Producing dopamine-making neurons from stem cells for transplantation into Parkinson’s disease patients remains challenging. Differentiating stem cells into dopaminergic neurons is not as efficient a process as we would like it to be. While several laboratories have managed to make pretty good batches of dopaminergic neurons, reliably producing large and pure batches of dopamine-making neurons from pluripotent stem cells is still somewhat problematic. Secondly, transplanting dopamine-making neurons into either the midbrain or the striatum of the brain represents another patch of problems because the production of too much dopamine can cause unwanted, uncontrollable movements. Preclinical assessments of stem cell-derived dopamine neurons in laboratory animals have produced positive, but highly varied results, even though the transplanted cells are very similar at the time of transplantation.
“This has been frustrating and puzzling, and has significantly delayed the establishment of clinical cell production protocols,” said Malin Parmar, who led the study at Lund University.
To address this issue, Parmar and his colleagues used modern global gene expression studies to gain a better understand the molecular changes that drive the differentiation of stem cells into dopamine-making neurons. Parmar conducted these experiments in collaboration with a team of scientists at Karolinska Institute. In their paper, which appeared in the journal Cell Stem Cell, Parmar and his colleagues used single-cell RNA seq to construct the neuronal development of dopaminergic neurons.
These neurons are characterized by the expression of a gene called LMX1a. However, it turns out that LMX1a-expressing neurons includes not only midbrain dopaminergic neurons (see below at the substantia nigra), but also subthalamic nuclear neurons.
These findings reveal that markers used to identify midbrain dopaminergic neurons do not specifically isolate midbrain dopaminergic neurons, but isolate a mixture of cells. Is there a way to separate these two populations?
Indeed, there is. Parmar and his colleagues in the laboratory of Thomas Perlmann showed that although dopaminergic neurons from the midbrain and subthalamic nuclear neurons are related, they do express a distinct profile of genes that are specific to the two cell types. The authors argue that the application of these distinct marker genes can help optimize those protocols that differentiate dopaminergic neurons from pluripotent stem cells.
See Nigel Kee and others, “Single-Cell Analysis Reveals a Close Relationship between Differentiating Dopamine and Subthalamic Nucleus Neuronal Lineages,” Cell Stem Cell, 2016; DOI: 10.1016/j.stem.2016.10.003.
The Australian government has recently given its approval for a clinical trial of what is almost certainly a medical first. The Carlsbad-based stem cell company, International Stem Cell Corp. (ISCO), a publicly traded biotechnology company, has developed a unique stem cell technology to address particular conditions.
The clinical trial that has been approved will examine the use the ISCO’s unique stem cell products in the treatment of Parkinson’s disease. Twelve Parkinson’s patients will receive implantations of these cells sometime in the first quarter of 2016, according to Russell Kern, ISCO’s chief scientific officer. The implanted cells will be neural precursor cells, which are slightly immature neurons that will complete their maturation in the brain, hopefully into dopamingergic neurons, which are the precise kind of neurons that die off in patients with Parkinson’s disease.
Parkinson’s disease (PD) is a progressive disorder of the nervous system that affects voluntary movement. PD develops gradually and sometimes begins with a slight tremor in only one hand, but PD may also cause stiffness or slowing of movement. PD worsens over time.
PD patients suffer from tremor, or shaking of the limbs, particularly when it is relaxed and at rest. Over time, PD reduces the ability to move and slows movement (bradykinesis) which makes simple tasks difficult and time-consuming. Muscle stiffness may occur and this limits the range of motion and causes pain. PD patients also suffer from stooping posture and balance problems and a decreased ability to perform unconscious movements. For example, they have trouble swinging their arms while they walk, blinking, or smiling. They might also experience speech problems that can range from slurring of the speech to monotone speech devoid of inflexions, or softer speech with hesitations before speaking. Writing might also become problematic.
PD is caused by the gradual death of neurons in the midbrain that produce a chemical messenger called dopamine. The drop in dopamine levels in the system of the brain that controls voluntary movement leading to the signs and symptoms of Parkinson’s disease.
