility, neurons are not the only cells in the nervous system. Glial cells or just glia support neurons, hold them in place, and supply neurons with oxygen and nutrients and protect them from pathogens.

Glial Cells

When mouse neural stem cells were grown in culture, Wange Lu, associate professor of biochemistry and molecular biology at the Keck School of Medicine, and his colleagues came upon a protein called SMEK1 that promotes the differentiation of neural stem and progenitor cells. SMEK1 also keeps neural stem cells in check by preventing them from dividing uncontrollably.

When Lu and others took a more detailed look at the role of SMEK1, they discovered that it does not work alone, but in concert with a protein called Protein Phosphatase 4 (PP4) to suppress the function of a third protein called PAR3. PAR3 discourages the birth of new neurons (neurogenesis), and PAR3 inhibition leads to the differentiation of neural stem progenitor cells into neurons and glia.

“These studies reveal the mechanisms of how the brain keeps the balance of stem cells and neurons when the brain is formed,” said Wange. “If this process goes wrong, it leads to cancer, or mental retardation or other neurological diseases.”

Neural stem and progenitor cells offer tremendous promise as a future treatment for neurodegenerative disorders, and understanding their differentiation is the first step towards co-opting the therapeutic potential of these cells. This could offer new treatments for patients who suffer from Alzheimer’s, Parkinson’s and many other currently incurable diseases.

This work is interesting. It was published in Cell Reports 5, 593–600, November 14, 2013. My only criticism of some of the thinking in this paper is that neural stem cell lines are usually made from aborted fetuses. I realize that some of these neural stem cell lines come from medical abortions in which the baby had already died, but many of them come from aborted babies. If we are going to use neural stem cells for therapeutic purposes, then we should make them from induced pluripotent stem cells and take them from aborted babies.

Possible Treatment for Brain Disorders

Tuberous Sclerosis is a rare genetic disease that causes the growth of tumors in the brain and other vital organs and may also lead to other conditions such as autism, epilepsy, and cognitive impairment; all of which result from the abnormal generation of neurons.

Tuberous sclerosis is also called tuberous sclerosis complex or TSC, and it is a rare genetic disease that affects multiple organ systems. TSC causes the growth of benign tumors in the brain and on other vital organs such as the kidneys, heart, eyes, lungs, and skin. TSC typically affects the central nervous system and results in a combination of symptoms including seizures, developmental delay, behavioral problems, skin abnormalities, and kidney disease.

TSC affects as many as 25,000 to 40,000 individuals in the United States and about 1 to 2 million individuals worldwide. The estimated prevalence of this disease is one in 6,000 newborns, and it occurs in all races and ethnic groups, and in both genders.

TSC derives its name from the characteristic tuber or potato-like nodules in the brain. These growths calcify with age and become hard or sclerotic.

Many TSC patients show evidence of the disorder in the first year of life. However, clinical features can be subtle initially, and many signs and symptoms take years to develop. As a result, TSC can be unrecognized or misdiagnosed for years.

TSC is caused by defects, or mutations, on two genes-TSC1 and TSC2. Only one of the genes needs to be affected for TSC to be present. The TSC1 gene, discovered in 1997, is on chromosome 9 and produces a protein called Hamartin. The TSC2 gene, discovered in 1993, is on chromosome 16 and produces the protein Tuberin. These proteins combine to form a complex that suppresses cell growth by preventing activation of a master control protein called mTOR. Loss of regulation of mTOR occurs in cells lacking either Hamartin or Tuberin, and this leads to abnormal differentiation and development, and to the generation of enlarged cells, as are seen in TSC brain lesions.

Since Tuberous Sclerosis affect stem cell activity, scientists at Clemson University are examining how neurons are formed from neural stem cells and this research is vital to providing a treatment to Tuberous Sclerosis, which affects how neurons are formed in the brain.

David M Feliciano, assistant professor of biological sciences at Clemson University, said: “Current medicine is directed at inhibiting the mammalian target of rapamycin (mTOR), a common feature within these tumors that have abnormally high activity. However, current treatments have severe side effects, like due to mTOR’s many functions and playing an important role in cell survival, growth and migration.”

mTOR pathway

Feliciano continued: “Neural stem cells generate the primary communicating cells of the brain called neurons through the process of neurogenesis, yet how this is orchestrated is unknown.”

