Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders
Funding Type: 
Early Translational IV
Grant Number: 
TR4-06693
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$2 278 080
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

ALS is a progressive neurodegenerative disease that primarily affects motor neurons (MNs). It results in paralysis and loss of control of vital functions, such as breathing, leading to premature death. Life expectancy of ALS patients averages 2–5 years from diagnosis. About 5,600 people in the U.S. are diagnosed with ALS each year, and about 30,000 Americans have the disease. There is a clear unmet need for novel ALS therapeutics because no drug blocks the progression of ALS. This may be due to the fact that multiple proteins work together to cause the disease and therapies targeting individual toxic proteins will not prevent neurodegeneration due to other factors involved in the ALS disease process. We propose to develop a novel ALS therapy involving small molecule drugs that stimulate a natural defense system in MNs, autophagy, which will remove all of the disease-causing proteins in MNs to reduce neurodegeneration. We previously reported on a class of neuronal autophagy inducers (NAIs) and in this grant will prioritize those drugs for blocking neurodegeneration of human iPSC derived MNs from patients with familial and sporadic ALS to identify leads that will then be tested for efficacy in vivo in animal models of ALS to select a clinical candidate. Since all of our NAIs are FDA approved for treating indications other than ALS, our clinical candidate could be rapidly transitioned to testing for efficacy and safety in treating ALS patients near the end of this grant.

Statement of Benefit to California: 

Neurodegenerative diseases such as ALS as well as Alzheimer’s (AD), Parkinson’s (PD) and Huntington’s Disease (HD) are devastating to the patient and family and create a major financial burden to California (CA). These diseases are due to the buildup of toxic misfolded proteins in key neuronal populations that leads to neurodegeneration. This suggests that common mechanisms may be operating in these diseases. The drugs we are developing to treat ALS target this common mechanism, which we believe is an impairment of autophagy that prevents clearance of disease-causing proteins. Effective autophagy inducers we identify to treat ALS may turn out to be effective in treating other neurodegenerative diseases. This could have a major impact on the health care in CA. Most important in our studies is the translational impact of the use of patient iPSC-derived neurons and astrocytes to identify a new class of therapeutics to block neurodegeneration that can be quickly transitioned to testing in clinical trials for treating ALS and other CNS diseases. Future benefits to CA citizens include: 1) development of new treatments for ALS with application to other diseases such as AD, HD and PD that affect thousands of individuals in CA; 2) transfer of new technologies to the public realm with resulting IP revenues coming into the state with possible creation of new biotechnology spin-off companies and resulting job creation; and 3) reductions in extensive care-giving and medical costs.

Progress Report: 
  • ALS is a progressive neurodegenerative disease that primarily affects motor neurons (MNs). It results in paralysis and loss of control of vital functions, such as breathing, leading to premature death. Much scientific evidence indicates that ALS is due to the buildup of toxic misfolded proteins in key neuronal populations that leads to neurodegeneration. In this CIRM-funded project, we are developing drugs that can improve a cellular process called “autophagy” by which cells, including neurons, clear out built-up toxic misfolded proteins and increase their longevity. We had discovered that a series of FDA-approved drugs already on the market for other indications happen to induce autophagy in a manner that is independent of their original purpose. Our goal is to show that these FDA-approved drugs can induce autophagy and slow neurodegeneration in ALS patient-derived neurons, and to repurpose these drugs for ALS. In the last year, we have made significant progress towards testing these drugs on neurons that we derived from induced pluripotent stem cells engineered from skin cells taken from ALS patients. We have built robotic microscopes that can rapidly image ALS patient neurons that are treated with drugs in the lab and determine whether any of these “autophagy-inducing” FDA-approved drugs slowdown the rate of neurodegeneration. We have optimized large-scale methods to grow patient neurons, treat them with drugs, image them over many days, and analyze the images to measure neurodegeneration. In August 2014, we published a paper in the journal Nature Chemical Biology that showed two FDA-approved drugs can in fact induce autophagy and increase the clearing of an ALS-related protein called TDP43 in neurons. The drugs were also able to slow neurodegeneration in neurons and astrocytes derived from a familial ALS patient with an altered version of the TDP43 gene. We have now obtained stem cells from broader types of familial ALS as well as sporadic ALS patients, have made neurons from their stem cells, and have treated their stem cell-derived neurons with more than 10 autophagy-inducing drugs at varying concentrations to determine whether autophagy-inducers can slow neurodegeneration in neurons from broader forms of ALS. These neurons are currently being imaged using our robotic microscope. In addition, we have started to make astrocytes from patient stem cells and plan to test the drugs on astrocytes in the coming months.
Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06093
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$1 264 248
Disease Focus: 
Neurological Disorders
Pediatrics
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 

White matter is the infrastructure of the brain, providing conduits for communication between neural regions. White matter continues to mature from birth until early adulthood, particularly in regions of brain critical for higher cognitive functions. However, the precise timing of white matter maturation in the various neural circuits is not well described, and the mechanisms controlling white matter developmental/maturation are poorly understood. White matter is conceptually like wires and their insulating sheath is a substance called myelin. It is clear that neural stem and precursor cells contribute significantly to white matter maturation by forming the cells that generate myelin. In the proposed experiments, we will map the precise timing of myelination in the human brain and changes in the populations of neural precursor cells that generate myelin from birth to adulthood and define mechanisms that govern the process of white matter maturation. The resulting findings about how white matter develops may provide insights for white matter regeneration to aid in therapy for diseases such as cerebral palsy, multiple sclerosis and chemotherapy-induced cognitive dysfunction.

Statement of Benefit to California: 

Diseases of white matter account for significant neurological morbidity in both children and adults in California. Understanding the cellular and molecular mechanisms that govern white matter development the may unlock clues to the regenerative potential of endogeneous stem and precursor cells in the childhood and adult brain. Although the brain continues robust white matter development throughout childhood, adolescence and young adulthood, relatively little is known about the mechanisms that orchestrate proliferation, differentiation and functional maturation of neural stem and precursor cells to generate myelin-forming oligodendrocytes during postnatal brain development. In the present proposal, we will define how white matter precursor cell populations vary with age throughout the brain and determine if and how neuronal activity instructs neural stem and precursor cell contributions to human white matter myelin maturation.

Disruption of white matter myelination is implicated in a range of neurological diseases, including cerebral palsy, multiple sclerosis, cognitive dysfunction from chemotherapy exposure, attention deficit and hyperactivity disorder (ADHD) and even psychiatric diseases such as schizophrenia. The results of these studies have the potential to elucidate clues to white matter regeneration that may benefit hundreds of thousands of Californians.

Progress Report: 
  • Formation of the insulated fiber infrastructure of the human brain (called "myelin") depends upon the function of a precursor cell type called "oligodendrocyte precursor cells (OPC)". The first Aim of this study seeks to determine how OPCs differ from each other in different regions of the brain, and over different ages. Understanding this heterogeneity is important as we explore the regenerative capacity of this class of precursor cells. We have, in the past year, isolated OPCs from various regions of the human brain from individuals at various ages and are studying the molecular characteristics of these precursor cells at the single cell level in order to define distinct OPC subpopulations. We have also begun to study the functional capabilities of OPCs isolated from different brain regions. The second Aim of this study seeks to understand how interactions between electrically active neurons and OPCs affect OPC function and myelin formation. We have found that when mouse motor cortex neurons "fire" signals in such a way as to elicit a complex motor behavior, much as would happen when one practices a motor task, OPCs within that circuit respond and myelination increases. This affects the function of that circuit in a lasting way. These results indicate that neurons and OPCs interact in important ways to modulate myelination and supports the hypothesis that OPC function may play a role in learning.
  • Sending neural impulses quickly down a long nerve fiber requires a specialized type of insulation called myelin, made by a cell called an oligodendrocyte that wraps itself around neuronal projections. Myelin-insulated nerve fibers make up the “white matter” of the brain, the vast tracts that connect one information-processing area of the brain to another. We have now shown that neuronal activity prompts oligodendrocyte precursor cell (OPC) proliferation and differentiation into myelin-forming oligodendrocytes. Neuronal activity also causes an increase in the thickness of the myelin sheaths within the active neural circuit, making signal transmission along the neural fiber more efficient. This was found to be true in both juvenile and in adult brains Metaphorically, it’s much like a system for improving traffic flow along roadways that are heavily used. And as with a transportation system, improving the routes that are most productive makes the whole system more efficient.
  • Interestingly, some parts of the neural circuit studied showed evidence of myelin-forming precursor cell response to neuronal activity, while other parts of the active circuit did not. In related work, we are making progress towards understanding how OPCs differ in various regions of the brain, examining the molecular heterogeneity of human OPCs at a single cell level.
Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02040
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 933 022
Disease Focus: 
Spinal Muscular Atrophy
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 

