Activation of Dormant Viruses May Cause ALS


Inactive viruses that litter the human genome may become reactivated and contribute to the development of motor neuron disease, according to new research published today in the journal Science Translational Medicine.

Human endogenous retroviruses (HERVs) are the flotsam and jetsam of ancient viruses that integrated into our chromosomes long ago as the results of retrovirus infections that occurred over several million years of our history.  These HERV sequences account for about 8% of human DNA and the vast majority of them have acquired multiple genetic mutations that made rendered them innocuous.  Therefore, HERVs are sometimes referred to as “junk” DNA, although some of these sequences have been shown to have function (for example, see Dupressoir A, Lavialle C, Heidmann T. Placenta. 2012 Sep;33(9):663-7).

In 2011, Avindra Nath, the intramural clinical director of the National Institute of Neurological Disorders and Stroke, and his colleagues reported that proteins synthesized by one such HERV known as HERV-K are found in very high concentrations in the brains of patients who died of amyotrophic lateral sclerosis (ALS), which is a progressive and fatal neurodegenerative disease that destroys those motor neurons that control speech, movement, swallowing and breathing, which leads to death between three to five years after the symptoms first appear.

In their new study, Nath’s research group investigated the toxicity of viral proteins to nerve cells. They examined samples of nervous tissue from 11 patients who had died of ALS, 10 Alzheimer’s patients, and 16 people who showed no signs of neurological disease as controls.  They used RNA sequencing to confirm that transcripts of three HERV-K genes are present in tissue samples from the ALS patients but not in those from the Alzheimer’s patients or control patients.  In their next set of experiments, Nath and his coworkers showed that the proteins encoded by these viral genes localized to motor neurons in the brains and front halves of the spinal cords of ALS patients.  This is significant, since the ventral or font portions of the spinal cord contains the cell bodies of motor neurons that send their axonal fibers to the body’s skeletal muscles where they synapse with those muscles.  Thus the presence of the viral proteins strongly correlates with the tendency of these cells to die.

To definitively test the toxicity of these viral proteins to neurons, Nath and others transfected either the entire viral genome, or just the viral env gene, which encodes the virus’s coat protein, into cultured human neurons.  Once integrated into the genomes of the cultured cells, the viral genes were fully activated and used the cell’s molecular machinery to synthesize their respective proteins.  Expression of these viral genes killed off significant numbers of cells and caused them to retract their neural fibers.  Furthermore expression of only the env gene in these cultured neurons was sufficient to kill them.

To test their hypothesis in a living animal, Nath and others generated a strain of genetically engineered mice whose neurons express high levels of the HERV-K env gene.  Behavioral tests showed that these HERV-K env+ animals developed motor function abnormalities; they had difficulty walking and balancing compared to healthy mice.  These symptoms progressed rapidly between 3 and 6 months of age, and half of the animals had died before or shortly after reaching 10 months of age.

Closer examination revealed that neurons in the motor cortex had degenerated.  They also showed a decrease in the length, branching and complexity of dendrites, and a reduction in the number of dendritic spines (small, finger-like extensions that receive chemical signals from other cells).

All of these data strongly suggest that reactivation of dormant HERV-K contributes to neurodegeneration in the brain and spinal cord.  The absence of this virus in the brains of Alzheimer’s patients supports the conclusion that reactivation of it causes degeneration, rather than being a consequence of it, and further suggests that it is specific to ALS.

ALS is associated with genetic mutations in more than 50 different genes.  However, as is the case for Alzheimer’s, these inherited forms of the disease, which account for just 10-15% of cases. But this study only examined patients with sporadic, or non-inherited, ALS, the cause of which have been much harder to pin down.

Further genetic analyses may identify DNA sequence variations, in the HERV-K genes themselves, and others that interact with them, which might make the virus more prone to reactivation.  More work will need to be done to determine exactly how the reactivated virus genes contribute to the disease.

Meanwhile, Nath and his colleagues are collaborating with researchers at Johns Hopkins University to determine if anti-retroviral drugs might alleviate disease symptoms in subsets of ALS patients.

See Li, W., et al. (2015). Human endogenous retrovirus-K contributes to motor neuron disease. Sci. Trans. Med., 7: 307ra153.

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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.

Compound from Sully Putty Might Advance Neural Stem Cell Therapies


According to a University of Michigan engineering team, human pluripotent stem cells differentiate differently in response to the sponginess of the surface upon which they grow.

