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.

Human Neural Stem Cell Line Heals Spinal Cord-Injured Rats


Spinal cord injuries represent one of the most intractable problems for regenerative medicine. When the spinal cord is injured, a tissue that is normally isolated from the bloodstream, now comes into contact with a variety of inflammatory factors and cells that increase the destruction of the original lesion. The spinal responds with a glial scar that plugs the lesion and prevents further exposure of the spinal cord to damaging inflammation, but the scar is also filled with molecules that repel neuronal axon growth cones. This spells curtains for neuronal regeneration, and finding a cell type that can negotiate around the glial scar and find the original muscle is a genuine tour de force.

Given this to be the case, there have been many experiments in rodents to examine the efficacy of various stem cell populations to as treatments for spinal cord injuries. A recent paper in Stem Cell Research and Therapy (van Gorp et al., 2013, 4:57) has examined human fetal spinal cord-derived neural stem cells (HSSCs) and their ability to restore motor function in rats with spinal cord injuries to the lower back. Because this group examined movement and spinal cord tissue samples, this paper contributes something significant to our knowledge of HSSC-mediate healing of spinal cord injuries.

The HSSC line used in this paper is neural stem cell line NSI566RSC, which was extracted from the spinal cord of an 8-week old “fetus.” I have placed fetus in quotes because at eight weeks, the fetus is actually a very old embryo, since the end of the eighth week is end of embryonic development. I realize that these types of age calculations have room for error, and therefore, the baby might very well have been at the early fetal stage. However, the baby’s mother terminated her pregnancy (yes it was an abortion and no I am not cool with that) and donated the dead baby’s tissue to UC San Diego for research purposes.

Sprague-Daley rats were subjected to spinal cord injuries at the level of the third lumbar vertebra. Three days later, half of the rats were given saline injections into their spinal cord and the other half were given HSSC injections into their spinal cords. The animals were evaluated for two months after the treatments on a daily basis. After two months, the rats were sacrificed (put down) and the spinal cord tissue was extensively analyzed.

Of the 35 animals employed in this study, 3 were excluded because of paw injuries or drug toxicity. Eight weeks after the cells were implanted, the rats were tested with a CatWalk apparatus to determine their gait. The rats injected with HSSCs showed a much more normal gait than those injected with saline. To give you some idea of the improvement, the rats that were not injured had a RCHPP or rostro-caudal hind paw positioning score of 0+/- 1.7mm, and the saline injected animals had an average RCHPP of -18 +/- 3.1 mm, and those injected with HSSCs had an RCHPP of -9.0 +/- 1.9 mm.

Despite these improvements, there were no significant differences in ladder climbing, stride length, overall coordination, or single-frame motion.

Next, Marsala and colleagues showed that the muscle spasms associated with spinal cord injury were slightly decreased by the implantation of HSSCs and not by injection of saline. To measure spasticity, the ankle or front paw is rotated and the electromyograph of the muscle is measured. The electromyograph or EMG measures the electrical activity of the muscle showed modest improvements in the HSSC-injected animals

Sensory sensitivity was improved in the HSSC-injected animals, and this improvement was progressive. When the rats were prodded below the level of the injury, where they should have no feeling, the HSSC-injected rats showed better response to the stimulation. This was the case with mechanical stimulation and thermal stimulation.

Post-mortem analysis also showed something interesting. When the fluid-filled cavity of the damaged spine was examined, the HSSC-injected animals had a significantly small cavity. Because the injected cells had been labeled with green fluorescent protein, they glowed under UV light and any neuronal cells derived from the injected HSSCs glowed green too. The lesioned areas in the HSSC-injected mice were repopulated with cells. Motorneurons, interneurons and glial cells were detected.

What to make of this study? The repopulation of the spinal cord and the growth of spinal nerve elements within the fluid-filled cavity is remarkable, but the lack of better motor function is disappointing. The recovery of sensory ability is significant, especially, since it is pretty clearly not due to spinal hypersensitivity.

There are two possibilities for the low motor recovery. First, there is a possibility that the these experiments were not conducted for as long a time period as they needed to be. Since the sensory ability improvement was progressive, maybe the motor recovery was too, perhaps? Secondly, maybe the grow and connection of motor neurons had trouble with the glial scar. Why the sensory nerves did not have such a problem and the motor neurons would is inexplicable at this time. However, another possibility is that the muscular targets of motor neurons are not as obvious in adult animals as they are in a developing animal. Finding ways to “paint” the muscles might be a way to increase motor neuron innervation in the future.

Thus, this cell line, NSI-566 RSC is certainly a potential treatment for spinal cord patients. A phase I trial is in the works.