Spinal Muscular Atrophy (SMA) is one of the most common lethal genetic diseases in children. One in thirty five people carry a mutation in a gene called survival of motor neurons 1 (SMN1) which is responsible for this disease. If two carriers have children together they have a one in four chance of having a child with SMA. Children with Type I SMA seem fine until around 6 months of age, at which time they begin to show lack of muscular development and slowly develop a "floppy" syndrome over the next 6 months. Following this period, SMA children become less able to move and are eventually paralyzed by the disease by 3 years of age or earlier. We know that this mutation causes the death of motor neurons - which are important for making muscle cells work. Interestingly, there is a second gene which can lessen the severity of the disease process (SMN2). Children with more copies of this modifying gene have less severe symptoms and can live for longer periods of time (designated Type II, III and IV and living longer periods respectively).

There is no therapy for SMA at the current time. One of the roadblocks is that there are no human models for this disorder as it is very difficult to make the motor neurons that die in the disease in the laboratory. The researchers in the current proposal have recently created pluripotent stem cells from a patient with Type I SMA (the most severe) and shown that motor neurons grown out from the pluripotent stem cells also die in the culture dish just like they do in children. This is an important model for SMA.

The proposed research takes this model of SMA and extends it to Type II and Type III children in order to have a wider range of disease severity in the culture dish (Type IV is very rare and difficult to get samples from). It then develops new technologies to produce very large numbers of motor neurons and perform large scale analysis of their survival profiles. Finally, it will explore whether novel compounds can slow down the degeneration of motor neurons in this model which should lead to the discovery of dew drugs that then may be used to treat the disease.

Statement of Benefit to California: 

The aim of this research is to develop novel drugs to treat a lethal childhood disease - SMA. There would be three immediate benefits to the state of California and its citizens.

1. Children in California would have access to novel drugs to slow or prevent their disease.
2. SMA is a world wide disease. The institutions involved with the research would be able to generate income from any new drugs developed and the profit from this would come back to California.
3. The project will employ a number of research staff in Californian institutions

Progress Report: 
  • This year we have created a large number of new SMA lines, developed ways to differentiate them into motor neurons using high content dishes, and begun to analyze the health of the motor neurons over time. We have also submitted a new paper showing that much of the cell death seen in the dying motor neurons is due to apoptosis - a form of cell death that is treatable with specific types of drug. We are now using these new lines to begin setting up screening runs with drug libraries and should be able to start these in the new year of funding.
  • In this year we have made more induced pluripotent stem (iPSC) cell lines from Spinal Muscular Atrophy patients also using blood cells in addition to skin cells. Blood cells from patients are usually more readi;y accessible. As such, this technique can be used to make larger bank of similar cell lines. We have also rigorously tested all the iPSCs them for their quality. These lines are now available for distribution to other California researchers along with a certificate of analysis.
  • Motor neurons are a type of neuron that control muscle movement and are markedly destroyed in SMA patients. In order for these powerful iPS cells form patients to be useful for discovering new drugs for SMA it is very important that we can make motor neurons from iPSCs in large quantities of millions to billions in number. Only then will testing of thousands to millions of new drugs would be feasible in neurons from SMA patients. To this end, we have created a method for making a predecessor cell type to human motor neurons from human iPSCs in a petri dish. These predecessor cells, known as motor neuron precursor spheres (iMNPS), are grown as clumps of floating spherical balls, each containing thousands such cells that are grown in large numbers repeatedly for long periods of time. We have made these iMNPS now from many SMA patients as well as healthy humans. These spheres can be preserved for long period of time by freezing them at very low temperatures. They are then awoken at a later time making it convenient for testing large numbers of drugs.
  • Since iPSCs have the power to make any cell type in the human body, they can also be contaminated with other unwanted types of cells. Typically such a technique is very difficult to accomplish in pluripotent stem cells such as embryonic and iPSCs. Therefore, we have designed a more efficient scheme to generate iPSC lines from SMA patients that will become fluorescent color (green, red or blue) when then motor neurons are made from iPSCs. These types of cells are known as reporter cell lines. This will aid in picking out the desired cell type from patient iPSCs, in this case a motor neuron, and discard any unwanted cell types. This will enormously simplify testing of new drugs in SMA patient motor neurons.
  • Deficiency of an important protein in SMA patients is one of the key causes to the course of the disease. We have also designed an automated method for identifying new drugs in patient motor neurons that will test for correction of SMN protein levels in motor neurons.
  • In Year 3 we completed making all iPSC lines from Spinal Muscular Atrophy patients. We rigorously tested all the iPSCs for quality. These lines are now available for distribution to other California researchers along with a quality control certificate.
  • Motor neurons are a type of neuron that control muscle movement and are markedly destroyed in SMA patients. In order for these powerful iPS cells form patients to be useful for discovering new drugs for SMA it is very important that we can make motor neurons from iPSCs in billions and repeatedly. Only then will testing of thousands to millions of new drugs would be feasible in neurons from SMA patients.
  • To this end, we have created a method for making a predecessor cell type to human motor neurons from human iPSCs in a petri dish. These predecessor cells, known as motor neuron precursor spheres (iMPS), are grown as clumps of floating spherical balls, each containing thousands such cells that are grown in large numbers repeatedly for long periods of time. We have now tested our method in multiple patient cells and characterized these spheres. The iMPS have now been produced from many SMA patients as well as healthy humans. The next step we have developed is to take the iMPS to make motor neurons that are similar to those that are affected in SMA children. We have then discovered a method for creating them quickly. These aggregate spheres and spinal cord motor neurons from them can be preserved for long period of time by freezing them at very low temperatures. They are then awoken at a later time making it convenient for testing large numbers of drugs.
  • Since iPSCs have the power to make any cell type in the human body, they can also be contaminated with other unwanted types of cells. Typically such a technique is very difficult to accomplish in pluripotent stem cells such as embryonic and iPSCs. Therefore, we have designed a more efficient scheme to generate iPSC lines from SMA patients that will become fluorescent color (green, red or blue) when then motor neurons are made from iPSCs. These types of cells are known as reporter cell lines. This will aid in picking out the desired cell type from patient iPSCs, in this case a motor neuron, and discard any unwanted cell types. This will enormously simplify testing of new drugs in SMA patient motor neurons. Using new technologies that can edit, cut, copy, and paste new DNA in the stem cell genome, we are also developing ways to engineer iPS cell lines that will tag the motor neurons when they are made. This will allow us another method for making pure motor neurons and tracking them in a dish among other types of cells while they are alive.
  • Deficiency of an important SMN protein in SMA patients is one of the key causes to the course of the disease. An automated method has been developed for identifying what causes the SMA neurons to become sick and test new drugs in motor neurons. We are now gearing up to test some ~1400 known compounds on patient motor neurons to determine whether we can raise SMN protein levels in motor neurons.
  • The goal of this project has now been reached. We have developed a new screening platform using motor neurons from induced pluripotent stem cells taken from children with spinal muscular atrophy. Through this technology we have screened thousands of compounds and have shown a small sub set that active gene expression and enhance motor neuron survival in this model. These compounds will now be moved to the next stage for validation. This funding has allowed us to complete the development of this tool/technology and put us in a strong position to continue these studies and the drug development process to move interesting drugs to the market for spinal muscular atrophy.
Funding Type: 
Early Translational II
Grant Number: 
TR2-01767
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 708 549
Disease Focus: 
Neurological Disorders
Trauma
Collaborative Funder: 
Maryland
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 

Traumatic brain injury (TBI) affects 1.4 million Americans a year; 175,000 in California. When the brain is injured, nerve cells near the site of injury die due to the initial trauma and interruption of blood flow. Secondary damage occurs as neighboring tissue is injured by the inflammatory response to the initial injury, leading to a larger area of damage. This damage happens to both neurons, the electrically active cells, and oligodendrocytes, the cell which makes the myelin insulation. A TBI patient typically loses cognitive function in one or more domains associated with the damage (e.g. attention deficits with frontal damage, or learning and memory deficits associated with temporal lobe/hippocampal damage); post-traumatic seizures are also common. Currently, no treatments have been shown to be beneficial in alleviating the cognitive problems following even a mild TBI.