University of Michigan assistant professor of mechanical engineering, Jianping Fu, and his colleagues, efficiently directed human embryonic stem cells to differentiate into working spinal cord cells by growing the cells on a carpet of poly(dimethylsiloxane), which is one of the main ingredients in the toy known as “Silly Putty.” This study established the importance of physical signals in the control of stem cell differentiation.

According to Fu, these data could be the beginning of a series of investigations that uncovers the most efficient way to guide pluripotent stem cells to differentiate into nervous tissues that can be used to replace diseased cells in patients with Alzheimer’s disease, Huntington’s disease or amyotrophic lateral sclerosis (Lou Gehring’s disease).

In Fu’s system, he and his co-workers engineered the poly(dimethylsiloxane) carpets by using this compound to form fine threads that were strung between microscopic posts. By varying the height of the posts, Fu discovered that he could vary the stiffness of the surface. Shorter posts gave a more rigid, stiff carpet and longer posts gave softer more plush carpets.

When embryonic stem cells were grown on poly(dimethylsiloxane) carpet strung between tall posts, they differentiated into neurons much more quickly and at a higher percentage than when they were grown on the more rigid and stiffer poly(dimethylsiloxane) carpets.  After 23 days, colonies of spinal cord motor neurons that control how muscles move grew on the softer micropost carpets.  These cell assemblages were four times more pure and 10 times larger than those growing on either traditional plates or rigid carpets.

“To realize promising clinical applications of human embryonic stem cells, we need a better culture system that can reliably produce more target cells that function well,” said Fu.  He added: “Our approach is a big step in that direction, by using synthetic micro-engineered surfaces to control mechanical environmental signals.”

Fu is presently collaborating with U-M Medical School professor of neurology, Eva Feldman.  Dr. Feldman is an expert in amyotrophic lateral sclerosis (ALS), and firmly believes in the power of stem cells to help ALS patients grow new stem cells that can replace the diseased, death or damaged nerve cells.  Feldman is also applying Fu’s ingenious technique to make neurons from a patient’s own cells.  Mind you, these results are purely exploratory at this point, since Feldman simply wants to determine the feasibility of this procedure.

Even if this technique does not pan out for regenerative treatments, it provides a very workable model system to study the electrical behavior of neurons from ALS patients in comparison to neurons from non-ALS individuals.

Fu’s system also has identified a cell signaling pathway that is involved in the regulation of mechanically sensitive behaviors.  This signaling pathway – the Hippo/Yap pathway – is also involved in controlling organ size and suppression of tumor formation.

Corresponding proteins in Drosophila and mammals are shown in the same colours. When organs are growing (Hippo pathway OFF), nuclear Yki/Yap binds to unknown DNA-binding factor(s) X and regulates the transcription of growth targets. When organs have reached the correct size (ON), the Hippo signalling pathway is activated (unknown ligand Y–Fat– Merlin–Expanded–Hippo interactions, in the Drosophila case; ligand Y–FatJ–NF2–FDM6–Mst½–Lats½ in mammals), and Yki and YAP is inactivated by localizing to the cytoplasm in response to Wts phosphorylation and 14-3-3 binding. ? indicates regulatory relationships that still need to be investigated. Figure adapted from reference 2.
Corresponding proteins in Drosophila and mammals are shown in the same colors. When organs are growing (Hippo pathway OFF), nuclear Yki/Yap binds to unknown DNA-binding factor(s) X and regulates the transcription of growth targets. When organs have reached the correct size (ON), the Hippo signalling pathway is activated (unknown ligand Y–Fat– Merlin–Expanded–Hippo interactions, in the Drosophila case; ligand Y–FatJ–NF2–FDM6–Mst½–Lats½ in mammals), and Yki and YAP is inactivated by localizing to the cytoplasm in response to Wts phosphorylation and 14-3-3 binding. ? indicates regulatory relationships that still need to be investigated. Figure adapted from reference 2.

The work of Fu and Feldman could certainly provide significant advances in our understanding of how pluripotent stem cells differentiate in the body.  This work also suggests that physical signals are important in patterning the nervous system, especially since the cells of the nervous system become specialized for specific tasks according to their physical location within the body and nervous system in general.

An Improved Way to Make Motor Neurons in the Laboratory from Stem Cells


A research team from the University of Illinois at Urbana-Champaign has reported that they can produce human motor neurons from stem cells much more quickly and efficiently than previous methods allowed. This finding was published in the journal Nature Communications and it will almost certainly provide new ways to model human motor neuron development, diseases of the nervous system, and ways to treat spinal cord injuries.

