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.

Stem Cell Trial for ALS Patients


Two patients afflicted with amyotrophic lateral sclerosis have received stem cell injections into their spinal cords at the University of Michigan Health System. These are the first two subjects in a national clinical trial.

Both of these volunteers have returned home and will continue to receive medical follow-up and monitoring in order to assess the safety of this procedure and to detect any potential improvements in the condition of these patients.

Additional patients with this condition, which is also known as Lou Gehring’s disease, are being evaluated for possible participation in the trial at U-M and Emory University. This phase 2 trial is approved by the US Food and Drug Administration (US FDA) and is being funded by a Maryland-based company called Neuralstem, Inc., the proprietor of this stem cell product.

Neuralstem, Inc., has developed a neural stem cell line called NSI-566. When injected into the central nervous system of a living animal, these cells will divide up to 60 times and differentiate into a variety of neural cells (neurons, glial cells, etc.). Several publications have shown that injected NSI-566 cells survive when injected into the spinal cord, differentiate into several different neural cell types, and successfully integrate into the presently existing neural network.

In ALS patients, motor neurons progressively die off in the spinal cord, which limits voluntary movement.  ALS is a progressive neurodegenerative disease that affects nerve cells in the brain and spinal cord, leading to complete paralysis, and eventually, death. According to the ALS Association, as many as 30,000 Americans have the disease, and about 5,600 people in the U.S. are diagnosed with ALS each year.  The goal of this treatment strategy is to stabilize ALS patients and to replace dead or dying neurons and to slow the progressive decline and loss of movements, walking, and eventually breathing.

Eva Feldman, professor of neurology at the U-M Medical School, is the principal investigator for this clinical trial, and serves as an unpaid consultant to Neuralstem, Inc.  Dr. Feldman led the analysis of the results from the Phase 1 trial, which ended in 2012.  In this Phase 1 trial, 100,000 cells were delivered to each patient, and the patients tolerated them well and experienced to severe side effects.  One subgroup of patients seemed to experience interruption of the progression of ALS symptoms.

Feldman commented, “We’re going to be permitted to give more injections and more stem cells, in Phase 2.  We’re very excited that we have been able to bring this important work to the University of Michigan.”

Parag Patil, a neurosurgeon and biomedical engineer, performed both operations on the trial participants.  In each case, the patient’s spinal column was unroofed and the spinal cord exposed to receive the cells.  The stem cells are then introduced by means of a custom-designed delivery device that is affixed to the subject’s spinal bones so that it moves with the patient’s breathing throughout the process.

Neuralstem spinal cord injection device

Patil, as assistant professor, also serves as a paid engineering consultant to Neuralstem, Inc., in order to further prefect the injection device.  A third participant in this clinical trial received a stem cell injection in September at Emory University in Atlanta, Georgia.  This Phase 2 dose escalation trial is designed to treat up to 15 ambulatory patients in five different dosing cohorts, and will do so under an accelerated dosing and treatment schedule.  The first 12 patients will be divided into four cohorts and each will receive injections only in the cervical region of the spinal cord, where breathing function is controlled.

The first cohort of three patients received 10 cervical region injections of 200,000 stem cells per injection.  The trial will now progress to a maximum of 20 cervical injections of up to 400,000 stem cells per injection.  The last three Phase 2 patients will receive injections into the cervical and lumbar spinal regions, and will receive 20 injections of 400,000 cells in the lumbar region in addition to the cervical injections they have already received.  The trial also accelerates the treat schedule, and is designed to progress at the rate of one cohort per month with one month observations periods between cohorts.  Researchers expect all of the patients could be treated by the end of the second quarter in 2014.
Lumbar and Cervical

Neural Stem Cells Improve Spinal Injuries in Rats


Disclaimer:  I am reporting on this experiment because of its significance for people with spinal cord-injuries even though I remain appalled at the manner in which the stem cells were acquired.

An international research team has reported that a single set of injections of human neural stem cells had provided significant neuronal regeneration and improvement of function in rats impaired by acute spinal cord injury.