Neural stem cells (NSCs) provide a cell population that is promising as a therapeutic for neurotrauma. One idea is that transplanting NSCs into an injury would provide “cell replacement”; the stem cells would differentiate into new neurons and new oligodendrocytes and fill in for lost host cells. We have successfully used “sorted“ human NSCs in rodent models of spinal cord injury, showing that hNSCs migrate, proliferate, differentiate into oligodendrocytes and neurons, integrate with the host, and restore locomotor function. Killing the NSCs abolishes functional improvements, showing that integration of hNSCs mediates recovery. Two Phase I FDA trials support the potential of using sorted hNSC for brain therapy and were partially supported by studies in my lab. NSCs may also improve outcome by helping the host tissue repair itself, or by providing trophic support for newly born neurons following injury. Recently, transplantation of rodent-derived NSCs into a model of TBI showed limited, but significant improvements in some outcome measures. These results argue for the need to develop human-derived NSCs that can be used for TBI.

We will establish and characterize multiple “sorted” and “non-sorted“ human NSC lines starting from 3 human ES lines. We will determine their neural potential in cell culture, and use the best 2 lines in an animal model of TBI, measuring learning, memory and seizure activity following TBI; then correlating these outcomes to tissue modifying effects. Ultimately, the proposed work may generate one or more human NSC lines suitable to use for TBI and/or other CNS injuries or disorders. A small reduction in the size of the injury or restoration of just some nerve fibers to their targets beyond the injury could have significant implications for a patient’s quality of life and considerable economic impact to the people of California. If successful over the 3-year grant, additional funding of this approach may enable a clinical trial within the next five years given success in the Phase I FDA approved trials of sorted hNSCs for other nervous system disorders.

Statement of Benefit to California: 

The Centers for Disease Control and Prevention estimate that traumatic brain injury (TBI) affects 1.4 million Americans every year. This equates to ~175,000 Californian’s suffering a TBI each year. Additionally, at least 5.3 million Americans currently have a long-term or a lifelong need for help to perform activities of daily living as a result of suffering a TBI previously. Forty percent of patients who are hospitalized with a TBI had at least one unmet need for services one year after their injury. One example is a need to improve their memory and problem solving skills. TBI can also cause epilepsy and increases the risk for conditions such as Alzheimer's disease, Parkinson's disease, and other brain disorders that become more prevalent with age. The combined direct medical costs and indirect costs such as lost productivity due to TBI totaled an estimated $60 billion in the United States in 2000 (when the most recent data was available). This translates to ~$7.5 billion in costs each year just to Californians.

The proposed research seeks to generate several human neural-restricted stem cell lines from ES cells. These “sorted” neural-restricted stem cell lines should have greatly reduced or no tumor forming capability, making them ideally suited for clinical use. After verifying that these lines are multipotent (e.g. they can make neurons, astrocytes and oligodendrocytes), we will test their efficacy to improve outcomes in TBI on a number of measures, including learning and memory, seizure activity, tissue sparing, preservation of host neurons, and improvements in white matter pathology. Of benefit to California is that these same outcome measures in a rodent model of TBI can also be assessed in humans with TBI, potentially speeding the translational from laboratory to clinical application.

A small reduction in the size of the injury, or restoration of just some nerve fibers to their targets beyond the injury, or moderate improvement in learning and memory post-TBI, or a reduction in the number or severity of seizures could have significant implications for a patient’s quality of life and considerable economic impact to the people of California. Additionally, the cell lines we have chosen to work with are unencumbered with IP issues that would prevent us, or others, from using these cell lines to test in other central nervous system disorders. Two of the cell lines have already been manufactured to “GMP” standards, which would speed up the translation of this work from the laboratory to the clinic. Finally, if successful, these lines would be potentially useful for treating a variety of central nervous system disorders in addition to TBI, including Alzheimer’s disease, Parkinson’s disease, stroke, autism, spinal cord injury, and/or multiple sclerosis.