The new protocol described in the Nature Communications paper includes adding critical signaling molecules to precursor cells a few days earlier than specified by previous methods. This innovation increases the proportion of healthy motor neurons derived from stem cells from 30 to 70 percent. It also cuts in half the time required to make motor neurons.

“We would argue that whatever happens in the human body is going to be quite efficient, quite rapid,” said University of Illinois cell and developmental biology professor Fei Wang, who led the study with visiting scholar Qiuhao Qu and materials science and engineering professor Jianjun Cheng. “Previous approaches took 40 to 50 days, and then the efficiency was very low – 20 to 30 percent. So it’s unlikely that those methods recreate human motor neuron development.”

The new method designed by Qu generated a larger population of mature, functional motor neurons in 20 days. According to Wang, this new approach will allow scientists to induce mature human motor neuron development in cell culture, and to identify the factors that drive this process

Because stem cells can differentiate into a wide variety of cell types, they are unique compared to mature, adult cells. Making neurons from either embryonic stem cells or induced pluripotent stem cells requires the addition of signaling molecules to the cells at critical moments in culture.

Previously, Wang and his colleagues discovered a molecule called compound C that converts stem cells into “neural progenitor cells,” or NPCs. NPCs represent an early stage in neuronal development, and further manipulation of NPCs can drive them to become neurons, but differentiating NPCs into motor neurons presents another set of problems.

Other published studies have established that the addition of two important signaling molecules, six days after exposure to compound C, to NPCs in culture can generate motor neurons, but at rather poor efficiencies. In this newly published study, Qu showed that adding the signaling molecules at Day 3 worked better: The NPCs differentiated into motor neurons quickly and efficiently. Thus, Day 3 represents a previously unrecognized NPC cell stage.

This new approach has immediate applications in the laboratory. Amyotrophic lateral sclerosis or ALS is a neurological disease that causes motor neurons to die off. By using Wang and Qu’s cell culture system to make neurons from the skin cells of ALS, and watching them develop into motor neurons, scientists and physicians will divine other new insights into disease processes. Therefore, any method that improves the speed and efficiency of generating the motor neurons will be a boon to neuroscientists. These cells can also be used to screen for drugs to treat motor neuron diseases, and might even be used to therapeutically restore lost function in patients someday.

“To have a rapid, efficient way to generate motor neurons will undoubtedly be crucial to studying – and potentially also treating – spinal cord injuries and diseases like ALS,” Wang said.

New Method Derived Skeletal Muscle Cells from Pluripotent Stem Cells


A University of Wisconsin research team led by Masatoshi Suzuki has devised a new protocol for the production of large quantities of skeletal muscle cells from pluripotent stem cells.

Suzuki and his team used embryonic stem cells lines and induced pluripotent stem cells to generate large quantities of muscles and muscle progenitor.

Suzuki adapted a technique used to make brain cells to derive his muscle cells in culture. He grew the stem cells as floating spheres in high concentrations of two growth factors: fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF). This combination of growth factors directed the stem cells to differentiate into skeletal muscle cells and muscle progenitors.

To replace damaged or diseased muscles in the clinic, physicians will require large quantities of muscle cells. Therefore, there was an ardent search to design a technique that was efficient, but also fast and relatively simple. Even though several protocols have been devised to differentiate pluripotent stem cells into muscle cells, not all of these protocols are practical for clinical use. For example, some protocols are simply too cumbersome for clinical use. Still others make use of genetically engineered cells that have not been approved for clinical use.

Earlier, Suzuki transplanted lab-engineered skeletal muscle into mice that had a form of amyotrophic lateral sclerosis. These animals had better muscle function and survived better than the control animals.

The muscle progenitors generated in Suzuki’s laboratory could potentially play a similar role in human patients with Lou Gehring’s disease. Suzuki’s method can grow muscle progenitor cells, which can grow in culture, from induced pluripotent stem cells, which are derived from the patient’s own cells. Such cells could be used as a model system to study the efficacy of particular treatments on the patient’s muscles, or they could be used to treat patients who have muscle defects.

“Our protocol can work in multiple ways and so we hope to provide a resource for people who are exploring specific neuromuscular diseases in the laboratory,” said Suzuki.

The advantages of Suzuki’s protocol are manifold. First, the cells are grown in a defined medium devoid of animal products. Secondly, the stem cells are grown as spheres, and these grow faster when grown as spheres than they do with other techniques. Third, 40-60 percent of the cells grown in this culture system differentiate into skeletal muscle cells or muscle progenitor cells. This is a very high proportion of muscle cells when compared to other protocols.

