Nerve Growth Factor-Secreting Mesenchymal Stem Cells To Treat Huntington’s Disease

Vicki Wheelock at the UC Davis Medical Center has registered clinical trial number NCT01937923, which is otherwise known as “PRE-CELL.” This clinical trial will use various imaging techniques, laboratory tests, and clinical evaluations of Huntington’s disease (HD) patients to map the disease progression over 12-18 months. This trial will then hopefully identify candidates for a new trial in which these patients will be implanted with mesenchymal stem cells that secrete nerve growth factors. This represents one of the first clinical trials to examine the use of mesenchymal stem cells in the treatment of HD

The rationale for this study comes from a 2012 study in mice. Ofer Sadan, Eldad Melamed, and Daniel Offen from the Rabin Medical Center in Tel Aviv University, Israel, used R6/2 mice to test the efficacy of nerve growth factor-secreting mesenchymal stem cells isolated from bone marrow . In this paper, Sadan and others isolated mesenchymal stem cells from the bone marrow of healthy human volunteers and mice and then cultured them in special growth media that induces these cells to secrete special nerve growth factors. These so-called NTF+ cells were then transplanted into the striatum of R6/2 mice.

R6/2 mice express part of the human HTT gene; specifically the part that causes HD. Since HD is an inherited disease, there is a specific gene responsible for the vast majority of HD cases, and that gene is the human HTT gene, which encodes the Huntington protein. The function of the Huntington protein is uncertain, but it is found at high levels in neurons, even though it is found in other tissues as well, and dysfunctional Huntington protein affects neuron health.

Huntingtin Function

The HTT gene in HD patients contains the insertion of extra copies of the CAG triplet. The more CAG triplets are inserted into the HTT gene, the more severe the HD caused by the mutation. The hitch is that normal copies of the HTT gene has multiple copies of this CAG repeat. CAG encodes the amino acid glutamine, and Huntington contains a stretch of glutamine residues that seem to allow the protein to interact with other proteins found in neurons. When this glutamine stretch becomes too long, the protein is toxic and it begins to kill the cells. How long is too long? Research has pretty clearly shown that people whose HTT genes contain less than 28 CAG virtually never develop HD. People with between 28–35 CAG repeats, are usually unaffected, but their children are at increased risk of developing HD. People whose HTT genes contain 36–40 CAG repeats may or may not show HD symptoms, and those who have over 40 copies almost always are afflicted with HD.


Now, back to R6/2 mice. These animals contain a part of the human HTT gene that has 150 CAG triplets. These mice show the characteristic cell death in the striatum and have behavioral deficits. In short R6/2 mice are pretty good model systems to study HD.

Sadan and others implanted MSCs that had been conditioned in culture to express high levels of nerve growth factors. Then these cells were transplanted into the striatum of R6/2 mice. R6/2 mice were also injected with buffer as a control.

The results showed that injections of NTF+ MSCs before the onset of symptoms did little good. The mice still showed cell death in the brains and behavioral deficits. However, NTF+ MSCs injected later (6.5 weeks), resulted in temporary improvement in the ability of the R6/2 mice to move and these cells also extended their life span. These results were published in the journal PLoS Currents (2012 Jul 10;4:e4f7f6dc013d4e).

Other work, also by Sadan and others, showed that injected MSCs tended to migrate to the damaged areas. When the injected cells were labeled with iron particles, they could be robustly observed with MRIs, and MRIs clearly showed that the injected cells migrated to the damaged areas in the brain (Stem Cells 2008; 26(10):2542-51). Another paper by Sadan and others also demonstrated that the striatum of NTF+ MSC-injected mice show less cell death than control mice (Sadan, et al. Exp Neurol. 2012; 234(2): 417-27). Other workers have also shown that implanted MSCs can provide improve symptoms in R6/2 mice and that they primary means by which they do this is by the secretion of nerve growth factors (Lee ST, et al. Ann Neurol 2009; 66(5): 671-81).

Thus, there is ample reason to suspect the PRECELL trial may lead to a stem cell-based clinical trial that will yield valuable clinical information. The animal data shows definite value in using preconditioned MSCs as a treatment for HD, and if the proper patients are identified by the PRE-CELL trials, then hopefully it will lead to a “CELL” trial in which HD patients are treated with NTF+ MSCs.

Mind you, this treatment will only delay HD at best and buy them time. Such treatments will not cure them. The NTF+ MSCs survive for a finite period of time in the hostile environment of the striatum of the HD patient, and the relief they will provide will be temporary. MSCs do not differentiate into neurons in this case, and they do not replace dead neurons, but they only help spare living neurons from suffering the same fate.