Progress Report: 
  • In the first year of this Early Translation Award for traumatic brain injury (TBI), our goal was to develop the stem cells lines necessary to begin testing of stem cells in an animal model of TBI in year 2. If we are fortunate to demonstrate that the stem cell products are effective in animal models of TBI, these cells will need to be grown in a way that is acceptable to the FDA for future use in man. Xenofree means that the cells are not exposed to possible animal product contaminants (e.g. serum or blood products) and that every component that the cells were exposed to is chemically defined and can be traced to the original source.
  • First, we obtained three separate embryonic stem (ES) cell lines from Sheffield, UK and imported them to the United States. These lines where then thawed and grown in “xenofree” cell culture conditions. Many labs have had difficulty transitioning human ES cells to xenofree conditions without introducing genetic defects in the cell lines or killing the cells. We were able to work out the correct conditions for all three ES cell lines to be grown xenofree. We were also successful in converting two of the three ES lines into neural stem cells (the subtype of stem cell needed for transplanting into brain tissue). These neural stem cells (NSCs) were further purified by labeling them for a stem cell surface marker present on NSCs (called CD133) and then magnetically sorting out just the CD133 positive cells and continuing to grow them. This approach is thought to enrich the stem cell population for NSCs and eliminate any remaining non-differentiated ES cells (which have an added risk of forming tumors if injected into animals or man). We successfully “sorted” both Shef cell lines and we now have four candidate populations of sorted and unsorted Shef4 and Shef6 cells. We grew these cells in culture and tested whether they differentiated into neuronal precursor or glial precursor cells. Quantification of the type of cells they turn into after 2 weeks showed that the four cell populations were different. These differences were even more apparent when looking at the cells in a microscope. At the end of year one, we have four different populations of neural stem cells which are growing in defined xenofree conditions, are frozen down in master cell banks, and which are genetically normal. There are sufficient quantities of these human neural stem cells (hNSC) to complete the remaining aims of the ETA grant over the remaining two years.
  • In the first year we also trained staff in the surgical procedures required to produce controlled cortical impact injuries in Athymic nude rats (ATNs), a type of rat that has no immune system. These procedures were necessary because no one has ever used ATN rats to model TBI. Our goal in year two is to transplant hNSCs into rats with TBI. If the rats had a normal immune system, their bodies would detect the foreign human cells and reject them. Also, because no one has ever tested TBI in ATN rats, we needed to find out if ATN rats respond like regular rats to the injury and if they have similar, predictable deficits on the cognitive tasks we plan to use in year 2 to measure whether hNCSs improve the animal’s recovery or not. This training and these pilot tests in ATN rats were completed successfully. Finally, the hypothesis is that by “sorting” the hNSCs to be CD133 positive, we are making the stem cell population safer for transplantation. This will be tested in year 2 using a tumorigenicity assay. We worked out how to conduct these assays in year 1 using a population of ES cells known to cause tumors so that we will have a positive control to compare the hNSCs to in year 2.
  • In summary, we met all of our goals and milestones for year 1 and are poised to make good progress in year 2.
  • The goal of this project is to take three human embryonic stem cell lines (Shef3, Shef4, and Shef6), transition them to multipotent neural stem cell (hNSC) populations, sort/enrich these hNSC stem/progenitor populations, and then test these cell lines for efficacy in a rat model of controlled cortical impact (CCI) model of traumatic brain injury (TBI). Our strategy is to develop xenofree culture methods for the transition of hESC to NSCs, use magnetic activated cell sorting (MAC) for the cell surface markers CD133+/CD34- to enrich the hNSC populations for stem/progenitor cells, test these sorted vs unsorted cell lines in tumorigenicity assays, and use the best two non-tumorigenic lines in a CCI model of TBI. Efficacy will be assessed on a battery of cognitive tests, via a reduction in spontaneous seizure, and in histological outcomes.
  • At the Two Year time-point in the grant, we have (A) generated 6 hNSC populations, (B) completed short-term teratoma assays which demonstrate that none of our hNSC populations form teratomas in either of two transplantation sites (sub cutaneous into the leg or intracranially into the brain, (C) established parameters for graded contusion traumatic brain injuries in ATN rats that (D) yield long-term (≥8 weeks) deficits in both learning and memory on the Morris Water Maze. (E) We have also determined that TBI yields an altered response on a conditioned taste aversion task (neophobia) and on the elevated plus maze compared to sham controls. (F) Determined that unsorted hNSCs (both Shef4 and Shef6) do not survive long-term in uninjured brain and (G) transplanted two large cohorts of TBI injured animals with Shef6 sorted NSCs of high passage, Shef6 sorted hNSCs of low passage, sham animals, and animals with a vehicle control. These two cohorts are too large to run simultaneously, so they are being run in parallel. Animals from both cohorts will complete functional all assessments by the end of June 2013.
  • Summary: We have very promising preclinical efficacy data in a rodent model of traumatic brain injury (TBI) using stem cells as a potential therapeutic. We have found that intra-cranial transplants of Shef-6 derived human neural stem cells (hNSCs) appear to induce improvement on two different behavioral domains after long-term (>2 months) survival. Importantly, Shef-6 hNSCs did not form tumors when transplanted at high doses into naïve brain. Shef-6 hNSCs are xenofree, GMP compatible, suitable for use in man (the donor and cells were certified to be free of HIV, Hepatitis A, B, C, HTLV, EBV, CMV, and are mycoplasma free). Furthermore, Shef-6 is on the FDA embryonic stem cell registry, enabling future Federal funding of their clinical testing in man if warranted. Specifically, we have demonstrated long-term efficacy in a moderate to severe controlled cortical impact (CCI) model of TBI using Shef-6 derived hNSCs on both a cognitive task (MWM Reversal Learning) and an emotional task (Elevated Plus Maze for anxiety). This dual improvement across cognitive and emotional domains is unique to the field and supports external validity of the model. These behavioral findings need to be correlated with quantification of the total number of surviving human cells and their terminal cell fate (whether the hNSCs differentiated into neurons, oligodendrocytes, or astrocytes) to confirm efficacy. Stereological quantification is currently ongoing and very labor intensive. If the correlation between surviving cells and cognitive improvements holds up after the quantification is complete, these findings will support a future Preclinical Development Award application to CIRM. Additionally, we are the first group to couple kindling and TBI to model the critical complication of post-TBI seizures. Traditional TBI models yield seizures in less than 20% of rodents, making hNSC studies cost prohibitive. Coupling kindling with TBI ensures that all animals start with a hypersensitive neural circuit so hNSCs can be tested in a more relevant environment; we will be ready to begin this important kindling test coupled with hNSCs in the Spring of 2014. These studies have paved new ground for a field with huge economic costs, no treatments, and no GMP qualified ES based solutions on the horizon.
  • Traumatic Brain Injuries (TBI) are the leading cause of death and disability in the young population. Falls resulting in injury to the brain are also a major problem in the elderly. The rate of TBI is greater than the number of people diagnosed with brain, breast, colon, lung, and prostate cancers combined, yet nationally the US invests 95% more research dollars on cancer compared to TBI. 1.7 million new cases of TBI occur each year, at an economic cost of $60 billion. Extrapolating to California (12% of US population), there are ~210,000 new cases of TBI a year in our state, with a yearly cost that exceeds $7 billion. TBI results in permanent long-term deficits, including memory impairments and emotional disfunction, that affect both the patient and their families. There are no treatments to alleviate the long-term consequences of TBI. Yet a small reduction in damage, restoration of just some nerve fibers to their targets beyond the injury, or moderate improvement in learning, memory, or emotional outcomes could have significant implications for an individual’s quality of life. Our hypothesis was that human neural stem cells (hNCSs) might alleviate some impairments associated with TBI in a new animal model of neurotrauma. Our first goal was to grow hNSCs under cell culture conditions free from contamination of non-human products (referred to as “xenofree”), and then sort these cells based on cell surface markers known to be present in high concentrations on migratory neural stem cells (and not other byproducts of the culture conditions). Our second goal was to develop an animal model of TBI with long-lasting cognitive and emotional deficits; this animal model had to be “immuno-deficient”, or lacking a functional immune system, so that “foreign” human cells would not be rejected. Long-lasting deficits were need so that there would be a sufficient time window of dysfunction to allow the hNSCs to divide, migrate through the brain, and possibly restore function. If animals recover function too quickly on their own (as happens in some models of neurotrauma), then there would not be a large enough difference between control animals and injured animals to detect an effect of the hNSCs or not. Goal three was to test the therapeutic effects of hNSCs in this model. Finally, because a large number of people with TBI also experience seizures long after the initial injury, our forth goal was to combine “kindling” with TBI and ask whether hNSCs could alter kindling. Kindling involves implanting an electrode in the brain and very gently stimulating the brain every day until seizures occur. One can then measure how strong the seizure are and their duration (called after-discharge).
  • As the result of receiving CIRM Early Translation funding, we successfully generated two “xenofree” human neural stem cell lines (hNSCs) which are suitable for future therapeutic use in a variety of human neurological conditions (Goal 1). We also developed an athymic nude rat (ATN) model of controlled cortical impact TBI which exhibits sustained (2-months or longer) cognitive and emotional deficits. ATN rats lack T-cells, and thus have a sufficiently impaired immune system that they do not completely reject transplanted human cells. These ATN rats show deficits on novel place recognition (NPR), acquisition and memory of location on the Morris Water Maze, and disturbances on an Elevated Plus Maze (EPM) task in comparison to sham controls (Goal 2). We also found that sorted hNSCs survive and are not rejected in this model and that performance on the NPR task, learning on the Morris Water Maze and exploration on the EPM are all improved in the hNSC treated group compared to sham controls (Goal 3). Finally, when we repeated a therapeutic transplantation test of sorted hNSCs, but in seizure/kindled animals with TBI we found three interesting results (Goal 4). First, we replicated our earlier finding that hNSCs are efficacious in restoring memory function on the NPR task prior to kindling. Second, we found that after kindling, the improvement found with hNSCs was lost. And finally, we found that hNSCs reduce the number of After Discharge events in TBI+Kindled animals in comparison to TBI+Kindled animals that received a vehicle control injection.
  • In summary, we have successfully met all of our goals: (1) we generated a new human neural stem cell line suitable for future clinical trials in humans. (2) We developed an immunodeficient animal model of traumatic brain injury with sustained behavioral deficits. (3) We found very promising preclinical efficacy of our hNSCs in TBI. And (4), we have shown that hNSCs may play a role in reducing the number or severity of seizures following TBI, but if seizure activity is severe, that activity may interfere with hNSC mediated improvements on memory. With additional funding, we hope to complete the full range of preclinical studies required to translate these positive findings into an FDA approved human trial.
Funding Type: 
Research Leadership
Grant Number: 
LA1-08015
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$6 368 285
Disease Focus: 
Heart Disease
Neurological Disorders
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Directly Reprogrammed Cell
Public Abstract: 

Tissues derived from stem cells can serve multiple purposes to enhance biomedical therapies. Human tissues engineered from stem cells hold tremendous potential to serve as better substrates for the discovery and development of new drugs, accurately model development or disease progression, and one day ultimately be used directly to repair, restore and replace traumatically injured and chronically degenerative organs. However, realizing the full potential of stem cells for regenerative medicine applications will require the ability to produce constructs that not only resemble the structure of real tissues, but also recapitulate appropriate physiological functions. In addition, engineered tissues should behave similarly regardless of the varying source of cells, thus requiring robust, reproducible and scalable methods of biofabrication that can be achieved using a holistic systems engineering approach. The primary objective of this research proposal is to create models of cardiac and neural human tissues from stem cells that can be used for various purposes to improve the quality of human health.

