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.

Hippocampus

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.