Huntington disease striatum

There is an MSC cell line that does make neurons, and if this cell line were used in combination with NTF+ MSCs, then perhaps neural replacement could be a possibility.  Also neural precursor cells could be used in combination with NTF+ MSCs to increase their survival.  Even then, as long as diseased neurons are producing toxic products, until gene therapy is perfected to the point that the actual genetic lesion in the striatal neurons is fixed, the deterioration of the striatum is inevitable. However, treatments like this could, potentially, delay this deterioration. This clinical trial should give us more information on exactly that question.

Two more points are worth mentioning.  When fetal striatal grafts were implanted into the brains of HD patients, the grafts underwent disease-like degeneration, and actually made the patients worse (see Cicchetti et al. PNAS 2009; 106(30): 12483-8 and Cicchetti F, et al. Brain 2011; 134(pt 3): 641-52).  Straight fetal implants do not seem to work.  Please let’s put the kibosh on these gruesome experiments.  Secondly, when neuronal precursor cells differentiated from human embryonic stem cells were implanted into HD rodents, the implanted cells formed some neurons and improved behavior to some extent, but non-neuronal differentiation remained a problem (Song J, et al., Neurosci Lett 2007; 423(1): 58-61).  Having non-brain cells in your brain is a significant safety problem.  Thus, embryonic stem cell-derived neuronal precursor cells do not seem to be the best bet to date either.  So, this present clinical trial seems to be making the most of what is presently safely available.

Mouse Model of Huntington’s Disease Shows Replacement of Lost Neurons by Endogenous Stem Cell Populations

Huntington’s disease (HD) is a debilitating and invariably fatal disease that results from mutations in the IT15 gene. IT151 stands for “interesting transcript 15,” but it is more commonly referred to as the “huntingtin” gene. Mutations in the front of the gene (exon 1 for those who are interested) expand a run of CAG codons, and these mutations are probably the result of DNA polymerase slippage. Because CAG codons encode the amino acid glutamine, the mutant proteins contain long polyglutamine repeats and these repeats tend to clump inside neurons.

These protein aggregates form in neurons of the “striatum.” The striatum is a region of the brain that is also called the striate nucleus of the striate body. The striatum receives its name from the fact that it is organized in striped layers of gray and white matter. The striate nucleus is part of the cerebrum or forebrain.


Mutant Huntington (Htt) protein has a toxic that causes cell death by means of unknown mechanisms. Clinically, the most obvious symptoms of HD involve involuntary movements of the arms, legs, and face. But the severe cognitive and personality changes are the most devastating to HD patients and most troubling for their caregivers.

Researchers are using animal models of HD to study the disease pathogenesis, to elucidate areas of the brain involved in structural and functional decline, and to evaluate potential therapeutic interventions. These animal models include injecting toxins into the brain to kill off those populations of neurons that typically die in HD patients, and transgenic models in which animals are bred with either extra mutant copies of the Htt gene or a pair of copies of the mutant Htt gene that have replaced the original, normal copies. All of these model systems have limitations, but they are all useful in some way for assessing the pathology of HD.

This long introduction leads us to new data from the laboratory of Steve Goldman, the co-director of the University of Rochester Medical Center’s Center for Translational Medicine. Goldman and his colleagues triggered the production of new neurons in mice that had a rodent form of HD. These new neurons successfully integrated into the brain’s existing neural networks and dramatically extended the survival of the mice.

“This study demonstrates the feasibility of a completely new concept to treat Huntington’s disease, by recruiting the brain’s endogenous neural stem cells to regenerate cell lost the disease,” said Goldman.

One of the types of neurons most commonly affected in HD patients is the medium spinal neuron, which is critical to motor control. Goldman banked on findings from previous studies in his laboratory on canaries. Songbirds such as canaries have the ability to lay down new neurons in the adult brain when mating season comes. The male birds, in response to a flush of male sex hormones,, grow a gaggle of new neurons in the vocal control centers of the brain, and this provides the bird the means to sing specific songs in order to attract mates. This event is known as adult neurogenesis, and Goldman and Fernando Nottebohm of the Rockefeller University discovered this phenomenon in the early 1980s.

“Our work with canaries essentially provided us with the information we needed to understand how to add new neurons to adult brain tissue,” said Goldman. Once we mastered how this happened in birds, we set about how to replicate the process in the adult mammalian brain.”

Humans possess the ability to make new neurons, but Goldman’s lab demonstrated in the 1990s that a font of neuronal precursor cells exist in the lining of the ventricles (these are structures at the very center of the brain and spinal cord that are filled with cerebrospinal fluid). In early development, these cells are actively producing neurons.