Statement of Benefit to California: 

California has become internationally renowned as home to the world's most cutting-edge stem cell biology and a global leader of clinical translation and commercialization activities for stem cell technologies and therapies. California has become the focus of worldwide attention due in large part to the significant investment made by the citizens of the state to prioritize innovative stem cell research as a critical step in advancing future biomedical therapies that can significantly improve the quality of life for countless numbers of people suffering from traumatic injuries, congenital disorders and chronic degenerative diseases. At this stage, additional investment in integration of novel tissue engineering principles with fundamental stem cell research will enable the development of novel human tissue constructs that can be used to further the translational use of stem cell-derived tissues for regenerative medicine applications. This proposal would enable the recruitment of a leading biomedical engineer with significant tissue engineering experience to collaborate with leading cardiovascular and neural investigators. The expected result will be development of new approaches to engineer transplantable tissues from pluripotent stem cell sources leading to new regenerative therapies as well as an enhanced understanding of mechanisms regulating human tissue development.

Funding Type: 
Research Leadership
Grant Number: 
LA1-06535
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$6 718 471
Disease Focus: 
Parkinson's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 

Protection and cell repair strategies for neurodegenerative diseases such as Parkinson’s Disease (“PD”) depend on well-characterized candidate human stem cells that are robust and show promise for generating the neurons of interest following stimulation of inherent brain stem cells or after cell transplantation. These stem cells must also be expandable in the culture dish without unwanted growth and differentiation into cancer cells, they must survive the transplantation process or, if endogenous brain stem cells are stimulated, they should insinuate themselves in established brain networks and hopefully ameliorate the disease course.
The studies proposed for the CIRM Research Leadership Award have three major components that will help better understand the importance and uses of stem cells for the treatment of PD, and at the same time get a better insight into their role in disease repair and causation. First, we will characterize adult human neural stem cells from control and PD brain specimens to distinguish their genetic signatures and physiological properties of these cells. This will allow us to determine if there are stem cells that are pathological and fail in their supportive role in repairing the nervous system. Next, we will investigate a completely novel disease initiation and propagation mechanism, based on the concept that secreted vesicles from cells (also known as “exosomes”) containing a PD-associated protein, alpha-synuclein, propagate from cell-to cell. Our hypothesis is that these exosomes carry toxic forms of alpha-synuclein from cell to cell in the brain, thereby accounting disease spread. They may do the same with cells transplanted in patients with PD, thereby causing these newly transplanted cells designed to cure the disease, to be affected by the same process that causes the disease itself. This is a bottleneck that needs to be overcome for neurotransplantation to take its place as a standard treatment for PD.
Our studies will address disease-associated toxicity of exosomal transmission of aggregated proteins in human neural precursor stem cells. Importantly, exosomes in spinal fluid or other peripheral tissues such as blood might represent a potentially early and reliable disease biomarker as well as a new target for molecular therapies aimed at blocking transcellular transmission of PD-associated molecules.
Finally, we have chosen pre-clinical models with α-synucleinopathies to test human neural precursor stem cells as cell replacement donors for PD as well as interrogate, for the first time, their potential susceptibility to PD and contribution to disease transmission. These studies will provide a new standard of analysis of human neural precursor cells at risk for and contributing to pathology (so-called “stem cell pathologies”) in PD and other neurodegenerative diseases via transmission of altered or toxic proteins from one cell to another.

Statement of Benefit to California: 

According to the National Institute of Health, Parkinson’s disease (PD) is the second most common neurodegenerative disease in California and the United States (one in 100 people over 60 is affected) second only to Alzheimer’s Disease. Millions of Americans are challenged by PD, and according to the Parkinson’s Action Network, every 9 minutes a new case of PD is diagnosed. The cause of the majority of idiopathic PD is unknown. Identified genetic factors are responsible for less than 5% of cases and environmental factors such as pesticides and industrial toxins have been repeatedly linked to the disease. However, the vast majority of PD is thought to be etiologically multi-factorial, resulting from both genetic and environmental risk factors. Important events leading to PD probably occur in early or mid adult life. According to the Michael J. Fox Foundation, “…there is no objective test, or reliable biomarker for PD, so rate of misdiagnosis is high, and there is a seriously pressing need to develop better early detection approaches to be able to attempt disease-halting protocols at a non-symptomatic, so-called prodromal stage.”
The proposed innovative and transformative research program will have a major direct impact for patients who live in California and suffer from PD and other related neurodegenerative diseases. If these high-risk high-pay-off studies are deemed successful, this new program will have tackled major culprits in the PD field. They could lead to a better understanding of the role of stem cells in health and disease. Furthermore they could greatly advance our knowledge of how the disease spreads throughout the brain which in turn could lead to entire new strategies to halt disease progression. In a similar manner these studies could lead to ways to prevent the disease from spreading to cells that have been transplanted to the brain of Parkinson’s patients in an attempt to cure their disease. This is critical for neurotransplantation to thrive as a therapeutic approach to treating PD. In addition, if we extend the cell-to-cell transmissible disease hypothesis to other neurodegenerative diseases, and cancer, the studies proposed here represent a new diagnostic approach and therapeutic targets for many diseases affecting Californians and humankind in general.
This CIRM Research Leadership Award will not only have an enormous impact on understanding the cause of PD and developing new therapeutic strategies using stem cells and its technologies, this award will also be the foundation of creating a new Center for Translational Stem Cell Research within California. This could lead to further growth at the academic level and for the biotechnology industry, particularly in the area regenerative medicine.

Funding Type: 
Research Leadership
Grant Number: 
LA1-05735
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$5 609 890
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 

One of the most exciting and challenging frontiers in neuroscience and medicine is to repair traumatic injuries to the central nervous system (CNS). Most spinal cord and head injuries result in devastating paralyses, yet very limited clinical intervention is currently available to restore the lost abilities. Traumatic injuries of the spine cause fractures and compression of the vertebrae, which in turn crush and destroy the axons, long processes of nerve cells that carry signals up and down the spinal cord between the brain and the rest of the body. It follows that the best chance for promoting functional recovery is identifying strategies that enable lesioned axons to regenerate and reconnect the severed neural circuits. Even minor improvements in voluntary motor functions after spinal cord injury could be immensely helpful for increasing the quality of life, employability, and independence, especially for patients with injuries at the upper spinal level. Thus, our overall research program centers on axon regeneration in general, with a focus on regenerating descending axons from the brain that control voluntary motor and other functions.

We recently made breakthrough discoveries in identifying key biological mechanisms stimulating the re-growth of injured axons in the adult nervous system, which led to unprecedented extents of axon regeneration in various CNS injury models. While our success was compelling, we found that many regenerated axons were stalled at the lesion sites by the injury-induced glial scars. Furthermore, it is unclear whether the regenerated axons can form functional synaptic connections when they grow into the denervated spinal cord. This proposed research program is aimed at solving these obstacles by using human stem cell technologies. In the first aim, we will use human neural stem cells to engineer “permissive cell bridges” that can guide the maximum number of regenerating axons to grow across injury sites. In the second aim, we will test the therapeutic potential of human stem cell-derived neurons in forming “functional relays” that could propagate the brain-derived signals carried by regenerating axons to the injured spinal cord. Together, our research program is expected to develop a set of therapeutic strategies that have immediate clinical implications for human SCI patients.

