Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders

Banking transplant ready dopaminergic neurons using a scalable process

Funding Type: 
Early Translational II
Grant Number: 
TR2-01856
ICOC Funds Committed: 
$6 016 624
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Collaborative Funder: 
Maryland
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons (a type of nerve cells in the central nervous system) present in the midbrain. These neurons secrete dopamine (a signaling molecule) and are a critical component of the motor circuit that ensures movements are smooth and coordinated. All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy (that is, transplantation of dopaminergic neurons into the brain to replace lost cells and restore function) as proposed in this application attempts to use cells as small pumps of dopamine that will be secreted locally and in a regulated way, and will therefore avoid the complications of other modes of treatment. Indeed, cell therapy using fetal tissue-derived cells have been shown to be successful in multiple transplant studies. Work in the field has been limited however, partially due to the limited availability of cells for transplantation (e.g., 6-10 fetuses of 6-10 weeks post-conception are required for a single patient). We believe that human embryonic stem cells (hESCs) may offer a potentially unlimited source of the right kind of cell required for cell replacement therapy. Work in our laboratories and in others has allowed us to develop a process of directing hESC differentiation into dopaminergic neurons. To move forward stem cell-based therapy development it is important to develop scale-up GMP-compatible process of generating therapeutically relevant cells (dopaminergic neurons in this case). The overall goal of this proposal is to develop a hESC-based therapeutic candidate (dopaminergic neurons) by developing enabling reagents/tools/processes that will allow us to translate our efforts into clinical use. We have used PD as a model but throughout the application have focused on generalized enabling tools. The tools, reagents and processes we will develop in this project will allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects. In addition, the processes we will develop would be of benefit to the CIRM community.
Statement of Benefit to California: 
Parkinson’s disease affects more than a million patients United States with a large fraction being present in California. California, which is the home of the Parkinson’s Institute and several Parkinson’s related foundations and patient advocacy groups, has been at the forefront of this research and a large number of California based scientists supported by these foundations and CIRM have contributed to significant breakthroughs in this field. In this application we and our collaborators in California aim propose to develop a hESC-based therapeutic candidate (dopaminergic neurons) that will allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects for this currently non-curable disorder. We believe that this proposal includes the basic elements that are required for the translation of basic research to clinical research. We believe these experiments not only provide a blueprint for moving Parkinson’s disease towards the clinic for people suffering with the disorder but also a generalized blueprint for the development of stem cell therapy for multiple neurological disorders including motor neuron diseases and spinal cord injury. The tools and reagents that we develop will be made widely available to Californian researchers. We expect that the money expended on this research will benefit the Californian research community and the tools and reagents we develop will help accelerate the research of our colleagues in both California and worldwide.
Progress Report: 
  • Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons (a type of nerve cells in the central nervous system) present in the midbrain. These neurons secrete dopamine (a signaling molecule) and are a critical component of the motor circuit that ensures movements are smooth and coordinated.
  • All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy (that is, transplantation of dopaminergic neurons into the brain to replace lost cells and restore function) as proposed in this application attempts to use cells as small pumps of dopamine that will be secreted locally and in a regulated way, and will therefore avoid the complications of other modes of treatment. Indeed, cell therapy using fetal tissue-derived cells have been shown to be successful in multiple transplant studies. Work in the field has been limited however, partially due to the limited availability of cells for transplantation (e.g., 6-10 fetuses of 6-10 weeks post-conception are required for a single patient).
  • We believe that human embryonic stem cells (hESCs) may offer a potentially unlimited source of the right kind of cell required for cell replacement therapy. Work in our laboratories and in others has allowed us to develop a process of directing hESC differentiation into dopaminergic neurons. To move forward stem cell-based therapy development it is important to develop scale-up GMP-compatible process of generating therapeutically relevant cells (dopaminergic neurons in this case).
  • The overall goal of this proposal is to develop a hESC-based therapeutic candidate (dopaminergic neurons) by developing enabling reagents/tools/processes that will allow us to translate our efforts into clinical use. We have used PD as a model but throughout the application have focused on generalized enabling tools. The tools, reagents and processes we will develop in this project will allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects. In addition, the processes we will develop would be of benefit to the CIRM community.
  • Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons (a type of nerve cells in the central nervous system) present in the midbrain. These neurons secrete dopamine (a signaling molecule) and are a critical component of the motor circuit that ensures movements are smooth and coordinated.
  • All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy (that is, transplantation of dopaminergic neurons into the brain to replace lost cells and restore function) as proposed in this application attempts to use cells as small pumps of dopamine that will be secreted locally and in a regulated way, and will therefore avoid the complications of other modes of treatment. Indeed, cell therapy using fetal tissue-derived cells have been shown to be successful in multiple transplant studies. Work in the field has been limited however, partially due to the limited availability of cells for transplantation (e.g., 6-10 fetuses of 6-10 weeks post-conception are required for a single patient).
  • We believe that human pluripotent stem cells (PSC) may offer a potentially unlimited source of the right kind of cell required for cell replacement therapy. Work in our laboratories and in others has allowed us to develop a process of directing PSC differentiation into dopaminergic neurons. To move forward stem cell-based therapy development it is important to develop scale-up GMP-compatible process of generating therapeutically relevant cells (dopaminergic neurons in this case).
  • During this grant, we have optimized a step-wise scalable process for generating authentic dopaminergic neurons in defined media from human PSC, and have determined the time point at which dopaminergic neurons can be frozen, shipped, thawed and transplanted without compromising their ability to mature and provide therapeutic benefit in animal models. Our process has been successfully transferred to a GMP facility and we have manufactured multiple lots of GMP-equivalent cells using this process. Importantly, we have shown functional equivalency of the manufactured cells in appropriate models. The tools, reagents and processes we have developed in this project allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects. In addition, the processes we have developed would be of benefit to the CIRM community.
  • CIRM Progress Report Part A: Scientific Progress
  • I. Project Overview
  • During the past three years (36 months) we have successfully completed the milestones defined in the NGA for this grant. In brief, we have selected 1 clinically compliant ESC line H14 (and a back-up line H9), which have shown reproducible, efficient differentiation to dopaminergic neurons at lab scale. We have performed in vitro and in vivo characterization as defined in the NGA and guided by our discussion with our program officer at CIRM. We have determined the time point at which dopaminergic precursors (14 days after the NSC stage) can be frozen, shipped, thawed and transplanted without compromising their ability to mature and provide therapeutic benefit in animal models. Importantly, we have evaluated efficacy of cryopreserved dopaminergic precursors manufactured by the GMP-compatible process in a rodent PD model and shown functional recovery up to 6 months post transplantation as well as survival of dopaminergic neurons.
  • In the meanwhile we have successfully transferred the process of generating transplant ready dopaminergic neurons to the manufacture facilities at City of Hope (COH). They have adapted and optimized our protocols and have established GMP-compatible protocols for the culture of ESC-NSC and for differentiating NSC to Stage 3, Day 14 DA precursors for transplantation. During this reporting period (36 month), we have tested the equivalency of these lots and confirmed that lots manufactured at COH are consistent and similar to cells produced in the laboratory.
  • Our effort resulted in two important manuscripts in Cytotherapy:
  • 1. Liu, Q., Pedersen, OZ., Peng, J., Couture, LA., Rao, MS., and Zeng, X. Optimizing dopaminergic differentiation of pluripotent stem cells for the manufacture of dopaminergic neurons for transplantation. Cytotherapy. 2013 Aug;15(8):999-1010.
  • 2. Peng, J., Liu, Q., Rao, MS., and Zeng, X. Survival and engraftment of dopaminergic neurons manufactured by a GMP-compatible process. Cytotherapy. 2014 Sep;16(9):1305-12.