Dr. Martin Marsala, who is professor of anesthesiology at the University of California, San Diego, with colleagues from academic institutions in Slovakia, the Czech Republic, and the Netherlands, used neural stem cells derived from an aborted human fetus to treat spinal cord-injured rats.

Sprague-Dawley rats received spinal cord injuries at the level of the third lumbar vertebra by means of compression. Such injuries render the rats incapable of using their hind legs. They cannot climb a ladder, walk a catwalk or perform other tasks that require the effective use of their hind legs.

The stem cells that were transplanted into the spinal cords of these rats were NSI-566RSC cells, which were provided by the biotechnology company Neuralstem. These cells were initially isolated from the spinal cord of an eight-week old human fetus whose life was terminated through elective abortion. These cells have been grown in culture and split many times. They are a neural stem cell culture that has the capacity to form neurons and glia.

The rats were broken into six groups, and four of these groups received spinal cord injuries. One of these spinal cord-injured groups received injections of were injured NSI-566RSC cells (12 injections total, about 20,000 cells per microliter of fluid injected), another received injections of only fluid, and the third group received no injections. The final spinal cord-injured group of rats received injections of NSI-566RSC cells that had been genetically engineered to express a green glowing protein. Another group of rats were operated on, but no spinal cord injury was given to these animals, and the final group of rats were never operated on.

All rats that received injections of cells were administered powerful drugs to prevent their immune systems from rejecting the administered human cells before the injections (methylprednisolone acetate for those who are interested at 10 mg / kg), and after the stem cell injections (tacrolimus at 1.5 mg / kg).

The results were significant and exciting. In the words of Marsala, “The primary benefits were improvement in the positioning and control of paws during walking tests and suppression of muscle spasticity.” Spasticity refers to an exaggerated muscle tone or uncontrolled spasms of muscles. Spasticity is a serious and common complication of traumatic injury. It can cause severe cramping and uncontrolled contractions of muscles, which increases the patient’s pain and decreases their control.

First, it is clear from several control experiments that the injection procedure did not affect the spinal cord function of these animals, since the sham injected rats had perfectly normal use of their hind limbs and normal sensory function of their limbs. Thus the injection procedure is innocuous. Also, the use of the drugs to suppress the immune response were also equally unimportant when it came to the spinal cord health of the rats.

Two months after the stem cell injections, the rats were subjected to the “catwalk test,” in which the animals walked a narrow path and their paw position was assessed. As you can see in the figure below, the stem cell-injected rats have a paw position that is far more similar to the normal rats than to the spinal cord injured rats.