Several different animal experiments with a variety different cell types have established that transplantation to dopamine-making neuronal precursors into the midbrains of laboratory animals with artificially-induced PD can reverse the symptoms of PD. Dopaminergic neurons can be derived from embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), umbilical cord blood hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), and NSCs (see Petit G. H., Olsson T. T., Brundin P. Neuropathology and Applied Neurobiology. 2014;40(1):60–67). Also, since the 1980s, various cell sources have been tested, including autografts of adrenal medulla, sympathetic ganglion, carotid body-derived cells, xenografts of fetal porcine ventral mesencephalon, and allografts of human fetal ventral mesencephalon (fVM) tissues have been implanted into the midbrains of PD patients (Buttery PC, Barker RA. J Comp Neurol. 2014 Aug 15;522(12):2802-16). While the results of these trials were varied and not terribly reproducible, these studies did show that the signs and symptoms of PD could be reversed, in some people, by implanting dopamine-making neurons into the midbrains of PD patients.
ISCO has derived neural precursor cells from a completely new source. ISCO scientists have taken unfertilized eggs from human egg donors and artificially activated them so that they self-fertilize, and then begin dividing until they form a blastocyst-stage embryo from which stem cells are derived. This new class of stem cells, which were pioneered by ISCO, human parthenogenetic stem cells (hpSCs) have the best characteristics of each of the other classes of stem cells. Since these stem cells are created by chemically stimulating the oocytes (eggs) to begin division, the oocytes are not fertilized and no viable embryo is created or destroyed. This process is called parthenogenesis and parthenogenetic stem cells derived from the parthenogenetically-activated oocytes, are produced from unfertilized human egg cells.
Why did ISCO decide to do this trial in Australia? According to Kern, ISCO chose to conduct their clinical trial in Australia because its clinical trial system is more “interactive,” which allows for better collaboration with Australia’s Therapeutic Goods Administration on trial design. This clinical trial, in fact, is the first stem cell trial for PD according to the clinical trial tracking site clinicaltrials.gov. The test will be conducted by ISCO’s Australian subsidiary, Cyto Therapeutics.
The approach pioneered in this clinical trial might cure or even provide an extended period of relief from the symptoms of PD. If this clinical trial succeeds, the stem cell clinical trial dam might very well break and we will see proposed clinical trials that test stem cell-based treatments for other neurodegenerative diseases such as Huntington’s disease, Lou Gehrig’s disease (ALS), frontotemporal dementia, or even Alzheimer’s disease.
ISCO has spent many years developing their parthenogenetic technology with meager financing. However the company’s total market value amounts to something close to $11.1 million, presently.
hpSCs are pluripotent like embryonic stem cells. Because they are being used in the brain, they will not be exposed to the immune system. Therefore an exact tissue type match is not necessary for this type of transplantation. In their publications, ISCO scientists have found their cells to be quite stable, but other research groups who have worked with stem cells derived from parthenogenetically-activated embryos have found such cells to be less stable than other types of pluripotent stem cells. The stability of the ISCO hpSCs remains an open question. The lack of a paternal genome might pose a safety challenge for the use of hpSCs.
Rita Vassena and her colleagues in the laboratory of Juan Carlos Izpisua Belmonte at the Salk Institute for Biological Studies in La Jolla, CA examined the gene expression patterns of mesenchymal stem cells derived from hpSCs and found that the overall gene expression patterns were similar to MSCs made from embryonic stem cells or induced pluripotent stem cells. However, upon further differentiation and manipulation, the gene expression patterns of the cells began to show more variability and further depart from normal gene expression patterns (Vassena R, et al Human Molecular Genetics 2012; 21(15): 3366-3373). Therefore, the derivatives of hpSCs might not be as stable as cellular derivatives from other types of stem cells. The good news about hpSCs established from parthenogenetic ESCs were reported to be morphologically indistinguishable from embryonic stem cells derived from fertilized embryos, and seem to show normal gene expression or even correct genomic imprinting in chimeras, when pESCs were used in tissue contribution (T.Horii, et al Stem Cells, vol. 26, no. 1, pp. 79–88, 2008).
For those of us who view the early embryo as the youngest members of the human community who have the right not to be harmed, hpSCs made by ISCO remove this objection, since their derivation does not involve the death of any embryos.
