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.

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.

Stem Cells For Better Drug Assays

Moving a drug from the laboratory to the clinic is terrifically expensive and slow. Even after extensive tests in cultured cells and laboratory animals, the drug may still fail in its clinical tests. Such failures cost drug companies massive amounts of money, and this drives up the cost of those drugs that succeed in clinical trials and secure FDA approval. If scientists could design drug assays that better predict whether a compound will succeed in human trials could help pharmaceutical companies identify the most promising drugs in which to invest their resources.

Fortunately, a study published this week in Cell Stem Cell might represent such a breakthrough. In this paper, researchers reported that a new stem cell-based assay was actually able to pinpoint a potential small molecule treatment for amyotrophic lateral sclerosis (ALS, also known as known as Lou Gehrig’s disease). In follow-up experiments, the drug promoted better cell survival better than two other drug candidates that had recently failed in phase III clinical trials.

One of the study’s co-authors, Clifford Woolf, the director of the F. M. Kirby Neurobiology Center at Boston Children’s Hospital, said that this new strategy “could either be used in the late preclinical stage to confirm the cellular actions of particular leads, or even better as a driver of early exploratory preclinical testing, revealing new targets and pathways.”

The laboratory of Lee Rubin at Harvard Medical School developed this new cell-bases assay by using embryonic stem cells derived from both healthy mice and those with a mutation in the gene SOD1. Mutations in SOD1 are known to cause ALS in people. After differentiating the stem cells into motor neurons, which are the cells that die off in ALS patients — the group exposed these embryonic stem cell-derived motor neurons to 5000 different small molecular-weight compounds. The cultured cells were also deprived of essential chemicals from the culture medium in order to accelerate their death.

In these experiments, a molecule called kenpaullone, which is an inhibitor of the enzyme GSK-3, stood out in their initial screen. GSK-3 controls cell growth and death, and kenpaullone strongly promoted the survival of both normal and mutated motor neurons and kept them morphologically healthy. In a different experiment, the group showed that the drug decreased levels of SOD1, which is thought to aggregate in the motor neurons of people with the disorder.

In further tests, Rubin and his colleagues treated motor neurons made from induced pluripotent stem cells derived from adult cells that had been taken from two ALS patients with kenpaullone. One of these ALS patients had mutations in the SOD1 gene, while the other harbored mutations in the TDP-43 gene. TDP-43 is yet another gene associated with ALS, since mutations in it also cause ALS. Rubin’s team found that the small molecule substantially boosted motor neuron survival by some 2- to 4-fold, and this effect was dose-dependent and was observed in both healthy and diseased cells.

In contrast, two drug candidates that recently failed phase III clinical trials were less effective when tested in the same assay. The drug dexpramipexole had no effect on patient-derived motor neurons, and olesoxime had a variable but only moderately positive effect.

It’s not clear how the SOD1 mutation causes the degeneration of motor neurons. According to Alysson Muotri, assistant professor of pediatrics and cellular and molecular medicine at the University of California, San Diego, who was not involved in the new study; knowing the pathological mechanism of inactive SOD1 on motor neurons could inform additional endpoints for the stem cell assays
To date, kenpaullone has only been tested in on cultured neurons and not in living mice to date. And, as with all cell culture assays, it is an open question as to “the extent to which changes in neurons in a dish phenocopy complex diseases that may take many years to manifest, and if rescue of the phenotype by a hit in a screen will translate into therapeutic benefit in patients,” Woolf noted.

However Muotri sees great potential for stem cell-based assays and their use for drug discovery. “Stem cell based screens will definitely speed up drug discovery, bringing more powerful candidates to clinical trial,” said Muotri. “I can see this going into personalized medicine—we will be testing drugs and doses in motor neurons derived from each patient to personalize treatment.”

Yang, Y. M., S. K. Gupta, K. J. Kim, B. E. Powers, A. Cerqueira, B. J. Wainger, H. D. Ngo, K. A. Rosowski, P. A. Schein, C. A. Ackeifi, et al. 2013. A small molecule screen in Stem-Cell-derived motor neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. Cell Stem Cell (April).

Making Preneurons from White Blood Cells for ALS Patients

ALS or amyotrophic lateral sclerosis is a disease that results in he death of motor neurons. Motor neurons enable skeletal muscles to contract, which drives movement. The death of motor neurons robs the patient of the ability to move and ALS patients suffer a relentless, progressive, and sad decline that culminates in death from asphyxiation. Treatments are largely palliative, but stem cells treatments might delay the onset of the disease, or even regenerate the dead neurons.

To this end a Mexican group from Monterrey has used a protocol to isolate white blood cells from the circulating blood of ALS patients, and differentiate a specific population of stem cells from peripheral blood into preneurons. Although these cells were not used to treat the patients in this study, such cells do show neuroprotective features and using them in a clinical study does seem to be the next step.

In this study, CD133 cells were isolated from peripheral blood and subjected to a special culture system called a neuroinduction system. After 2-48 hours in this system, the cells showed many features that were similar to those of neurons. The cells express a cadre of neural genes (beta-tubulin III, Oligo 2, Islet-2, Nkx6.1, and Hb9). Some of the ells also grew extensions that resemble the axons of true neurons.

Interestingly, the conversion of the CD133 cells into preneurons showed similar efficiency regardless of the age, sex, or health of the individual. Even those patients with more advanced ALS had CD133 cells that differentiated into preneurons with efficiencies equal to those of their healthier counterparts. While each patient showed variation with regards to the efficiency at which their CD133 cells differentiated into preneurons, these variations could not be correlated with the age, health or sex of the patient.

The fact that these preneurons expressed Oligo2, suggests that they could differentiate into motor neurons. Therefore, even though this study was small (13 patients), it certainly shows that cells that might provide treatment possibilities for ALS patients can be made from the patient’s own blood cells.

See Maria Teresa Gonzalez-Garza et al., Differentiation of CD133+ Stem Cells from Amyotrophic Lateral Sclerosis Patients into Preneuron Cells. Stem Cells Translational Medicine 2013;2:129-35.