New Drug Discovery for SMA using Patient-derived Induced Pluripotent Stem Cells

Funding Type: 
Early Translational II
Grant Number: 
TR2-01844
Investigator: 
ICOC Funds Committed: 
$5 665 887
Disease Focus: 
Spinal Muscular Atrophy
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Spinal muscular atrophy (SMA) is the leading genetic cause of infant death in the U.S. This devastating disease affects 1 child in every 6,000-10,000 live births, with a North American prevalence of approximately 14,000 individuals. The disease is characterized by the death of spinal cord cells called motor neurons that connect the brain to muscle. Death of these cells causes muscle weakness and atrophy, which progresses to paralysis, respiratory failure and frequently death. The three different types of SMA differ in severity and prognosis, with Type I being the most severe. SMA is caused by a genetic defect that leads to reduced levels of a single protein called SMN. There are currently no approved therapies for the disease. The existing treatments for SMA consist of supportive care for the respiratory and nutritional deficits, for example ventilation and feeding tubes. Previous attempts to develop drugs using conventional technologies, such as cultured cancer cells or cells derived from animals have been unsuccessful. These failures are likely due the fact that previous attempts used cell types that don’t reflect the disease or aren’t affected by low levels of the SMN protein. Our approach uses patient-derived motor neurons, the specific cell type that dies. We will conduct drug discovery experiments using these motor neurons to find potential therapeutics that increase the levels of the SMN protein in these diseased cells. Induced pluripotent stem cell (iPSC) technology allows us to take skin cells from patients with SMA, grow them in a dish, and turn them into motor neurons. We are conducting high-throughput screens of potential drugs with these cells to identify drug candidates that increase SMN protein levels in motor neurons derived from SMA patients. An added advantage to our approach is that we can test our drug candidates in motor neurons from many different patients, with different disease subtypes and from different ethnic backgrounds. We have generated iPSCs from many patients with SMA and we will test compounds for effectiveness against this cohort. These studies will give us an indication of the effectiveness of our compounds across patients before moving into costly and lengthy clinical trials. If our drug candidate is successful, it could be the first effective therapeutic available for SMA. It will increase the amount of SMN protein and prevent motor neuron death. Halting the death of spinal cord motor neurons prevents the progressive weakness and muscle atrophy. We anticipate that this would prevent disability in Type III patients. For Type I and II patients, we believe such a therapy would mitigate respiratory and feeding challenges and allow lifespan increase. The sponsoring institution has integrated iPSC-based drug discovery capabilities, ranging from stem cell line production, high throughput drug screening and medicinal chemistry. Accordingly, this institution is uniquely positioned to achieve the aims of this grant.
Statement of Benefit to California: 
Spinal muscular atrophy (SMA) is the second-most common autosomal-recessive disorder and leading genetic cause of death of infants in the U.S. We estimate that there are up to 1,500 SMA patients currently living in California, with 100 new cases diagnosed in California every year. The CIRM Early Translational II Awards is intended to fund studies that will propel drug discovery forward for many devastating diseases. In keeping with this mission, we propose to leverage iPSC technology to generate disease-relevant cell types from patients themselves for a high throughput drug screen. A successful therapy for SMA would lead to significant cost savings to California’s health care system, and would provide relief to families of patients with this devastating disorder. Given that there are not many successful drugs in the making for neurological diseases such as ALS, SMA, Parkinson’s disease or Alzheimer’s disease, our project should significantly impact drug discovery in this area by introducing iPSC technologies as a valid drug discovery and development platform. The application of iPSC-based disease modeling and drug discovery to SMA is highly innovative and represents the opportunity to establish worldwide leadership for California in this emerging field. Furthermore, the sponsoring institution will fund over 70% of the direct costs during the timeframe of this award. Accordingly, the 3:1 leverage provides great opportunity to magnify the effect of a CIRM award. Our research program will also create new, high-paying jobs in California, and will stimulate California’s economy by creating new research and clinical tools. These activities will continue to strengthen California’s leadership position at the forefront of the stem cell and regenerative medical revolution of the 21st century.
Progress Report: 
  • Spinal muscular atrophy (SMA) is the leading genetic cause of infant death in the U.S. This devastating disease affects 1 child in every 6,000-10,000 live births, with a North American prevalence of approximately 14,000 individuals. The disease is characterized by the death of spinal cord cells called motor neurons that connect the brain to muscle. Death of these cells causes muscle weakness and atrophy, which progresses to paralysis, respiratory failure and frequently death. The three different types of SMA differ in severity and prognosis, with Type I being the most severe. SMA is caused by a genetic defect that leads to reduced levels of a single protein called SMN. There are currently no approved therapies for the disease.
  • Existing treatments for SMA consist of supportive care for the respiratory and nutritional deficits, for example ventilation and feeding tubes. Previous attempts to develop drugs using conventional technologies, such as cultured cancer cells or cells derived from animals have been unsuccessful. These failures are likely due to the fact that previous attempts used cell types that do not reflect the disease or are not affected by low levels of the SMN protein. Our approach uses patient-derived motor neurons, the specific cell type that dies in SMA.
  • An added advantage to our approach is that we can test our drug candidates in motor neurons from many different patients and different disease subtypes. We have generated iPSCs from many patients with SMA and we will test compounds for effectiveness against this cohort. These studies will give us an indication of the effectiveness of our compounds across patients before moving into costly and lengthy clinical trials. It will increase the amount of SMN protein and prevent motor neuron death. Halting the death of spinal cord motor neurons prevents the progressive weakness and muscle atrophy. We anticipate that this would prevent disability in Type III patients. For Type I and II patients, we believe such a therapy would mitigate respiratory and feeding challenges and allow an increase in lifespan.
  • In the past year, we conducted drug discovery experiments using these motor neurons to find potential therapeutics that increase the levels of the SMN protein in these diseased cells. Induced pluripotent stem cell (iPSC) technology allows us to take skin cells from patients with SMA, grow them in a dish, and turn them into SMA motor neurons. We conducted high-throughput screens of potential drugs with these cells to identify drug candidates that increase SMN protein levels in motor neurons derived from SMA patients. Despite the high quality of these screens, no suitable drug candidate was identified. We have modified our strategy and developed a method to identify, in parallel, all targets in the “druggable” genome that regulate SMN protein levels. An exhaustive screen currently is being performed to identify such a target and will be completed by end April 2012. Once a target is identified, it will be developed into a lead and validated in animals.