The ISCO approach to Parkinson’s is similar to that of a San Diego group called Summit for Stem Cell, which is going to use induced pluripotent stem cell derivatives. This nonprofit organization is presently raising money for a clinical trial to test the efficacy of their treatment.
Both groups intend to transplant the cells while they are still slightly immature, so that they can complete their development in the brain. Animal studies suggest that implanting immature precursors are better than transplanting mature dopaminergic neurons into the midbrain. The precursors then differentiate into dopamine-making neurons, and other cells differentiate into supportive glial cells, which support the dopamine-making neurons.
“It’s a dual action,” Kern said. “Also, neural stem cells reduce inflammation, and inflammation is huge in Parkinson’s.”
Summit 4 Stem Cell will also take a similar approach, according to stem cell scientist Jeanne Loring, a leader of the Summit 4 Stem Cell project. The cells make proper connections with the brain better when they are still maturing, said Loring, who’s also head of the regenerative medicine program at The Scripps Research Institute in La Jolla. This is all provided that Summit 4 Stem Cell can raise the millions of dollars required for the clinical trial and secure the required approvals from the U.S. Food and Drug Administration.
Loring said she views ISCO as a partner in fighting Parkinson’s. One of her former students is working for the company, she said. “The whole idea is to treat patients by whatever means possible,” Loring said.
ISCO’s choice of Australia for its streamlined regulatory process makes sense, Loring said. Her team, with U.S.-based academics and medical professionals, doesn’t have the same flexibility as ISCO in looking for clinical trial locations, she said.
For the first time, German stem cell scientists from the University of Bielefeld and Dresden University of Technology have used adult human stem cells to “cure” rats with Parkinson’s disease.
Parkinson’s disease results from the death of dopamine-using neurons in the midbrain, and the death of these midbrain-based, dopamine-using neurons causes a loss of control of voluntary motion. Presently, no cure exists for Parkinson disease.
In this study, which was published in STEM CELLS Translational Medicine, the German team produced mature dopamine-using neurons from inferior turbinate stem cells (ITSCs). ITSCs are stem cells taken from tissues that are normally discarded after an adult patient undergoes sinus surgery. The German team tested how ITSCs would behave when transplanted into a group of rats with a chemically-induced form of Parkinson’s disease. Prior to transplantation, the animals showed severe motor and behavioral abnormalities. However, 12 weeks after transplantation of the ITSCs, the cells had not only migrated into the animals’ brains, but their functional ability was fully restored and significant behavioral recovery was also observed. Additionally, none of the treated animals shows any signs of tumors after the transplantations, something that also has been a concern in stem cell therapy.
“Due to their easy accessibility and the resulting possibility of an autologous transplantation approach, ITSCs represent a promising cell source for regenerative medicine,” said UB’s Barbara Kaltschmidt, Ph.D., who led the study along with Alexander Storch, M.D., and Christiana Ossig, M.D., both of Dresden University. “The lack of ethical concerns associated with human embryonic stem cells is a plus, too.”
“In contrast to fighting the symptoms of Parkinson’s disease with medications and devices, this research is focused on restoring the dopamine-producing brain cells that are lost during the disease,” said Anthony Atala, M.D., Editor-in-Chief of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine. These cells are easy to access and isolate from nasal tissue, even in older patients, which adds to their attraction as a potential therapeutic tool.”
This is certainly a very exciting animal study, but treating chemically-induced Parkinson’s disease in rodents and treating Parkinson’s disease in aged human patients is two very different things. Thus while this study is important, work in human wild require more testing and studies in larger animals.
Living Cell Technologies (LCT) is a Australasian biotechnology company with offices in Australia and New Zealand. One of the products pioneered by LCT is NTCELL; a capsule coated with alginate (a porous compound extracted from seaweed) that contains clusters of choroid plexus cells from newborn pigs. NTCELL transplantation allows them to function as a biological factory that produces growth factors and other small molecules that promote new central nervous system growth and repair disease induced by nerve degeneration.