Improvement in hind paw positioning and muscle spasticity in SCI animals grafted with HSSC. A: CatWalk gait analysis of hind paw positioning at two months after treatment. In comparison to SCI control animals, a significant improvement was seen in HSSC-grafted animals. B1-B3: An example of paw step images taken from the CatWalk software in naïve (B1), SCI-control (B2) and SCI-HSSC-treated animals (B3). Note a large paw footprint overlap between the front and hind paws in naïve animals (B1) but a substantial dissociation in footprint overlap in SCI controls (B2). An improvement in paw placement in SCI-HSSC-treated animals can be seen (B3). C: Statistical analysis showed significant suppression of spasticity response (expressed as a muscle resistance ratio: values at two months versus seven days post injury in ‘HIGH spasticity’ HSSC-treated animals if compared to ‘HIGH spasticity’ controls). D: To identify the presence of muscle spasticity in fully awake animals, the hind-paw ankle is rotated 40° at a velocity of 80°/second. Spasticity is identified by exacerbated EMG activity measured in the gastrocnemius muscle and corresponding increase in muscle resistance. In control SCI animals with developed spasticity (that is, ‘high spasticity’/HIGH group), no change in spasticity response if compared to seven days post-vehicle injection was seen at two months (compare D1 to D3). In contrast to SCI control animals, a decrease in spasticity response was seen in SCI-HSSC-treated animals at two months after cell injections (compare D4 to D6). To identify mechanical resistance, animals are anesthetized with isoflurane at the end of the recording session and the contribution of mechanical resistance (which is, isoflurane non-sensitive) is calculated. (D2, D5: data expressed as mean ± SEM; one-way ANOVAs). ANOVA, analysis of variance; EMG, electromyography; HSSC, human fetal spinal cord-derived neural stem cells; SCI, spinal cord injury; SEM, standard error of the mean.
Improvement in hind paw positioning and muscle spasticity in SCI animals grafted with HSSC. A: CatWalk gait analysis of hind paw positioning at two months after treatment. In comparison to SCI control animals, a significant improvement was seen in HSSC-grafted animals. B1-B3: An example of paw step images taken from the CatWalk software in naïve (B1), SCI-control (B2) and SCI-HSSC-treated animals (B3). Note a large paw footprint overlap between the front and hind paws in naïve animals (B1) but a substantial dissociation in footprint overlap in SCI controls (B2). An improvement in paw placement in SCI-HSSC-treated animals can be seen (B3). C: Statistical analysis showed significant suppression of spasticity response (expressed as a muscle resistance ratio: values at two months versus seven days post injury in ‘HIGH spasticity’ HSSC-treated animals if compared to ‘HIGH spasticity’ controls). D: To identify the presence of muscle spasticity in fully awake animals, the hind-paw ankle is rotated 40° at a velocity of 80°/second. Spasticity is identified by exacerbated EMG activity measured in the gastrocnemius muscle and corresponding increase in muscle resistance. In control SCI animals with developed spasticity (that is, ‘high spasticity’/HIGH group), no change in spasticity response if compared to seven days post-vehicle injection was seen at two months (compare D1 to D3). In contrast to SCI control animals, a decrease in spasticity response was seen in SCI-HSSC-treated animals at two months after cell injections (compare D4 to D6). To identify mechanical resistance, animals are anesthetized with isoflurane at the end of the recording session and the contribution of mechanical resistance (which is, isoflurane non-sensitive) is calculated. (D2, D5: data expressed as mean ± SEM; one-way ANOVAs). ANOVA, analysis of variance; EMG, electromyography; HSSC, human fetal spinal cord-derived neural stem cells; SCI, spinal cord injury; SEM, standard error of the mean.

Secondly, when muscle spasticity was measured, the stem cell-injected rats showed definite decreases in muscle spasticity. The spinal cord-injured rats that received no stem cell injections showed no such changes.

Sensory assessments also showed improvements in the stem cell-treated rats, but the improvements were not stellar. Nevertheless, the stem cell-treated rats progressively improved in their sensory sensitivity whereas the non-treated spinal cord-injured rats consistently showed no such improvement.

Amelioration of hypoesthesia in SCI-HSSC-grafted animals. Baseline and biweekly assessments of perceptive thresholds for (A) mechanical and (B) thermal stimuli, applied below the level of injury, showed a trend towards progressive recovery in SCI-HSSC-grafted animals. C: When expressed as percentages of the maximal possible effect for mechanical and thermal perceptive thresholds improvements, SCI-HSSC-treated animals showed significant improvements in sensory function for both mechanical and thermal components. (A-C: data expressed as mean ± SEM; A-B: repeated measures ANOVAs; C: Student t-tests). ANOVA, analysis of variance; HSSC, human fetal spinal cord-derived neural stem cells; SCI, spinal cord injury; SEM, standard error of the mean.
Amelioration of hypoesthesia in SCI-HSSC-grafted animals. Baseline and biweekly assessments of perceptive thresholds for (A) mechanical and (B) thermal stimuli, applied below the level of injury, showed a trend towards progressive recovery in SCI-HSSC-grafted animals. C: When expressed as percentages of the maximal possible effect for mechanical and thermal perceptive thresholds improvements, SCI-HSSC-treated animals showed significant improvements in sensory function for both mechanical and thermal components. (A-C: data expressed as mean ± SEM; A-B: repeated measures ANOVAs; C: Student t-tests). ANOVA, analysis of variance; HSSC, human fetal spinal cord-derived neural stem cells; SCI, spinal cord injury; SEM, standard error of the mean.