Developing a therapeutic candidate for Canavan disease using induced pluripotent stem cell

Funding Type: 
Early Translational II
Grant Number: 
TR2-01832
ICOC Funds Committed: 
$1 835 983
Disease Focus: 
Genetic Disorder
Neurological Disorders
Pediatrics
Collaborative Funder: 
Germany
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Canavan disease is a devastating disease of infants which affects their neural development and leads to mental retardation and early death. It occurs in 1 in 6,400 persons in the U.S. and there is no treatment so far. We propose to generate genetically-repaired and patient-specific stem cells (called iPSCs) from patients’ skin cells, and then coax these stem cells into specific types of corrective neural precursors using methods established in our laboratories in order to develop a therapeutic candidate for this disease. By use of a mouse model of Canavan disease, we will determine the ability of these genetically corrected cells to successfully treat the disease. These results will form the basis for an eventual clinical trial in humans, and if successful, would be the first treatment for this terrible disease. There are many families affected by this disease, and other diseases similar to it. Results from this work could have applications to this and other similar genetic diseases. Through the proposed research, maybe no parents will have to watch their child suffer and die as a result of these dreadful diseases in one day. What a wonderful day that would be!
Statement of Benefit to California: 
It is estimated that California has ~12% of all cases of Canavan disease in the U.S. Besides the tremendous emotional and physical pain that this disease inflicts on families, it produces in California a medical and fiscal burden that is larger than any other states. Thus, there is a real need to develop a strategy of treatment for this disease. Stem cells provide great hope for the treatment of a variety of human diseases that affect the citizens of California. Combination of gene therapy and iPSC technology will enable the development of therapeutic candidates of human genetic diseases via the creation of genetically-corrected patient-specific iPSCs. Our proposal aims to establish a therapeutic development candidate for Canavan disease, a devastating neurodegenerative disease that leads to mental retardation and early death. The generation of genetically-repaired and patient-specific iPSC lines will represent great potential not only for California health care patients but also for pharmaceutical and biotechnology industries in California. Moreover, California is a strong leader in pre-clinical and clinical research developments. To maintain this position, we need to create patient-specific stem cells as autologous therapeutic candidates, in order to overcome the challenges of immune rejection faced by today’s cell therapy field. This proposal addresses the very issue by generating “disease-corrected” and patient-specific iPSCs as a therapeutic candidate with the potential to create safer and more effective cell replacement therapies.
Progress Report: 
  • Canavan disease is a devastating disease of infants which affects their neural development and leads to mental retardation and early death. It occurs in 1 in 6,400 persons in the U.S. and there is no treatment so far. We propose to generate genetically-repaired and patient-specific stem cells (called iPSCs) from patients’ skin cells, and then coax these stem cells into specific types of corrective neural precursors using methods established in our laboratories in order to develop a therapeutic candidate for this disease.
  • For the reporting period, we have obtained primary dermal fibroblasts from clinically affected Canavan disease patients and have derived Canavan disease patient iPSCs. We have demonstrated that these iPSCs exhibited typical human embryonic stem cell (ESC) like morphology, expressed human ESC cell surface markers and hold pluripotency potential. We are also optimizing methods to coax these cells into specific types of neural precursors. Either the patient iPSCs or their neural precursor derivatives will be genetically corrected in the following years to develop a therapeutic tool for Canavan disease patients.
  • There are many families affected by this disease, and other diseases similar to it. Results from this work could have applications to this and other similar genetic diseases. Through the proposed research, maybe no parents will have to watch their child suffer and die as a result of these dreadful diseases in one day.
  • Canavan disease is a devastating disease of infants which affects their neural development and leads to mental retardation and early death. It occurs in 1 in 6,400 persons in the U.S. and there is no treatment so far. We propose to generate genetically-repaired and patient-specific stem cells (called iPSCs) from patients’ skin cells, and then coax these stem cells into specific types of corrective neural precursors using methods established in our laboratories in order to develop a therapeutic candidate for this disease.
  • For the reporting period, we have demonstrated that the Canavan disease patient iPSCs hold pluripotency potential. We also genetically corrected the patient iPSCs and demonstrated that these genetically-corrected cells maintained human embryonic stem cell-like features. We coaxed these cells into specific types of neural precursors and showed that the genetically-corrected patient cells restored their cellular function. These genetically corrected cells will be tested for their therapeutic effect in the next year, in order to develop a therapeutic tool for Canavan disease patients.
  • There are many families affected by this disease, and other diseases similar to it. Results from this work could have applications to this and other similar genetic diseases. Through the proposed research, maybe no parents will have to watch their child suffer and die as a result of these dreadful diseases in one day.
  • Canavan disease is a devastating disease of infants which affects their neural development and leads to mental retardation and early death. It occurs in 1 in 6,400 persons in the U.S. and there is no treatment so far. We propose to generate genetically-repaired and patient-specific stem cells (called iPSCs) from patients’ skin cells, and then coax these stem cells into specific types of corrective neural precursors using methods established in our laboratories in order to develop a therapeutic candidate for this disease.
  • We have demonstrated that the Canavan disease patient iPSCs hold pluripotency potential. We also genetically corrected the patient iPSCs and demonstrated that these genetically-corrected cells maintained human embryonic stem cell-like features. We coaxed these cells into specific types of neural precursors and showed that the genetically-corrected patient cells restored their cellular function.
  • For the reporting period, we provided evidence that the genetically-corrected patient iPSC-derived neural precursors were able to produce myelin binding protein in an animal model. We also characterized the Canavan disease mice to show that they exhibited the characteristic Canavan disease patient phenotypes. The genetically corrected cells will be tested in Canavan disease mice for their therapeutic effect in the next funding period, in order to develop a therapeutic tool for Canavan disease patients.
  • There are many families affected by this disease, and other diseases similar to it. Results from this work could have applications to this and other similar genetic diseases. Through the proposed research, maybe no parents will have to watch their child suffer and die as a result of these dreadful diseases in one day.