The choroid plexus is the structure in the brain that produces cerebrospinal fluid. These cells also filter wastes from the brain and keeps the brain free of debris and other potentially deleterious material. Choroid plexus cells not only produce cerebrospinal fluid, but also a range of neurotrophins (nerve growth factors) that have been shown to protect against neuron (nerve) cell death in animal models of disease.
Several papers have reported on the use of implanted NTCELL capsules in animal model systems. Luo and others used NTCELLs in nonhuman primates that suffered from chemically induced Parkinson’s disease. This paper reported that the transplanted encapsulated choroid plexus clusters significantly improved neurological functions in these monkeys with Parkinson’s disease (J Parkinsons Dis. 2013 Jan 1;3(3):275-91). An earlier paper also showed that implanted improved the neurological function of rodents with a chemically induced form of Huntington’s disease (Borlongan CV and others, Cell Transplant. 2008;16(10):987-92).
On the strength of these successful animal studies, LCT launched human clinical trials in patients with Parkinson’s disease. On December 15th of last year, LCT announced that the final patient had been successfully implanted in its Phase I/IIa clinical trial of regenerative cell therapy NTCELL for Parkinsons disease. These implantations required a minor surgical procedure, which took place at Auckland City Hospital
This Phase I/IIa clinical trial is being led by Dr. Barry Snow, and is an open-label investigation of the safety and clinical effects of NTCELL in Parkinson’s patients who no longer respond to current therapy. Dr. Snow is the leader of the Auckland Movement Disorders Clinic at the Auckland District Health Board but is also an internationally recognized clinician and researcher in Parkinson’s disease.
These patients will be carefully tracked for improvements in the control of movement and balance. LCT hopes to present the results on this clinical trial, which will last 29 weeks) at the 19th International Congress of Parkinson’s Disease and Movement Disorders in San Diego in June 2015.
Dr Ken Taylor, chief executive, notes that the success of the implant procedure means that the time scale for the LCT clinical program remains intact.
“The treatment phase of the trial has been completed on schedule. We believe NTCELL has the potential to be the first disease-modifying treatment for patients who are failing the current conventional treatment for Parkinson’s disease,” said Dr Taylor.
Even though this Phase I/IIa clinical trial is meant to test the efficacy of NTCELL in Parkinson disease patients, NTCELL also has the potential to be used in a number of other central nervous system indications such as Huntington’s, Alzheimer’s and other types of diseases that affect motor neurons.
Let me emphasize that the huge number of posters and talks at the SfN conference made it impossible to attend all of them, so my recollections here are some of the high points that I was able to take in. There is a lot of terrific science going on out there and these conferences are windows into it.
One poster described a feeding study in rats. One group of rats received a diet rich in omega-3 fatty acids, which are found in fish oils and soy. Another group was fed a standard laboratory diet that tends to skim on the omega-3 fatty acids. In the brains of the omega-3-fed rats, the expression off the gene that encodes Brain Derived Neurotropic Factor or BDNF increased significantly.
This is significant because BDNF promotes the survival of nerve cells (neurons) by playing a role in the growth, maturation (differentiation), and maintenance of these cells. In the brain, BDNF protein is active at the connections between nerve cells (synapses), where cell-to-cell communication occurs. The synapses can change and adapt over time in response to experience, a characteristic called synaptic plasticity, and BDNF regulates synaptic plasticity, which is important for learning and memory.
When these researchers examined why the BDNF gene was unregulated in rats fed the omega-3-rich diet, they discovered that the starting point of the gene, which is called the promoter was nice and clear. In the standard diet rats, the promoter of the BDNF gene was chemically modified with methyl (-CH3) groups. In the absence of the methyl groups, the transcription factor CTCF was able to bind and increase the rate of transcription. If the promoter was chemically modified with methyl groups, then a protein called MeCP2 bound to the promoter and prevented expression of BDNF.
This group looked further and discovered that the omega-3-rich diet seemed to influence the expression of BDNF by means of the balance of reduced and oxidized versions of electron carriers in cells, in particular, the ratio of NAD+ to NADH. NAD is a major electron carrier in cells and the ratio of NAD+, the oxidized version of this molecule, to the reduced version of this molecule, NADH, is a measure of the energy charge of the cell and how well-fed the individual is. More importantly, NAD is a substrate for another regulator of gene expression called Sirtuins.