What were the implanted cells doing? To answer this question, Marsala and his co-workers examined tissue sections of spinal cords from the rats implanted with the glowing green stem cells. According to Marsala, the implanted neural stem cells are stimulating host neuron regeneration and partially replacing the function of lost neurons.

Marsala explained: “Grafted spinal stem cells are a rich source of different growth factors which can have a neuroprotective effect and can promote sprouting of nerve fibers of host neurons. We have demonstrated that grafted neurons can develop contacts with the host neurons and, to some extent, restore the connectivity between centers, above and below the injury, which are involved in motor and sensory processing.”

The implanted neural stem cells definitely showed extensive integration with the spinal nerves of the host rats. Again Marsala, “In all cell-grafted animals, there was a robust engraftment and neuronal maturation of grafted human neurons was noted.” Marsala continued: “Importantly cysts or cavities were not present in any cell-treated animal. The injury-caused cavity was completely filled by grafted cells.”

Effective cavity-filling effect by transplanted cells in SCI HSSC-injected animals. At the end of the two-month post-treatment survival, animals were perfusion fixed with 4% PFA, the spinal column dissected and MRI-imaged in situ before spinal cord dissection for further histological processing. A, B: Three-dimensional MRI images of spinal cord segments in animals with previous traumatic injury and treated with spinal HSSC (A) or media (B) injections. Note the near complete injected-cells cavity-filling effect in HSSC-treated animals. A1, A2, B1, B2: To validate the presence of grafted cells or cavitation at the epicenter of injury, the same region was histologically processed, semi-thin plastic sections prepared and compared to the corresponding MRI image (compare A1 to A2 and B1 to B2). C: Two-dimensional MRI image taken from a naïve-non-injured animal. D: Quantification of the cavity and scar volume from serial MRI images showed significantly decreased cavity and scar volumes in SCI-HSSC-injected animals if compared to media-injected SCI controls. (D: data expressed as mean ± SEM; Student t-tests), (Scale Bars: A, B: 5 mm; A1, A2, B1, B2, C: 3 mm). HSSC, human fetal spinal cord-derived neural stem cells; MRI, magnetic resonance imaging; PFA, paraformaldehyde; SCI, spinal cord injury; SEM, standard error of the mean.
Effective cavity-filling effect by transplanted cells in SCI HSSC-injected animals. At the end of the two-month post-treatment survival, animals were perfusion fixed with 4% PFA, the spinal column dissected and MRI-imaged in situ before spinal cord dissection for further histological processing. A, B: Three-dimensional MRI images of spinal cord segments in animals with previous traumatic injury and treated with spinal HSSC (A) or media (B) injections. Note the near complete injected-cells cavity-filling effect in HSSC-treated animals. A1, A2, B1, B2: To validate the presence of grafted cells or cavitation at the epicenter of injury, the same region was histologically processed, semi-thin plastic sections prepared and compared to the corresponding MRI image (compare A1 to A2 and B1 to B2). C: Two-dimensional MRI image taken from a naïve-non-injured animal. D: Quantification of the cavity and scar volume from serial MRI images showed significantly decreased cavity and scar volumes in SCI-HSSC-injected animals if compared to media-injected SCI controls. (D: data expressed as mean ± SEM; Student t-tests), (Scale Bars: A, B: 5 mm; A1, A2, B1, B2, C: 3 mm). HSSC, human fetal spinal cord-derived neural stem cells; MRI, magnetic resonance imaging; PFA, paraformaldehyde; SCI, spinal cord injury; SEM, standard error of the mean.

Marsala’s goal is to used a neuronal stem cell line derived from a patient-specific induced pluripotent stem cell line in a clinical trial. For now, the UC San Diego Institutional Review Board or IRB is reviewing a small phase 1 clinical trial to test the safety and efficacy of this neural stem cell line in patients with spinal cord injuries who have no feeling or motor function below the level of the spinal cord injury.