Suzuki hopes that by toying with the culture system, he and his colleagues can increase this proportion of muscle cells that form from the initial stem cell culture. This would enhance the potential of using these cells for clinical purposes.

Induced Pluripotent Stem Cells Recapitulate ALS in Culture and Suggest New Treatment


Induced pluripotent stem cells are made from the adult cells of an individual by means of genetic engineering techniques. After introducing four different genes into adult cells, some of the cells de-differentiate to form cells that grow indefinitely in culture and have most of the characteristics of embryonic stem cells. However, if iPSCs are made from a patient who suffers from a genetic disease, then those stem cells will have the same mutation as the patient, and any derivatives of those iPSCs will show the same behaviors and pathologies of the tissues from the patient. This strategy is called the “disease in a dish” model and it is being increasingly used to make seminal discoveries about diseases and treatment strategies.

Scientists from Cedars-Sinai Regenerative Medicine Institute have used iPSC technology to study Lou Gehrig’s disease, and their research has provided a new approach to treat this horrific, debilitating disease.

Because I have previously written about Lou Gehrig’s disease or Amyotrophic Lateral Sclerosis (ALS), I will not describe it further.

Cedar Sinai scientists isolated skin scrapings from each patient and used the skin fibroblasts from each sample to make iPSCs. According to Dhruv Sareen, the director of the iPSC facility and faculty research scientist with the Department of Biomedical Sciences and the first author on this article, skins cells of patients who have ALS were converted into motor neurons that retained the genetic defects of the disease, thanks to iPSC technology. Then they focused on gene called C9ORF72, which was found to be the most common cause of familial ALS and frontotemporal lobar disease, and is even responsible for some cases of Alzheimer’s and Parkinson’s disease.

Mutations in a gene that has the very non-descriptive name “chromosome 9 open reading frame 72” or C9ORF72 for short seems to play a central role in the onset of Lou Gehrig’s disease. Mutations in C9orf72 have been linked with familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). FTD is a brain disorder that typically leads to dementia and sometimes occurs in tandem with ALS.

Mutations in C9ORF72 result from the expansion of a hexanucleotide repeat GGGGCC. When the C9ORF72 gene is replicated, the enzyme that replicates DNA (DNA polymerase) has a tendency to slip when comes to this stretch of nucleotides and this polymerase slip causes the hexanucleotide GGGGCC sequence to wax and wane (expand and shrink). Normally, there are up to 30 repeats of this GGGCC sequence, but in people with mutations in C9ORF72, this GGGGCC repeat can occur many hundreds of times. Massive expansions of the GGGGCC repeat interferes with normal expression of the protein made by C9ORF72. The presence of messenger RNAs (mRNAs) with multiple copies of GGGGCC in the nucleus and cytoplasm is toxic to the cell, since it gums up protein synthesis, RNA processing and other RNA-dependent functions. Also the lack of half of the C9ORF72 protein contributes to the symptoms of this conditions.

Robert Baloh, director of Cedars-Sinai’s Neuromuscular Division and the lead researcher of this research project, said, “We think this buildup of thousands of copies of the repeated sequence GGGGCC in the nucleus of patient’s cells may become toxic by altering the normal behavior of other genes in the motor neurons. Because our studies supported the toxic RNA mechanism theory, we used to small segments of genetic material called antisense oligonucleotides – ASOs – to block the buildup and degrade the toxic RNA. One ASO knocked down overall C9ORF72 levels. The other knocked down the toxic RNA coming from the gene without suppressing overall gene expression levels. The absence of potentially toxic RNA, and no evidence of detrimental effect on the motor neurons, provides a strong basis for using this strategy to treat patients suffering from these diseases.”

Baloh continued: “In a sense, this represents the full spectrum of what we are trying to accomplish with patient-based stem cell modeling. It gives researchers the opportunity to conduct extensive studies of a disease’s genetic and molecular makeup and develop potential treatments in the laboratory before translating them into patient trials.”

Researchers from another institution recently began a phase one clinical trial that used a similar ASO strategy to treat ALS caused by a different mutation. No safety issues were reported in this clinical trial.

Clive Svendsen, director of the Regenerative Medicine Institute and one of the authors, has investigated ALS for more than a decade, said, “ALS may be the cruelest, most severe neurological disease, but I believe the stem cell approach used in this collaborative effort holds the key to unlocking the mysteries of the and other devastating disorders. Within the Regenerative Medicine Institute, we are exploring several other stem cell-based strategies in search of treatments and cures.”

ALS affects 30,000-50,000 people in the US alone, but unlike other neurodegenerative diseases, it is almost always fatal within three to five years.