Sirtuins are protein deacetylases, but they are unusual deacetylases since many of them they do not simply hydrolyze acetyl-lysine residues. Instead they couple lysine deacetylation to NAD hydrolysis. This hydrolysis produces O-acetyl-ADP-ribose, which is the deacetylated substrate and nicotinamide, which is an inhibitor of sirtuin activity. The dependence of sirtuins on NAD links their enzymatic activity directly to the energy status of the cell via the cellular NAD:NADH ratio.
The fact that a diet high in omega-3 fatty acids affects the NAD/NADH ratio is significant for Alzheimer’s disease because the sirtuin, SIRT1, deacetylates and coactivates the promoter for the gene that encodes the retinoic acid receptor beta gene, which subsequently upregulates the expression of alpha-secretase (ADAM10). Alpha-secretase is able to suppress beta-amyloid production. ADAM10 activation by SIRT1 also induces the Notch signaling pathway, which is known to repair neuronal damage in the brain. All of this begins with a dietary factor that actually protects the brain from Alzheimer’s disease by profound changes in gene expression.
Another poster from an Italian group used the 5XFAD mouse model of Alzheimer’s disease to test a growth factor called “painless Nerve Growth Factor” on mice with protein plaque formation in their brains. The growth factor was given by placing droplets of the growth factor in the noses of the mice while they were anesthetized. The results were stunning. Normally, 5XFAD mice get plaques quickly in their brains and lots of them. However, the growth factor was able to rescue the onset of behavioral deficits and reduces, although not eliminate, plaque formation. Other brain-specific pathologies found in these mice were reduced, such as astrocytosis. The wandering white cells in the brain known as microglia did a better job of gobbling up protein aggregates and clearing them from the brain, and the markers of inflammation were significantly reduced. I asked the investigator if there were plans to try to move this to clinical trials, and she said that she was unable to do so because of a lack of funding. Maybe someone will collaborate with this dear lady to make it so?
In another poster, the overexpression of an enzyme called heparanase in the brain decreased the burden of protein aggregates in the brains of mice with Alzheimer’s disease. I was not able to get into the details of this poster because of time.
In another poster, a very energetic young man told me about his very interesting work with a Parkinson’s disease model in rodents. If mice are administered a drug called MPTP (short for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), the dopamine-using neurons in the brain will specifically take up this drug in high concentrations and it will kill them. Therefore, this drug is an excellent model system to study Parkinson’s disease in mice.
Prokineticin-2 is a gene that is expressed in high quantities in the surviving dopamine-using neurons that came from the brains of Parkinson’s disease patients after their deaths. When Prokineticin-2 was overexpressed in cultured dopaminergic neurons, they unregulated a protein called Bcl-2. Bcl-2 is one of the group of proteins can protect cells from dying. Therefore, Prokineticin-2 is a prosurvival protein.
Next, this chap switched from a culture system to a “in a living animal” system or an in vivo system. By using genetically engineered viruses that overexpressed Prokineticin-2 in the brains of mice, he discovered that this viruses did not adversely affect the mice and he did in fact achieve high levels of Prokineticin-2 in the brains of mice with this recombinant viruses. The overexpression did not affect the mice in the least. When he did the same experiment with MPTP-treated mice – oh, just to be clear, he overexpressed Prokineticin-2 first and then administered the MPTP because it takes about 30 days for the viruses to properly upregulate Prokineticin-2 – he saw decreased inflammation in the brain, and increase in Bcl-2 and Pink1 expression in the brain (both of these genes are pro-survival genes), and the behavioral problems of the mice never emerged with the severity of the MPTP mice. When he examined TH – an enzyme that makes the neurotransmitter dopamine, he saw that levels of this enzyme were up too. This means that the dopamine-using neurons were surviving. Is this cool stuff or what?
According to the Japan Times, Kyoto University’s Jun Takahashi and his team have plans to launch a clinical study for Parkinson’ disease patients that will utilize cells derived from induced pluripotent stem cells made from the patient’s own cells.