Statement of Benefit to California: 

Approximately 1.9% of the U.S. population, roughly 5,596,000 people, report some forms of paralysis; among whom, about 1,275,000 individuals are paralyzed due to spinal cord injuries (SCI). The disabilities and medical complications associated with SCI not only severely reduce the quality of life for the injured individuals, but also result in an estimated economical burden of $400,000,000 annually for the state of California in lost productivity and medical expenses. Traumatic injuries of the spine cause fractures and compression of the vertebrae, which in turn crush and destroy the axons, long processes of nerve cells that carry signals up and down the spinal cord between the brain and the rest of the body. Thus, identifying strategies that enable lesioned axons to regenerate and reconnect the severed neural circuits is crucial for promoting functional recovery after SCI. In recent years, we made breakthrough discoveries in identifying key biological mechanisms stimulating the re-growth of injured axons in the adult nervous system. This proposed research program is aimed at developing human neural stem cell based therapeutic strategies that enable regenerated axons to grow through tissue cavities at the injury site, and establish functionally relays between the regenerating cortical axons and the spinal circuits below the injury site, thereby restore the lost sensory/motor functions in SCI patients. Success of these proposed studies could lead to immediate therapeutic applications for SCI patients. The first stem cell-based clinical trial for human SCI is started in California in which stem cells are used to provide support and stimulate remyelination. Our stem cell based therapeutic strategies are aimed at re-building neural connections, which will compliment the existing strategy nicely. As a result, Californians will be the first beneficiaries of these therapies.

Funding Type: 
Basic Biology V
Grant Number: 
RB5-07320
Investigator: 
ICOC Funds Committed: 
$598 367
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

Our goal is to use the mechanisms that generate neuronal networks to create neurons from stem cells, to either replace diseased and damaged tissue or as a source of material to study disease mechanisms. A key focus of such regenerative studies is to restore function to the spinal cord, which is particularly vulnerable to damage. However, although considerable progress has been made in understanding how to direct stem cells towards motor neurons that control coordinated movement, little progress has been made so far directing stem cells to form the sensory neurons that allow us to experience the environment around us.

Our proposed research will use insights from the mechanisms known to generate the sensory neurons during the development of the spinal cord, to derive these neurons from stem cells. We will initially use mouse embryonic stem cells in these studies, to accelerate the experimental progress. We will then apply our findings to human embryonic stem cells, and assess whether these cells are competent to repopulate the spinal cord. These studies will significantly advance our understanding of how to generate the full repertoire of neural subtypes necessary to repair the spinal cord after injury, specifically permitting patients to recover sensations such as pain and temperature. Moreover, they also represent a source of therapeutically beneficial cells for modeling debilitating diseases, such as the chronic insensitivity to pain.

Statement of Benefit to California: 

Millions of Californians live with compromised nervous systems, damaged by either traumatic injury or disease. These conditions can be devastating, stripping patients of their ability to move, feel and think, and currently have no cure. As well as being debilitating for patients, living with these diseases is also extremely expensive, costing both Californians and the state of California many billions of dollars. For example, the estimated lifetime cost for a single individual managing spinal paralysis is estimated to be up to $3 million.

Stem cell technology offers tremendous hope for reversing or ameliorating both disease and injury states. Stem cells can be used to replenish any tissue damaged by injury or disease, including the spinal cord, which is particularly vulnerable to physical damage. Our proposed studies will develop the means to produce the spinal sensory neurons that permit us to perceive the environment. We will also determine whether these in vitro derived sensory neurons are suitable for transplantation back into the spinal cord. The generation of these neurons will constitute an important step towards reversing or ameliorating spinal injuries, and thereby improve the productivity and quality of life of many Californians. Moreover, progress in this field will solidify the leadership role of California in stem cell research and stimulate the future growth of the biotechnology and pharmaceutical industries within the state.

Progress Report: 
  • A promising strategy to treat neurodegenerative diseases is to use embryonic stem cell (ESC)-derived neurons to replace damaged or diseased populations of neurons. In our CIRM-funded studies, we proposed to establish a protocol that will derive spinal sensory interneurons (INs) from ESCs. These INs are required to reestablish the sensory connections that would allow an injured patient to perceive external stimuli, such as pain and temperature. The existence of in vitro derived spinal sensory INs would also accelerate studies examining the basis of debilitating spinal dysfunctions, such as congenital pain insensitivity.
  • We have made significant progress towards this goal in year 1. We have identified that specific members of the Bone Morphogenetic Protein (BMP) family can direct mouse ESCs towards specific classes of spinal INs. These signals appear to be evolutionally conserved: our preliminary results suggest that BMPs have the same activity directing human ESCs towards spinal sensory IN fates. We are thus well poised to initiate our proposed studies in year 2: assessing whether stem cell derived INs can integrate in the spinal cord.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-02129
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$1 382 400
Disease Focus: 
Autism
Neurological Disorders
Rett's Syndrome
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 

Stem cells, including human embryonic stem cells, provide extraordinary new opportunities to model human diseases and may serve as platforms for drug screening and validation. Especially with the ever-improving effective and safe methodologies to produce genetically identical human induced pluripotent stem cells (iPSCs), increasing number of patient-specific iPSCs will be generated, which will enormously facilitate the disease modeling process. Also given the advancement in human genetics in defining human genetic mutations for various disorders, it is becoming possible that one can quickly start with discovery of disease-related genetic mutations to produce patient-specific iPSCs, which can then be differentiated into the right cell type to model for the disease in vitro, followed by setting up the drug screening paradigms using such disease highly relevant cells. In the context of neurological disorders, both synaptic transmission and gene expression can be combined for phenotyping and phenotypic reversal screening and in vitro functional (synaptic transmission) reversal validation. The missing gap for starting with the genetic mutation to pave the way to drug discovery and development is in vivo validation-related preclinical studies. In order to fill this gap, in this application we are proposing to use Rett syndrome as a proof of principle, to establish human cell xenografting paradigm and perform optogenetics and in vivo recording or functional MRI (fMRI), to study the neurotransmission/connectivity characteristics of normal and diseased human neurons. Our approach will be applicable to many other human neurological disease models and will allow for a combination of pharmacokinetic, and in vivo toxicology work together with the in vivo disease phenotypic reversal studies, bridging the gap between cell culture based disease modeling and drug screening to in vivo validation of drug candidates to complete the cycle of preclinical studies, paving the way to clinical trials. A success of this proposed study will have enormous implications to complete the path of using human pluripotent stem cells to build novel paradigms for a complete drug development process.

Statement of Benefit to California: 

Rett Syndrome (RTT) is a progressive neurodevelopmental disorder caused by primarily loss-of-function mutations in the X-linked MeCP2 gene. It mainly affects females with an incidence of about 1 in 10,000 births. After up to 18 months of apparently normal development, children with RTT develop severe neurological symptoms including motor defects, mental retardation, autistic traits, seizures and anxiety. RTT is one of the Autism Spectrum Disorders (ASDs) that affects many children in California. In this application, we propose to use our hESC-based Rett syndrome (RTT) model as a proof-of-principle case to define a set of core transcriptome that can be used for drug screenings. Human embryonic stem cells (hESCs) hold great potential for cell replacement therapy where cells are lost due to disease or injury. For the diseases of the central nervous system, hESC-derived neurons could be used for repair. This approach requires careful characterization of hESCs prior to utilizing their therapeutic potentials. Unfortunately, most of the characterization of hESCs are performed in vitro when disease models are generated using hESC-derived neurons. In this application, using RTT as a proof of principle study, we will bridge the gap and perform in vivo characterization of transplanted normal and RTT human neurons. Our findings will not only benefit RTT and other ASD patients, but also subsequently enable broad applications of this approach in drug discovery using human pluripotent stem cell-based disease models to benefit the citizens of California in a broader spectrum.