Shortly after birth, the neural stem cells stop generating neurons and produce support cells called glia. Some parts of the human brain continue to produce neurons into adulthood, the most prominent example is the hippocampus, where memories are formed and stored. However, the striatum, new neuron production is switched off in adulthood.

Goldman sought to switch neuron production back on in the striatum. He tested a cadre of growth factors that would switch the neural stem cells of the striatum (a region that is ravaged by HD) from producing new glia to producing new neurons. Goldman, however, had some help from his recent work in canaries. Namely that once mating season was upon the birds, targeted expression of brain-derived neurotrophic factor (BDNF) flared up in the vocal centers of the brain, where many new neurons were being produced.

Goldman used genetically engineered viruses to express BDNF and another protein called “Noggin” in the striatum. Goldman and others found that a single intraventricular injection of the adenoviruses expressing BDNF and Noggin triggered the sustained recruitment of new neurons in both normal of R6/2 (HD) mice. These treated mice also showed that the newly formed neurons were recruited to form new medium spiny neurons; the ones destroyed in HD. These new neurons also matured and achieved circuit integration.

Medium Spinal Neuron
Medium Spinal Neuron

Also the treated mice showed delayed deterioration of motor function and substantially increased survival.

When the same experiments were conducted in squirrel monkeys, there was a similar addition of new striatal neurons.

Thus, induced neuronal addition may therefore represent a promising avenue for decreasing the ravages of HD and increasing cognitive ability.

GABA-Making Neurons Made from Stem Cells Reverse Motor Defects in Mice with a Form of Huntington’s Disease

Huntington disease is a horrible, slow, relentless and progressive death sentence. This disease is inherited, and if one of your parents has Huntington’s disease (HD), you have a 50% chance of inheriting the disease. HD is cause by mutations in a gene found on human chromosome 4. This mutation resides in a gene that encodes the Huntingtin protein. However, these mutations are unusual in that they are due to excessive number of repeats of the triplet sequence, CAG. CAG codes for the amino acid glutamine, and normally, there is a stretch of 10-28 glutamines in normal versions of the Huntingtin protein. However, CAG repeats tend to cause the enzymes that make DNA to slip and resynthesize the repeat, thus causing the number of consecutive CAG triplets in this gene to expand. In persons with Huntington’s disease, the CAG triplet is repeated anyways from 36 to 120 times. This expands the stretch of glutamine residues and creates and toxic protein that is cut into smaller fragments that kill nerve cells.

The symptoms of Huntington’s disease usually begin with behavioral disturbances that show up before the onset of movement disorders. These behavioral symptoms can include hallucinations, irritability, moodiness, restlessness or fidgeting, paranoia, and even psychosis. Abnormal movements begin and these include facial movements, including grimaces, the turning of the head to shift eye position rather than moving the eyes, quick, sudden, sometimes wild jerking movements of the arms, legs, face, and other body parts, slow, uncontrolled movements, and an unsteady gait. The dementia slowly gets worse and other symptoms eventually emerge that include disorientation or confusion, loss of judgment, loss of memory, personality changes, and speech changes.

This disease has no treatments and no cure, but researchers have published a paper in the journal Cell Stem Cell that is a starting block of further research that might lead to a treatment. In this paper, a special type of brain cell generated from stem cells seems to help ameliorate the muscle coordination deficits that eventually lead to uncontrollable spasms (choreas) that are so characteristic of the disease.

Su-Chun Zhang, a neuroscientist at the University of Wisconsin-Madison and senior author of the new study said: “This is really something unexpected.” This work suggests that locomotion could be restored in mice with a Huntington’s-like condition.

Zhang’s laboratory has a great deal of experience and expertise at making different types of brain cells from human embryonic stem cells or induced pluripotent stem cells. In the newly published article, Zhang and his colleagues reported the production of neurons that use a neurotransmitter called “gamma-amino butyric acid,” which thankfully goes by the acronym “GABA.” GABA is one of the most heavily used neurotransmitters in the central nervous system, and GABA receptors come in many shapes and sizes, but virtually all of them are chloride channels. While this may not mean anything to you, to a neuron that is trying to generate a nerve impulse, chloride ions are inhibitory and they cut the neuron off at the knees. GABA, therefore, is an extremely important inhibitory neurotransmitter that shuts neurons down when they need to be shut down.

This significance of making GABA-using neurons in the laboratory cannot be lost on Huntington’s patients, because GABA-making neurons are the ones that take the biggest beating during the onset of Huntington’s disease. Without these GABA-using neurons, it is impossible for various portions of the brain to properly coordinate movement. According to Zhang, GABA-producing neurons produce one the key neurotransmitters for coordinating movement.