Crosstalk: Inflammation in Parkinson’s disease (PD) in a humanized in vitro model

Funding Type: 
Early Translational II
Grant Number: 
TR2-01778
ICOC Funds Committed: 
$2 472 839
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Collaborative Funder: 
Germany
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Parkinson’s Disease (PD) is the most common neurodegenerative movement disorder. It is characterized by motor impairment such as slowness of movements, shaking and gait disturbances. Age is the most consistent risk factor for PD, and as we have an aging population, it is of upmost importance that we find therapies to limit the social, economic and emotional burden of this disease. Most of the studies to find better drugs for PD have been done in rodents. However, many of these drugs failed when tested in PD patients. One problem is that we can only investigate the diseased neurons of the brain after the PD patients have died. We propose to use skin cells from PD patients and reprogram these into neurons and other surrounding cells in the brain called glia. This is a model to study the disease while the patient is still alive. We will investigate how the glial surrounding cells affect the survival of neurons. We will also test drugs that are protective for glial cells and neurons. Overall, this approach is advantageous because it allows for the study of pathological development of PD in a human system. The goal of this project is to identify key molecular events involved at early stages in PD and exploit these as potential points of therapeutic intervention.
Statement of Benefit to California: 
The goal of this proposal is to create human cell-based models for neurodegenerative disease using transgenic human embryonic stem cells and induced pluripotent stem cells reprogrammed from skin samples of highly clinically characterized Parkinson’s Disease (PD) patients and age-matched controls. Given that age is the most consistent risk factor for PD, and we have an aging population, it is of utmost importance that we unravel the cellular, molecular, and genetic causes of the highly specific cell death characteristic of PD. New drugs can be developed out of these studies that will also benefit the citizens of the State of California. In addition, if our strategy can go into preclinical development, this approach would most likely be performed in a pharmaceutical company based in California.
Progress Report: 
  • In the first year of our CIRM Early Translational II Award we have largely accomplished the first two aims put forth in our proposal “Crosstalk: Inflammation in Parkinson’s disease (PD) in a humanized in vitro model.” Dr. Juergen Winkler, in Erlangen, Germany, has enrolled 10 patients and 6 controls in this project, most of which have had a biopsy of their skin cells sent to The Salk Institute in La Jolla. In Dr. Gage’s lab at The Salk Institute these patient fibroblasts are being reprogrammed into induced pluripotent stem cells (iPSCs), and initial attempts at differentiation into dopaminergic neurons are underway. Additionally, patient blood cells have been sent from Dr. Winkler’s clinic to the lab of Dr. Glass at UC San Diego, where their gene expression profile is being determined. In this initial reporting period we are successfully building the cellular tools necessary to investigate the role of nuclear receptors and inflammation in Parkinson’s Disease.
  • In the second year of our CIRM Early Translational II Award we are making substantial progress towards completing all three aims put forth in our proposal. Dr. Juergen Winkler, our German collaborator, has completed the patient recruitment phase of this project, and skin cells from all 16 subjects (10 with PD and six controls) have been reprogrammed into induced pluripotent stem cells (iPSCs) at the Salk Institute in La Jolla. The patient-specific iPSCs have been differentiated into well-characterized neural stem cells, which the Gage lab is further differentiating into both dopaminergic neurons and astrocytes. In addition to collecting patient skin cells, Dr. Winkler’s group has collected blood cells which are currently being analyzed for gene expression differences by Dr. Glass’ lab at UCSD using state-of-the-art RNA sequencing technology. We have identified a compound that is anti-inflammatory in human cells that we will test on the patient-specific cells once we finish building the cellular tools required to investigate the role of nuclear receptors and inflammation in Parkinson’s Disease.
  • In the final year of our CIRM Early Translational II Award we made considerable progress towards completing all three aims put forth in our proposal. Dr. Juergen Winkler, our German collaborator, has completed the patient recruitment phase of this project, and skin cells from all 16 subjects (10 with PD and six controls) have been reprogrammed into induced pluripotent stem cells (iPSCs) at the Salk Institute in La Jolla. The patient-specific iPSCs have been differentiated into well-characterized neural stem cells, which the Gage lab is further differentiating into both dopaminergic neurons and astrocytes. In addition to collecting patient skin cells, Dr. Winkler’s group has collected blood cells which are currently being analyzed for gene expression differences by Dr. Glass’ lab at UCSD using state-of-the-art RNA sequencing technology. We have identified a compound that is anti-inflammatory in human cells that can reduce inflammation in patient-specific cells, and we are beginning to look at its effects on neuronal survival. This award has allowed us to build the cellular tools required to investigate the role of nuclear receptors and inflammation in Parkinson’s disease, which is a model with endless potential.