Neural stem cells lie at the very heart of brain development and repair, and alterations in the ability of these cells to self-renew and differentiate can have profound consequences for brain function at any stage of life, according to researchers.

In order to further elucidate the regulation of neurogenesis, Feliciano and his team delivered small pieces of DNA into the neural stem cells of the new-born mice. The team used electroporation to introduce the DNA into the mouse cells, and these small pieces of DNA allowed Feliciano’s team to express and control specific components of the mTOR pathway.

By using these tools, Feliciano and others showed that Increasing the activity of the mTOR pathway cause the neural stem cells to make more neurons at the expense of self-renewal. Increasing mTOR activity caused upregulation of 4E-BP2. 4E-BP2, also known as Eukaryotic translation initiation factor 4E-binding protein 2, binds to a component of the protein synthesis machinery and inhibits its function. Mice that lack functional EIF4EBP2 exhibit autism-like symptoms, including poor social interaction, altered communication and repetitive behaviors.

This work suggests that 4E-BP2 might be a new target for the treatment of TSC and that targeting this protein might cause fewer side effects than targeting mTOR. Future experiments hope to identify those proteins that are made due to the activation of this pathway in neural tissues.

Stem-Based Treatment of Stoke

When blood flow to the brain ceases as the result of a blood clot, trauma, or injury, the brain suffers from a shortage of oxygen. Such an incident is known as a stroke and it can result in the death of neurons and the loss of those functions to which the dead neurons contributed. Treatment for stroke is largely supportive, but regenerative treatments that replace the dead neurons would be the most ideal treatment.

A research consortium at Lund University in Lund, Sweden has found that neurons made from induced pluripotent stem cells integrate into the brains of mice that had suffered strokes. This experiment takes a closer step towards the development of a regenerative treatment for strokes.

Strategies for stem cell-based regenerative therapy in neurodegenerative diseases.
Strategies for stem cell-based regenerative therapy in neurodegenerative diseases.

In the aftermath of a stroke, nerve cells in the brain die. At the Lund Stem Cell Center, the research groups of Zaal Kokaia and Olle Lindvall teamed up to develop a stem cell-based method to treat stroke patients.

After a stroke, the cerebal cortex tends to take the bulk of the damage and neuron loss from the cerebral cortex underlies many of the symptoms following a stroke, such a paralysis and speech problems. The method developed by the Lund Institute scientists should make it possible to generate nerve cells for transplantation from the patient’s own skin cells.


First, the Lund team isolated skin fibroblasts from the afflicted mice and used genetic engineering techniques to convert them into induced pluripotent stem cells (iPSCs), which have many of the differentiation capabilities of embryonic stem cells. These iPSC lines were differentiated into cortical neurons, which tend to populate the cerebral cortex. However, transplanting fully differentiated neurons into the brain tend to not work terribly well because the mature neurons are unable to divide and have poor abilities to connect with other cells. Therefore, the neuron progenitor cells that will give rise to cortical neurons are a better candidate for transplantation.

After generating long-term self-renewing neuroepithelial-like stem cells from iPSCs in the laboratory, the Lund group scientists showed that these stem cells could give rise to neural progenitors that expressed the types of genes found in mature cortical neurons. When these neural progenitor cells were transplanted into rats that had suffered strokes, two months after transplantation, the cortically fated cells showed less proliferation and more efficient differentiation into mature neurons with the right shape, size, and structure of cortical neurons and expressed the same proteins as cortical neurons. These tranplanted cells also extended more axons than those cells that were not fated to form cortical neurons. Transplantation of both the cortical neuron-fated and non-cortical neuron-fated cells caused recovery of the impaired function in the stepping test in comparison to controls. At 5 months after stroke, there was no tumor formation and the grafted cells had all the electrophysiological properties of mature neurons and showed full evidence that they had integrated into the existing neural circuitry.

These results are very promising and represent a very early but important step towards a stem cell-based treatment for stroke in patients. Further experimental studies are necessary if these experiments are to be translated into the clinic in a responsible way.