At the UW-Madison Waisman Center, Zhang and his colleagues discovered how to make large quantities of GABA neurons from human embryonic stem cells. They then tested these neurons in mice that had an induced condition that resembled Huntington’s disease. They implanted these cells in the brains of mice, and they were very surprised to see that the implanted cells not only integrated into the brain, but also projected axons to the correct targets and effectively reestablished the broken communication network. This largely restored motor function.

Zhang noted that these results surprised so because GABA-making neurons are found in a part of the brain called the basal ganglia. The basal ganglia play a central role in voluntary motor coordination. However, GABA-making neurons, however, exert their influence at a distance on cells in the midbrain through neural circuits that are fueled by the GABA-making neurons.

Zhang explained it this way: “This circuitry is essential for motor coordination, and it is what is broken in Huntington patients. The GABA neurons exert their influence at a distance through this circuit. Their cell targets are far away.”

Zhang, however, did not stop there. Many neuroscientists do not think that the results Zhang and his co-workers observed are even possible. He explained further: “Many in the field feel that successful cell transplants would be impossible because it would require rebuilding the circuitry. But what we’ve shown is that the GABA neurons can remake the circuitry and produce the right neurotransmitter.”

This new study has profound implications for regenerative therapy of neurodegenerative disease. One day, it might be possible to treat Huntington’s disease with cell transplants that capitalize on the plasticity of the adult brain. Zhang noted that the adult brain is considered by some neuroscientists to be stable and not easily susceptible to therapies that try to correct things like broken neural circuits. For a therapy to work, it has to be engineered so that it targets only specific cells. Zhang added, “The brain is wired in such a precise way that if a neuron projects the wrong way, it could be chaotic.”

This new research is indeed promising, but it must be worked up and correlated from the mouse model to the condition found in human patients, and this type of very hard, tedious work will take a great deal of time, people hours, and a whole lot of trial and error. However, for a disease that now has no effective treatment, this work could become the next best hope for Huntington’s disease patients.

A caveat to this research is that the mice with Huntington’s disease-like symptoms were given the disease by means of the chemical called quinolinic acid. Administration of this chemical by means of “bilateral intrastriatal microinjections,” which is a fancy way of saying injecting really small amounts of this stuff into a specific part of the basal ganglia, generates mice that display the movement disorders similar to those seen in humans with this disease (see Sanberg PR, et al., Experimental Neurology 1989 Jul;105(1):45-53). Also, the pathology of the brains of these mice shows some similarity to that observed postmortem in the brains of Huntington’s disease patients.

The problem is this: implanting cells into the brains of mice that have been subjected to quinolinic acid results in those cells living and taking up residence in the brain of the mouse and somewhat reconstructing the striatum of the mouse brain (see Dunnett SB. Novartis Found Symp. 2000;231:21-41; discussion 41-52). This is due to the fact that quinolinic acid lesions in the brain specifically kill off particular parts of the brain, but the environment of the brain is still relatively normal. When similar experiments are attempted in human patients, the implanted tissue takes a beating and dies because the brains of Huntington’s disease patients are not chemically altered, but genetically altered. These brains are a toxic waste dump, so to speak, and implanted tissue or cells die (see Francesca Cicchetti, Denis Soulet, and Thomas B. Freeman. “Neuronal degeneration in striatal transplants and Huntington’s disease: potential mechanisms and clinical implications,” Brain (2011) 134 (3): 641-652. doi: 10.1093/brain/awq328).

It seems to me that the environment of the brain must be improved before cell therapy is going to work, and that is a much more difficult problem to address. Dying neurons spill their neurotransmitters into the intracellular space. Huge neurotransmitter overdose can kill nearby neurons and this contributes to the toxic environment in the brain of Huntington’s disease patients. Finding a way to quell the poisonous products released by dead neurons is the next great unanswered quest for these patients.

Stem Cells used to Transfer Inhibitory RNA Molecules to Neurons to Treat Huntington’s Chorea in Lab Animals

Huntington’s disease is an inherited brain disorder that causes progressive uncontrolled movements, dementia and culminates in death. The symptoms of Huntington’s disease are involuntary jerking or writhing movements (chorea), involuntary, sustained contraction of muscles (dystonia), muscle rigidity, slow, uncoordinated fine movements, slow or abnormal eye movements, impaired gait, posture and balance, difficulty with the physical production of speech, and difficulty swallowing.