Inhibitory Nerve Cell Precursors: Dosing, Safety and Efficacy

Funding Type: 
Early Translational II
Grant Number: 
TR2-01749
ICOC Funds Committed: 
$1 752 058
Disease Focus: 
Neurological Disorders
Epilepsy
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Many neurological disorders are characterized by an imbalance between excitation and inhibition. Our ultimate goal: to develop a cell-based therapy to modulate aberrant brain activity in the treatment of these disorders. Our initial focus is on epilepsy. In 20-30% of these patients, seizures are unresponsive to drugs, requiring invasive surgical resection of brain regions with aberrant activity. The candidate cells we propose to develop can inhibit hyperactive neural circuits after implantation into the damaged brain. As such, these cells could provide an effective treatment not just for epilepsy, but also for a variety of other neurological conditions like Parkinson's, traumatic brain injury, and spasticity after spinal cord injury. We propose to bring a development candidate, a neuronal cell therapy, to the point of preclinical development. The neurons that normally inhibit brain circuits originate from a region of the developing brain called the medial ganglionic eminence (MGE). When MGE cells are grafted into the postnatal or adult brain, they disperse seamlessly and form inhibitory neurons that modulate local circuits. This property of MGE cells has not been shown for any other type of neural precursor. Our recent studies demonstrate that MGE cells grafted into an animal model of epilepsy can significantly decrease the number and severity of seizures. Other "proof-of-principle" studies suggest that these progenitor cells can be effective treatments in Parkinson's. In a separate effort, we are developing methods to differentiate large numbers of human MGE (H-MGE) cells from embryonic stem (ES) cells. To translate this therapy to humans, we need to determine how many MGE cells are required to increase inhibition after grafting and establish that this transplantation does not have unwanted side-effects. In addition, we need simple assays and reagents to test preparations of H-MGE cells to determine that they have the desired migratory properties and differentiate into nerve cells with the expected inhibitory properties. At present, these issues hinder development of this cell-based therapy in California and worldwide. We propose: (1) to perform "dose-response" experiments using different graft sizes of MGE cells and determine the minimal amount needed to increase inhibition; (2) to test whether MGE transplantation affects the survival of host neurons or has unexpected side-effects on the behavior of the grafted animals; (3) to develop simple in vitro assays (and identify reagents) to test H-MGE cells before transplantation. Our application takes advantage of an established multi-lab collaboration between basic scientists and clinicians. We also have the advice of neurosurgeons, epilepsy neurologists and a laboratory with expertise in animal behavior. If a safe cell-based therapy to replace lost inhibitory interneurons can be developed and validated, then clinical trials in patients destined for invasive neurosurgical resections could proceed.
Statement of Benefit to California: 
This proposal is designed to accelerate progress toward development of a novel cell-based therapy with potentially broad benefit for the treatment of multiple neurological diseases. The potential to translate our basic science findings into a treatment that could benefit patients is our primary focus and our initial target disease is epilepsy. This work will provide benefits to the State of California in the following areas: * California epilepsy patients and patients with other neurological diseases will benefit from improved therapies. The number of patients refractory to available medications is significant: a recent report from the Center for Disease Control and Prevention [www.cdc.gov/epilepsy/] estimates that 1 out of 100 adults have epilepsy and up to one-third of these patients are not receiving adequate treatment. In California, it is one of the most common disabling neurological conditions. In most states, including California, epileptic patients whose seizures aren't well-controlled cannot obtain a driver's license or work certain jobs -- truck driving, air traffic control, firefighting, law enforcement, and piloting. The annual cost estimates to treat epilepsy range from $12 to $16 billion in the U.S. Current therapies curb seizures through pharmacological management but are not designed to modify brain circuits that are damaged or dysfunctional. The goals of our research program is to develop a novel cell-based therapy with the potential to eliminate seizures and improve the quality of life for this patient population, as well as decrease the financial burden to the patients' families, private insurers, and state agencies. Since MGE cells can mediate inhibition in other neurological and psychiatric diseases, the neural based therapy we are proposing is likely to have a therapeutic and financial impact that is much broader. * Technology transfer in California. Historically, California institutions have developed and implemented a steady flow of technology transfer. Based on these precedents and the translational potential of our research goals, both to provide bioassays and potentially useful markers to follow the differentiation of MGE cells, this program is likely to result in licensing of further technology to the corporate sector. This will have an impact on the overall competitiveness of our state's technology sector and the resulting potential for creation of new jobs. * Stem cell scientists training and recruitment in California. As part of this proposal we will train a student, technicians, and associated postdocs in MGE progenitor derivation, transplantation, and cell-based therapy for brain repair. Moreover, the translational nature of the disease-oriented proposal will result in new technology which we expect to be transferable to industry partners for facilitate development into new clinical alternatives.
Progress Report: 
  • Advances in stem cell research and regenerative medicine have led to the potential use of stem cell therapies for neurodegenerative, developmental and acquired brain disease. The Alvarez-Buylla lab at UCSF is part of a collaboration that is pioneering the investigation of therapeutic interneuron replacement for the correction of neurological disorders arising from defects in neural excitation/inhibition. Our preliminary data suggests that grafting interneuron precursors into the postnatal rodent brain allows for up to a 35% increase in the number of cortical interneurons. Interneuron replacement has been used in animal models to modify plasticity, prevent spontaneous epileptic seizures, ameliorate hemiparkinsonian motor symptoms, and prevent PCP-induced cognitive deficits. Transplantation of interneuron precursors therefore holds therapeutic potential for treatment of human neurological diseases involving an imbalance in circuit inhibition/excitation.
  • The goal of the research in progress here is to ultimately prepare human interneuron precursors for clinical trials. Towards the therapeutic development of inhibitory neuron precursor transplantation for human neurological disorders, we have made significant progress in the differentiation of these cells from human ESCs and will complete optimization of this protocol. We will continue our investigation of rodent-derived interneuron transplantation to obtain relevant preclinical data for dose response, safety and efficacy in animal models. These dosing and safety data will then serve as the baseline for comparison with human interneuron precursors and inform design of preclinical studies of these cells in immunosuppressed mice. Together, these data will provide essential information for developing a plan for clinical trials using human interneuron precursors.
  • During this first year, we have made considerable headway in the optimization of the human interneuron precursor differentiation protocol, verified functional engraftment of these cells in mice, and begun to collect dose, safety and efficacy data for rodent-derived interneuron transplantation. Importantly, we have achieved the development of a protocol that robustly generates interneuron-like progenitor cells from human ES cells and demonstrated that these progenitors mature in vitro and in vivo into GABAergic inhibitory interneurons with functional potential. We have also compared the behavior of primary fetal cells to these human interneuron precursor-like cells both in vivo and in vitro. As we continue to optimize our ES cell differentiation protocol, these primary interneuron precursors will enable initial human cell dose response and behavior experiments and, along with rodent-derived cells, will provide important baseline measures.
  • In sum, this work will provide essential knowledge for the therapeutic development of inhibitory neuron transplantation. The experiments underway will yield insights that will be critical to the development of a clinical trial using human interneuron precursors.
  • During the reporting period, we have developed methods to enable the optimization of inhibitory nerve precursor cell, or MGE cell, derivation from human pluripotent stem cells (hPSCs). Optimization encompassed increasing MGE cell motility, enhancing MGE cell maturation into inhibitory nerve subtypes, and elimination of tumors post-transplantation into the rodent brain. Furthermore, we demonstrated that the injected hPSC-MGE cells functionally matured into inhibitory nerves with advanced physiological properties that integrated into the rodent brain. In addition, we determined an optimum dose of injected mouse MGE cells in rodent. Moreover, following injection of either the optimum dose or a 10-fold higher dose of mouse MGE cells, we found no detectable behavioral side effects from MGE cell transplantation.
  • In this reporting period, we continued to improve the acquisition of migratory medial ganglionic eminence (MGE)-type interneurons from human embryonic stem cells (hESC). We compared alternative procedures by testing MGE marker expression, and we developed additional tests to measure cell division and migration of MGE cell made from hESCs. We also extended our methods to clinical-grade hESC lines. With these optimized procedures, both research and clinical grade lines were transplanted into the rodent brain. In addition, we completed an evaluation of human fetal MGE transplants after-injection into the rodent brain. Finally, we report safety, survival, and neuronal differentiation of both hESC-MGE and human Fetal-MGE grafts at three months post-injection into the brain of the non-human primate rhesus macaque. A manuscript is in preparation concerning this work. Our work has continued to show the viability of using a cell therapy technique in the potential treatment of brain-related disease.