Progress Report: 
  • The potential of stem cells, such as human embryonic stem cells and induced pluripotent stem cells (iPSCs), has been widely recognized for cell replacement therapy, modeling human diseases and serving as a platform for drug screening and validation. In this grant, we proposed to use Rett syndrome as a proof of principle, to establish a human cell xenografting paradigm (i.e., transplanting human cells into mouse/rat embryos) and perform in vivo analyses to study the neurotransmission characteristics of normal and diseased human neurons. We initially determined that it was feasible to use the lentiviral CamKII-ChR2 construct to drive excitatory neuronal-specific expression of ChR2 in mouse hippocampal pyramidal neurons as well as human embryonic stem cell derived neurons. Importantly, we have found that both ChR2 expressing mouse hippocampal neurons and human neurons derived from embryonic stem cells can spike action potentials when stimulated in vitro, indicating that exogenously expressed ChR2 is functional. Furthermore, we successfully transplanted human embryonic stem cell derived neural stem/progenitor cells into fetal rat forebrain at embryonic day 17. Our analysis of the recipient animals at postnatal day 21 showed that approximately 40-50% of the cells survived and began to express neuronal markers, such as NeuN, indicating the neuronal differentiation, as well as the long-term survival, of transplanted human cells in the recipient animals. As originally proposed, we will proceed with the documentation of the in vivo phenotype of Rett syndrome diseased neurons. Our approach will be particularly crucial to not only validate candidate drugs or other therapeutic interventions to treat Rett syndrome using xeno-transplanted human Rett neurons, but also to study the in vivo behavior of those neurons with and without the therapeutic intervention.
  • Stem cells, such as human embryonic stem cells and induced pluripotent stem cells (iPSCs), carry great potentials for cell replacement therapy, human diseases modeling and drug screenings. We proposed to use Rett syndrome (RTT) as a proof of principle, to establish a human cell xenografting paradigm (i.e., transplanting human cells into mouse/rat brains) to study the function of normal and diseased human neurons in vivo. During the 2nd year of funding, we gained new insights into the electrophysiological characteristics of RTT neurons. Specifically, we found that the neurotransmission phenotype of neurons derived from RTT patient-specific iPSCs was highly circuitry-dependent. On the other hand, when cell-intrinsic electrophysiological properties were measured, extremely stable abnormalities in action potential profiles, resting membrane potentials, etc. were observed, indicative of the validity of the culture system. Given that currently scientists have very limited control over the features of neuronal connections formed in culture conditions, our findings make the in vivo assessment of RTT neuronal properties even more desirable, because the circuitry features are more amenable in vivo, with anatomical cues. In light of aforementioned in vitro findings, we focused our attention to both cell-intrinsic electrophysiological characteristics of RTT neurons, as well as their connectivity or neural network properties, after neurons were integrated into host circuits in vivo following xenotransplantation. Our preliminary data demonstrated that the action-potential abnormalities of RTT neurons are preserved in vivo after xenotransplantation. So far we have established a relatively optimized system for studying human iPSC-derived RTT neurons integrated into mouse brains. We are poised to uncover not only the neuronal intrinsic electrophysiological properties but also the connectivity of wild type and RTT neurons with host circuits. Moreover, we have made substantial progress with regards to a novel technology, i.e., single neuron gene expression profiling coupled with electrophysiological recordings both in vitro and in vivo. Up to now, 8 RTT iPSC-derived neurons were profiled via RNA sequencing following electrophysiological recordings, and some interesting clues have already been revealed. Currently we are collecting more neurons and we expect to make unprecedented discoveries with mechanistic insights into RTT disease pathophysiology, which will facilitate the development of novel therapies for RTT. This paradigm is also generally applicable for studying other neurological disorders.
  • Over the last decade, the importance of the stem cells for cell replacement therapy, human disease modeling and drug toxicity/therapy screenings has been greatly appreciated by both the general public and the scientific community. In our application utilizing human embryonic stem cells and induced pluripotent stem cells (iPSCs), we proposed to use Rett syndrome (RTT) as a proof of principle to establish a human cell xenografting paradigm (i.e., transplanting human cells into mouse/rat brains) to study the function of normal and diseased human neurons in vivo. While we increased our knowledge about the electrophysiological characteristics of RTT neurons during Year 2 funding, we mainly focused on the transplantation of the normal and diseased cells, as well as the molecular signatures of transplanted cells at a single cell level, during Year 3 of the funding period. Following our initial transplantation experiments, we observed clustering of the transplanted cells at the injection site, even though there were number of cells integrating into the host brains. In order to circumvent this problem and answer our original questions, we developed an alternative approach. Specifically, we adopted the “transparent brain” methodology to better visualize the integration and the projections of the transplanted cells, as well as the circuitries that they participate, in the host environment to reveal the connectivity of wild type and RTT neurons with the host circuits. With this method, we’re able to follow the transplanted RTT neurons at a higher resolution -without the limitations of the conventional approaches- for studying human iPSC-derived RTT neurons integrated into mouse brains. As part of our last Specific Aim, we’ve performed single neuron gene expression profiling coupled with electrophysiological recordings both in vitro and in vivo. Specifically, we implemented electrophysiological recordings from the transplanted RTT iPSC-derived neurons and isolated the genomic material from the same cell to perform transcriptome analyses. We collected significant amount of data from RNA sequencing experiments and have been performing relevant bioinformatic analyses. In order to complete the gene expression profiling analysis, we obtained a no-cost-extension of the project, and upon completion of the no-cost-extension period, the relevant report will be filed outlining the outcomes of the single neuron transcriptome analysis coupled with electrophysiology. Collectively, our findings provide mechanistic insights into RTT disease pathophysiology, which will facilitate the development of novel therapies for RTT. Lastly, our approach is applicable for studying other neurological disorders in addition to RTT.
Funding Type: 
Disease Team Therapy Development - Research
Grant Number: 
DR2A-05416
Investigator: 
Institution: 
Type: 
PI
Type: 
Co-PI
ICOC Funds Committed: 
$20 000 000
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Closed
Public Abstract: 

Alzheimer’s disease (AD), the leading cause of dementia, results in profound loss of memory and cognitive function, and ultimately death. In the US, someone develops AD every 69 seconds and there are over 5 million individuals suffering from AD, including approximately 600,000 Californians. Current treatments do not alter the disease course. The absence of effective therapies coupled with the sheer number of affected patients renders AD a medical disorder of unprecedented need and a public health concern of significant magnitude. In 2010, the global economic impact of dementias was estimated at $604 billion, a figure far beyond the costs of cancer or heart disease. These numbers do not reflect the devastating social and emotional tolls that AD inflicts upon patients and their families. Efforts to discover novel and effective treatments for AD are ongoing, but unfortunately, the number of active clinical studies is low and many traditional approaches have failed in clinical testing. An urgent need to develop novel and innovative approaches to treat AD is clear.

We propose to evaluate the use of human neural stem cells as a potential innovative therapy for AD. AD results in neuronal death and loss of connections between surviving neurons. The hippocampus, the part of the brain responsible for learning and memory, is particularly affected in AD, and is thought to underlie the memory problems AD patients encounter. Evidence from animal studies shows that transplanting human neural stem cells into the hippocampus improves memory, possibly by providing growth factors that protect neurons from degeneration. Translating this approach to humans could markedly restore memory and thus, quality of life for patients.

The Disease Team has successfully initiated three clinical trials involving transplantation of human neural stem cells for neurological disorders. These trials have established that the cells proposed for this therapeutic approach are safe for transplantation into humans. The researchers in this Disease Team have shown that AD mice show a dramatic improvement in memory skills following both murine and human stem cell transplantation. With proof-of-concept established in these studies, the Disease Team intends to conduct the animal studies necessary to seek authorization by the FDA to start testing this therapeutic approach in human patients.