More than a quarter of a million Americans are affected by Huntington’s disease. Huntington’s disease is passed through families even if only one parent has the abnormal huntingtin gene, since it is inherited as an autosomal dominant. The huntingtin gene is found on the fourth chromosome, and Huntington’s disease-causing mutations result from the expansion of a trinucleotide (CAG) repeat (Jones L, Hughes A. Int Rev Neurobiol.2011;98:373-418 & Reiner A, Dragatsis I, Dietrich P. Int Rev Neurobiol. 2011;98:325-72). This trinucleotide repeat is normally repeated up to 28 times on the chromosome, but polymerase slip during DNA replication can expand the number of these repeats so that an abnormal form of the Huntingtin protein to be made. The abnormal Huntingtin protein accumulates in the brain and this cause the disease’s devastating progression. Individuals usually develop symptoms in middle age if there are more than 35 copies of the CAG repeats. A more rare form of the disease occurs in youth when the number of CAG repeats occurs many more times.

Huntington’s disease can be managed with medications. For example Terabenazine (Xenazine) suppresses the involuntary jerking and writhing movements associated with Huntington’s diseases. Antipsychotic drugs such as Haloperidol (Haldol) and Clozapine (Clozaril) can suppress movements but they can also increase muscle rigidity and involuntary contractions. Other medications like clonazepam (Klonopin) and diazepam (Valium) can suppress the chorea, dystonia and muscle rigidity.

Even though brain grafts in laboratory animals have shown some promise, these experiments used a chemically induced form of Huntington’s disease. Because the surrounding tissue was genetically normal, implanted brain tissue simply integrated into the damaged brain tissue and healed it. However, clinical Huntington’s disease is due to mutations in the huntingtingene, and the surrounding brain tissue is not genetically normal. Therefore grafted stem cells are killed off by the toxic environment in the brain (Clelland CD, Barker RA, Watts C. Neurosurg Focus.2008;24(3-4):E9 & Dunnett SB, Rosser AE. Exp Neurol. 2007 Feb;203(2):279-92). To overcome this problem, researchers have developed a technique for that used stem cells to deliver therapeutic agents that specifically target the genetic abnormality found in Huntington’s disease.

Scientists at the UC Davis Institute for Regenerative Cures have developed a novel, and promising approach that might prevent the disease from advancing. Jan A. Nolta, principal investigator of the study and director of the UC Davis stem cell program and the UC Davis Institute for Regenerative Cures, thinks that the best chance to halt the disease’s progression will be to reduce or eliminate the mutant Huntingtin (Htt) protein found in the neurons of those with the disease. RNA interference (RNAi) technology has been shown to be highly effective at reducing Htt protein levels and reversing disease symptoms in mouse models.

Nolta said: “For the first time, we have been able to successfully deliver inhibitory RNA sequences from stem cells directly into neurons, significantly decreasing the synthesis of the abnormal Huntingtin protein. Our team has made a breakthrough that gives families affected by this disease hope that genetic therapy may one day become a reality.” She continued: “Our challenge with RNA interference technology is to figure out how to deliver it into the human brain in a sustained, safe and effective manner,” said Nolta. “We’re exploring how to use human stem cells to create RNAi production factories within the brain.”

The research team from UC Davis showed for the first time that inhibitory RNA sequences are directly transferable from donor cells into target cells to greatly reduce unwanted protein synthesis from the mutant huntingtin gene. To transfer these inhibitory RNA sequences into their targets, Nolta’s team genetically engineered mesenchymal stem cells (MSCs) from bone marrow that had been collected from unaffected human donors. Over the past two decades, Nolta and her colleagues have shown MSCs are safe and effective vehicles for the transfer of enzymes and proteins to other cells. According to Nolta, MSCs can also transfer RNA molecules directly from cell to cell, in amounts sufficient to reduce levels of a mutant protein by over 50% in the target cells. This discovery has never been reported before and offers great promise for a variety of disorders.

Nolta has recently received a Transformative Research Grant from the National Institutes of Health (NIH) to study how MSCs can transfer microRNA and other factors into the cells of damaged tissues, and how that process can be harnessed to treat injuries and disease. Nolta said: “Not only is finding new treatments for Huntington’s disease a worthwhile pursuit on its own, but the lessons we are learning are applicable to developing new therapies for other genetic disorders that involve excessive protein development and the need to reduce it. We have high hopes that these techniques may also be utilized in the fight against some forms of amyotrophic lateral sclerosis (Lou Gehrig’s disease) as well as Parkinson’s and other conditions.”

Published – Scott D. Olson, Jan Nolta et al.; Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington’s disease affected neuronal cells for reduction of huntingtin;”  Molecular and Cellular Neuroscience,2011; DOI: 10.1016/j.mcn.2011.12.001.