Genetic Encoding Novel Amino Acids in Embryonic Stem Cells for Molecular Understanding of Differentiation to Dopamine Neurons

Funding Type: 
New Faculty I
Grant Number: 
RN1-00577
ICOC Funds Committed: 
$2 626 937
Disease Focus: 
Parkinson's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
Embryonic stem cells have the capacity to self-renew and differentiate into other cell types. Understanding how this is regulated on the molecular level would enable us to manipulate the process and guide stem cells to generate specific types of cells for safe transplantation. However, complex networks of intracellular cofactors and external signals from the environment all affect the fate of stem cells. Dissecting these molecular interactions in stem cells is a very challenging task and calls for innovative new strategies. We propose to genetically incorporate novel amino acids into proteins directly in stem cells. Through these amino acids we will be able to introduce new chemical or physical properties selectively into target proteins for precise biological study in stem cells. Nurr1 is a nuclear hormone receptor that has been associated with Parkinson’s disease (PD), which occurs when dopamine (DA) neurons begin to malfunction and die. Overexpression of Nurr1 and other proteins can induce the differentiation of neural stem cells and embryonic stem cells to dopamine (DA) neurons. However, these DA neurons did not survive well in a PD mouse model after transplantation. In addition, it is unclear how Nurr1 regulates the differentiation process and what other cofactors are involved. We propose to genetically introduce a novel amino acid that carries a photocrosslinking group into Nurr1 in stem cells. Upon illumination, molecules interacting with Nurr1 will be permanently linked for identification by mass spectrometry. Using this approach, we aim to identify unknown cofactors that regulate Nurr1 function or are controlled by Nurr1, and to map sites on Nurr1 that can bind agonists. The function of identified cofactors in DA neuron specification and maturation will be tested in mouse and human embryonic stem cells. These cofactors will be varied in combination to search for more efficient ways to induce embryonic stem cells to generate a pure population of DA neurons. The generated DA neurons will be evaluated in a mouse model of PD. Additionally, the identification of the agonist binding site on Nurr1 will facilitate future design and optimization of potent drugs.
Statement of Benefit to California: 
Parkinson’s disease (PD) is the second most common human neurodegenerative disorder, and primarily results from the selective and progressive degeneration of ventral midbrain dopamine (DA) neurons. Cell transplantation of DA neurons differentiated from neural stem cells or embryonic stem cells raised great hope for an improved treatment for PD patients. However, DA neurons derived using current protocols do not survive well in mouse PD models, and the details of DA neuron development from stem cells are unclear. Our proposed research will identify unknown cofactors that regulate the differentiation of embryonic stem cells to DA neurons, and determine how agonists activate Nurr1, an essential nuclear hormone receptor for DA neuron specification and maturation. This study may yield new drug targets and inspire novel preventive or therapeutic strategies for PD. These discoveries may be exploited by California’s biotech industry and benefit Californians economically. In addition, we will search for more efficient methods to differentiate human embryonic stem cells into DA neurons, and evaluate their therapeutic effects in PD mouse models. Therefore, the proposed research will also directly benefit California residents suffering from PD.
Progress Report: 
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. A nuclear hormone receptor named Nurr1 is found to regulate the differentiation process. To study the regulation mechanism, we proposed to genetically incorporate nonnatural amino acids into Nurr1 in stem cells, and use the novel properties of these amino acids to identify the interacting protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we made significant progress in genetically inserting nonnatural amino acids in stem cells. We are in the process of making stem cell lines that have this capacity. We also set up functional assays for studying Nurr1 and its mutants containing nonnatural amino acids. These results paved the way for our future identification of Nurr1 interacting networks in stem cells.
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. A nuclear hormone receptor named Nurr1 is found to regulate the differentiation process. To study the regulation mechanism, we proposed to genetically incorporate nonnatural amino acids into Nurr1 in stem cells, and use the novel properties of these amino acids to identify the interacting protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we figured out several mechanisms that prevent the efficient incorporation of nonnatural amino acids into proteins in stem cells. We now have developed new strategies to overcome these difficulties. In the meantime, we developed another complementary approach in order to detect unknown proteins that interact with Nurr1 during the differentiation process of stem cells. We are employing these new methods to identify Nurr1 interacting networks in stem cells.
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. The differentiation of embryonic stem cells to DA neurons has been found to be regulated by a nuclear hormone receptor Nurr1, but how Nurr1 involves in this complicated process remains unclear - no ligands or protein partners have been uncovered for Nurr1. To understand the regulation mechanism in molecular details, we proposed to incorporate non-natural amino acids into Nurr1 directly in stem cells, and use the novel properties of these amino acids to identify the protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, we figured out a right solution for generating stem cell lines capable of incorporating non-natural amino acids. We also created a novel bacterial strain for efficient producing Nurr1 proteins with the non-natural amino acids inserted. With these progresses we are now probing proteins that interact with Nurr1 during the differentiation of stem cells, which should eventually enable us to come up with new strategies for making DA neurons from embryonic stem cells.
  • Patients with Parkinson’s disease have malfunctioning or dying dopaminergic (DA) neurons. Human embryonic stem cells can be differentiated into DA neurons for transplantation with the potential to cure this disease, yet the differentiation mechanism is not very clear. The differentiation of embryonic stem cells to DA neurons has been found to be regulated by a nuclear hormone receptor Nurr1, but how Nurr1 is involved in this complicated process remains unclear - no ligands or protein partners have been uncovered for Nurr1. To understand the regulation mechanism in molecular details, we proposed to incorporate non-natural amino acids into Nurr1 directly in stem cells, and use the novel properties of these amino acids to identify the protein partners of Nurr1. Once these partners are discovered, effective protocols can be developed to generate high purity DA neurons for therapeutic purposes. In the past year, after testing numerous conditions in various cell lines, we discovered that photo-crosslinking is inefficient in capturing proteins interacting with Nurr1, possibly because the affinity between the unknown target protein and Nurr1 is too low. To overcome this challenge, we developed a new strategy of capture interacting proteins based on a novel class of non-natural amino acids, which do not require additional reagents nor external stimuli to function. We demonstrated the ability of these amino acids to crosslink proteins in the process of interacting with other proteins in live cells. We have also generated stable cell lines that are able to incorporate such non-natural amino acids. Using these new methods, we have been probing proteins that interact with Nurr1 during the differentiation of stem cells, which should eventually enable us to come up with new strategies for making DA neurons from embryonic stem cells.