This project will be conducted as a partnership between a biotechnology company with unique experience in clinical trials involving neural stem cell transplantation and a leading California-based academic laboratory specializing in AD research. The Disease Team also includes expert clinicians and scientists throughout California that will help guide the research project to clinical trials. The combination of all these resources will accelerate the research, and lead to a successful FDA submission to permit human testing of a novel approach for the treatment of AD; one that could enhance memory and save lives.

Statement of Benefit to California: 

The number of AD patients in the US has surpassed 5.4 million, and the incidence may triple by 2050. Roughly 1 out of every 10 patients with AD, over 550,000, is a California resident, and alarmingly, because of the large number of baby-boomers that reside in this state, the incidence is expected to more than double by 2025. Besides the personal impact of the diagnosis on the patient, the rising incidence of disease, both in the US and California, imperils the federal and state economy.

The dementia induced by AD disconnects patients from their loved ones and communities by eroding memory and cognitive function. Patients gradually lose their ability to drive, work, cook, and carry out simple, everyday tasks, ultimately losing all independence. The quality of life for AD patients is hugely diminished and the burden on their families and caregivers is extremely costly to the state of California. Annual health care costs are estimated to exceed $172 billion, not including the additional costs resulting from the loss of income and physical and emotional stress experienced by caregivers of Alzheimer's patients. Given that California is the most populous state and the state with the highest number of baby-boomers, AD’s impact on California families and state finances is proportionally high and will only increase as the AD prevalence rises.

Currently, there is no cure for AD and no means of prevention. Most approved therapies address only symptomatic aspects of AD and no disease-modifying approaches are currently available. By enacting Proposition 71, California voters acknowledged and supported the need to investigate the potential of novel stem cell-based therapies to treat diseases with a significant unmet medical need such as AD.

In a disease like AD, any therapy that exerts even a modest impact on the patient's ability to carry out daily activities will have an exponential positive effect not only for the patients but also for their families, caregivers, and the entire health care system. We propose to evaluate the hypothesis that neural stem cell transplantation will delay the progression of AD by slowing or stabilizing loss of memory and related cognitive skills. A single, one-time intervention may be sufficient to delay progression of neuronal degeneration and preserve functional levels of memory and cognition; an approach that offers considerable cost-efficiency.

The potential economic impact of this type of therapeutic research in California could be significant, and well worth the investment of this disease team proposal. Such an approach would not only reduce the high cost of care and improve the quality of life for patients, it would also make California an international leader in a pioneering approach to AD, yielding significant downstream economic benefits for the state.

Progress Report: 
  • Alzheimer’s disease (AD), the leading cause of dementia, results in profound loss of memory and cognitive function, and ultimately death. In the US, someone develops AD every 69 seconds and there are over 5 million individuals suffering from AD, including approximately 600,000 Californians. Current treatments do not alter the disease course. The absence of effective therapies coupled with the sheer number of affected patients renders AD a medical disorder of unprecedented need and a public health concern of significant magnitude. In 2010, the global economic impact of dementias was estimated at $604 billion, a figure far beyond the costs of cancer or heart disease. These numbers do not reflect the devastating social and emotional tolls that AD inflicts upon patients and their families. Efforts to discover novel and effective treatments for AD are ongoing, but unfortunately, the number of active clinical studies is low and many traditional approaches have failed in clinical testing. An urgent need to develop novel and innovative approaches to treat AD is clear.
  • We have proposed to evaluate the use of human neural stem cells as a potential innovative therapy for AD. AD results in neuronal death and loss of connections between surviving neurons. The hippocampus, the part of the brain responsible for learning and memory, is particularly affected in AD, and is thought to underlie the memory problems AD patients encounter. Evidence from previous animal studies shows that transplanting human neural stem cells into the hippocampus improves memory, possibly by providing growth factors that protect neurons from degeneration. Translating this approach to humans could markedly restore memory and thus, quality of life for patients.
  • In the first year of the loan, the Disease Team actively worked on 5 important milestones in our effort to develop the use of human neural stem cells for AD. Of those, 2 milestones have been completed and 3 are ongoing. Specifically, the team has initiated three animal studies believed necessary to seek authorization by the FDA to start testing this therapeutic approach in human patients; these studies were designed to confirm that transplantation of the neural stem cells leads to improved memory in animal models relevant for AD. We are currently collecting and analyzing the data generated in these mouse studies. We have also identified the neural stem cell line that will be used in patients and have made considerable progress in its manufacturing and banking. Finally, we have held a pre-IND meeting with the FDA in which we shared our plans for the preclinical and clinical studies; the meeting provided helpful guidance and assurances regarding our IND enabling activities.
  • This project is a partnership between a biotechnology company with unique experience in clinical trials involving neural stem cell transplantation and a leading California-based academic laboratory specializing in AD research. Together with expert clinicians and scientists throughout California, we continue to work towards a successful IND submission to permit human testing of a novel and unique approach for the treatment of AD.
  • Alzheimer’s disease (AD), the leading cause of dementia, results in profound loss of memory and cognitive function, and ultimately death. In the United States, someone develops AD every 69 seconds and there are over 5 million individuals suffering from AD, including approximately 600,000 Californians. Current treatments do not alter the disease course. The absence of effective therapies coupled with the sheer number of affected patients renders AD a medical disorder of unprecedented need and a public health concern of significant magnitude. Efforts to discover effective treatments for AD are ongoing, but unfortunately, the number of active clinical studies is low and many traditional approaches have failed in clinical testing. An urgent need to develop novel and innovative approaches to treat AD is urgent.
  • StemCells Inc., proposed to evaluate the use of human neural stem cells as a potential innovative therapy for AD. AD results in neuronal death and loss of connections between surviving neurons. The hippocampus, the part of the brain responsible for learning and memory, is particularly affected in AD. Evidence from previous animal studies shows that transplanting human neural stem cells into the hippocampus improves memory, possibly by providing growth factors that protect neurons from degeneration. Translating this approach to humans could markedly restore memory and thus, quality of life for patients.
  • In September 2012, the CIRM awarded a loan to StemCells Inc. to partially fund a program to test human neural stem cells in two animal models used by some researchers to study AD and the study was initiated in July of 2013. The goal of this study was chiefly to try to replicate earlier successful experiments with human neural stem cells in these mice in support of an IND filing with the U.S. FDA within four years.
  • In the first year of the study, the Disease Team actively worked on 5 important scientific milestones in our effort to develop human neural stem cells as a potential therapy for AD. We also held a pre-IND meeting with the FDA in which we shared our plans for the preclinical and clinical studies in AD; the meeting provided helpful guidance and assurances regarding our IND enabling activities.
  • As of the second year of the study, all of the first 5 scientific milestones have been completed. Specifically, the team conducted three animal studies believed necessary to start testing this therapeutic approach in human patients; these studies were designed to confirm that transplantation of the neural stem cells leads to improved memory in animal models relevant for AD.
  • Despite seeing a very exciting increase in the number of connections between key hippocampal neurons within the brains of mice treated with human neural stem cells, this did not appear to robustly and consistently improve memory in the animals. Without seeing a significant change in memory performance, the preclinical results of the study did not satisfy one or more of the specific “No/No Go” scientific milestones agreed to with the CIRM. Given this, the loan was subsequently terminated in December 2014 as a consequence of the unanticipated preclinical results.
  • This study was a partnership between a biotechnology company with unique experience in clinical trials involving neural stem cell transplantation and a leading California-based academic laboratory specializing in AD research. Although disappointing, the results of this study do not negate the potential of neural stem cell transplantation in AD; rather, having reviewed and discussed the data with our collaborators, we believe the data highlight the challenge of obtaining reliable and consistent behavior readouts of memory improvement in animals. Finally, the observed increases in the connections between hippocampal neurons are very interesting and may justify further efforts to improve pre-clinical development for this complex disorder.

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