In an interview with Takahashi, the Japan Times reported on Wednesday of this week that he hopes to develop the induced Pluripotent Stem Cell (iPSCs) treatment as soon as possible so that Kyoto University Hospital can provide this treatment by fiscal year 2018 as a designated advanced medical technique that can be used in combination with other conventional treatments and medicines already covered by various insurance policies. Takahashi also expressed his hope that by fiscal year 2023, public health insurance will pay for his treatment.
For this clinical study, Parkinson’s disease patients whose conditions have progressed to the point where their medications are no longer effective will be the primary targeted group. “It will take a long time” to establish an effective treatment for the progressive disorder, which is incurable at present, Takahashi said, stressing the importance of maintaining a positive attitude toward development and not losing hope.
Parkinson’s disease causes the nerve cells in the brain that utilize the neurotransmitter dopamine to die off. The death of these dopaminergic neurons adversely affects voluntary muscle movement.
The design of this clinical study will include the production of iPSCs from adult cells collected from participating patients. These stem cells will be differentiated into neural stem cells that make dopaminergic neurons. These dopaminergic neuron precursor cells will be transplanted back into the midbrains of the donors before they develop into nerve cells, according to Takahashi. This way, all injected cells will still have the capacity to divide and migrate once implanted into the brain, but they will still have the capacity to form dopaminergic neurons.
Takahashi’s team will also seek to develop a method for producing a nerve cell drug created from cells taken out of healthy people, to ease the financial burden on patients, he said, since the derivation of iPSCs remains prohibitively expensive.
Takahashi also said he aims to being clinical trials by March 2019.
Induced pluripotent stem cells (iPSCs) are made from mature adult cells by means of genetic engineering and cell culture techniques. These cells have embryonic stem cell-like capacities and can, potentially differentiate into any adult cell type. Because neurons made from iPSCs have sometimes not shown instability, the ability of neurons derived from iPSCs to stably integrate into brain has been questioned.
Schwamborn and Hemmer showed that six months after implantation, their iPSCs-derived neurons had become fully functionally integrated into the brain. This successful integration of iPSC-derived neurons into lastingly stable implants raises hope for future therapies that will replace sick neurons with healthy ones in the brains of patients with Parkinson’s disease, Alzheimer’s disease and Huntington’s chorea, for example. This work was published in the current issue of Stem Cell Reports.
The LCSB research group hopes to bring cell replacement therapy to maturity as a treatment for neurodegenerative diseases. The replacement of sick and/or dead neurons in the brain could one day cure disorders such as Parkinson’s disease. However, devising a successful therapy in human is a long, arduous process, and for good reasons. “Successes in human therapy are still a long way off, but I am sure successful cell replacement therapies will exist in future. Our research results have taken us a step further in this direction,” declared Schwamborn.
In their latest tests, the LCSB research group, in collaboration with colleagues from the Max Planck Institute and the University Hospital Münster and the University of Bielefeld, made stable neuronal implants in the brain from neurons that were derived from reprogrammed skin cells. They used a newer technique in which the neurons were produced from neural stem cells (NSCs). These NSCs or induced neural stem cells (iNSCs) had, in turn been made from iPSCs that were made from the host animal’s own skin cells, which considerably improves the compatibility of the implanted cells. Mice who received the neuronal implants showed no adverse side effects even six months after implantation. The new neurons were implanted into the hippocampus and cortex regions of the brain. Implanted neurons were fully integrated into the complex network of the brain and they exhibited normal activity and were connected to the original brain cells via newly formed connections known as synapses, which are the contact points between nerve cells.
These tests demonstrate that stem cells researchers are continuing to get a better handle on how to use cells derived from something other than human embryos in order to successfully replace damaged or dead tissue. “Building upon the current insights, we will now be looking specifically at the type of neurons that die off in the brain of Parkinson’s patients – namely the dopamine-producing neurons,” Schwamborn reports.
In future experiments, implanted neurons could provide the neurotransmitter dopamine (which is lacking in patients with Parkinson’s disease) directly into the patient’s brain and transport it to the appropriate sites. Such a result would herald an actual cure for the disease rather than a short-term fix. The first trials in mice are in progress at the LCSB laboratories on the university campus Belval.