In vitro reprogramming of mouse and human somatic cells to an embryonic state

Funding Type: 
New Faculty I
Grant Number: 
RN1-00564
ICOC Funds Committed: 
$2 229 427
Disease Focus: 
Rett's Syndrome
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Embryonic stem (ES) cells are remarkable cells in that they can replicate themselves indefinitely and have the potential to turn into all possible cell type of the body under appropriate environmental conditions. These characteristics make ES cells a unique tool to study development in the culture dish and put them at center stage for regenerative medicine. Two techniques, one called somatic cell nuclear transfer (SCNT) and the other in vitro reprogramming, have shown that adult cells from the mouse can be reverted to an ES like state. In SCNT, adult cell nuclei are transferred into oocytes and allowed to develop as early embryos from which ES cells can be derived, while in the in vitro method four genes are ectopically activated in the adult cell nucleus to induce an embryonic state in the culture dish. Key requirement for both processes is to erase the memory of the adult cell that specifies it as an adult cell and set up the ES cell program. How this happens remains unclear, and if it can be reproduced with human adult cells is an open question. Therefore, we will attempt to use the in vitro reprogramming method to generate human ES cells from adult cells and begin to understand the mechanism of the reprogramming process in both human and mouse cells. In addition to being integral to improving our understanding of how ES cells develop, if successful, this work will provide an important milestone for regenerative medicine. Many debilitating diseases and conditions are caused by damage to cells and tissue. In vitro reprogramming could provide a way to generate patient-specific stem cells that, in culture, could be turned into the type of cell or tissue needed to cure the patient’s disease or injury and transplanted back into the patient’s body. For example, Parkinson’s disease is caused by the loss or destruction of nerve cells. If reprogramming becomes possible, we could take a skin biopsy from a patient with Parkinson’s disease, induce the embryonic state in those skin cells to then be able to turn them into nerve cells and transplant them back into the same donor patient. Reprogramming could also be used to repair spinal cord injuries, allowing people who are paralyzed by accidents to walk again, or be helpful for patients with juvenile diabetes. One important advantage of patient-specific self-transplants is that they obviate the need for immunosuppression, which is often problematic for the patient. In addition, human cell reprogramming could be a new way to study how diseases progress at the cellular level as reprogramming could generate ES cells from patients with complex diseases that can be studied in detail for what makes them go awry during development. This knowledge could speed the search for new treatments and possibly cures for some of the most complex diseases that affect societies. We hope that the knowledge gained from our studies on reprogramming can, someday, support research that will help to put these idea to clinical use.
Statement of Benefit to California: 
Donated organs and tissues are often used to replace those that are diseased or destroyed, but unfortunately, the number of people needing a transplant exceeds the number of organs available for transplantation. Embryonic stem (ES) cells can be propagated in the laboratory for an unlimited period of time and can turn into all the specialized cell types that make us a human being. Therefore, ES cells offer the possibility of a renewable source of replacement cells and tissues to treat diseases, conditions, and disabilities such as Parkinson’s and Alzheimer’s, spinal cord injury, stroke, burns, heart disease, diabetes, osteoarthritis and rheumatoid arthritis. Our research is aimed to generate ES cells from adult cells through a method called in vitro reprogramming and to understand the mechanism by which the ES cell program can be reinstated in the adult cells. This work will not only provide the foundation for a better understanding of how human ES cells develop, but, if successful, be an important milestone for regenerative medicine. The advantage of using ES cells derived from adult cells by in vitro reprogramming would be that the patient’s own cells could be reprogrammed to an ES cell state and therefore, when transplanted back into the patient, not be attacked and destroyed by the body’s immune system. This would be beneficial to the people of California as tens of millions of Americans suffer from diseases and injuries that could benefit from research of in vitro reprogramming. Such advances would benefit the health as well as the economy of the state of California.
Progress Report: 
  • The discovery of induced pluripotent stem (iPS) cells by Shinya Yamanaka in 2006 marks a major landmark in the fields of stem cell biology and regenerative medicine. iPS cells can be obtained by co‐expression of four transcription factors in differentiated cells. The reprogramming process takes 2‐3 weeks and is very inefficient with about 1 in a 1000 somatic cells giving rise to an iPS cell. In previous work, we and others had demonstrated that mouse iPS cells are highly similar to ES cells in their molecular and functional characteristics as they for example can support adult chimerism with germline
  • contribution. The goal of the New Faculty Award proposal is to understand the molecular mechanisms underlying transcription factor‐ induced reprogramming of differentiated cells and to define the iPS cell state.
  • During this funding period, our efforts have focused on all three Aims. Within Aim 1, we have addressed a range of technical strategies to improve the reprogramming process. In Aim 2, we have analyzed human and mouse iPS cells in comparison to ES cells and attempted a better definition of the iPS cell state. In Aims 3, we are currently attempting to define barriers of the reprogramming process and begin to understand the transcriptional network that leads to reprogrammed cells.
  • The discovery of induced pluripotent stem (iPS) cells, which are derived from differentiated cells by simply overexpression a few transcription factors, by Shinya Yamanaka in 2006 marks a major landmark in the fields of stem cell biology and regenerative medicine. To unfold the full potential of reprogramming for disease studies and regenerative medicine, we believe that it is important to understand the molecular mechanisms underlying transcription factor‐ induced reprogramming and to carefully characterize the iPS cell state. To this end, during the third year of funding, we have devised a novel screen to identify factors important for the reprogramming process and allow replacement of the original reprogramming factors. We also studied the role of candidate transcriptional and chromatin regulators in the reprogramming process, which led us to identify novel barriers of the reprogramming process and to a better understanding of how chromatin interferes with the reprogramming process. We have also made progress in understanding the function of the reprogramming factors. Regarding human iPS cell lines, we have derived iPS cells from patients carrying X-linked diseases, and are beginning to characterize them molecularly. Together, we hope that our work will contribute to a better understanding of the reprogramming process.
  • Cellular reprogramming and the generation of induced pluripotent stem cells (iPSCs) from differentiated cells has enabled the creation of patient-specific stem cells for use in disease modeling. Reprogramming to the induced pluripotent state can be achieved through the ectopic expression of Oct4, Sox2, Klf4 and cMyc. Insight into the role that the reprogramming factors, various signaling pathways and epigenetic mechanisms play during the different stages of reprogramming remains limited, partly due to the low efficiency with which somatic cells convert to pluripotency. During the past year we have made great progress in (i) defining the molecular requirement for the reprogramming factors; (ii) gaining a better understanding of how repressive chromatin states control the reprogramming process; (iii) determining the differential regulation of chromatin states during reprogramming; (iv) identifying novel reprogramming stages; (v) assessing the three-dimensional organization of the genome during reprogramming; and (vi) determining the influence of a specific signaling pathway and its downstream effectors on different stages of the reprogramming process. Together, our findings provide novel mechanistic insights into the reprogramming process, which will form the basis of approaches to approve the efficiency of the process.
  • When this grant was awarded in 2008, reprogramming to the induced pluripotent state was just achieved by Shinya Yamanaka through the ectopic expression of Oct4, Sox2, Klf4 and cMyc in mouse fibroblasts. The overall goal of this proposal was to understand the molecular mechanisms underlying in vitro reprogramming of somatic cells of the mouse to iPSCs and to apply this knowledge to the reprogramming of human somatic cells. During the last funding period, our work particularly aimed at mechanistic questions: (i) determining the molecular origin of the spatio-temporal demarcation of the DNA binding sites of the reprogramming factors, and how the reprogramming factors induce chromatin changes, employing systematic and comprehensive mapping approaches; (ii) defining how the reprogramming factors induce a specific transcriptional output on target genes; (iii) identifying the steps of the reprogramming process to mouse iPSCs, which revealed an unprecedented detail of the reprogramming process and established that transition through a multitude of hierarchical stages is a fundamental feature of the reprogramming process; (iv) determining the dynamics of DNA methylation in reprogramming; (v) gaining a better understanding of how repressive Polycomb proteins control the reprogramming process; (vi) assessing the three-dimensional organization of the genome during reprogramming; and (vii) using the human iPSC approach for disease studies. Together, our findings provide novel mechanistic insights into the reprogramming process.

ES-Derived Cells for the Treatment of Alzheimer's Disease

Funding Type: 
New Faculty I
Grant Number: 
RN1-00538-A
ICOC Funds Committed: 
$2 120 833
Disease Focus: 
Aging
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Alzheimer’s disease is the most common cause of dementia in the elderly, affecting over 5 million people in the US alone. Boosting immune responses to beta-Amyloid (Aβ) has proven beneficial in mouse models and Alzheimer’s disease (AD) patients. Vaccinating Alzheimer’s mice with Aβ improves cognitive performance and lessens pathological features within the brain, such as Aβ plaque loads. However, human trials with direct Aβ vaccination had to be halted to brain inflammation in some patients. We have demonstrated that T cell immunotherapy also provides cognitive benefits in a mouse model for Alzheimer’s disease, and without any detectable brain inflammation. Translating this approach into a clinical setting requires that we first develop a method to stimulate the proliferation of Aβ-specific T cells without triggering generalized inflammatory response, as happens with vaccinations. Adaptive immune responses are provided by T cells and B cells, which are regulated by the innate immune system through antigen presenting cells, such as mature dendritic cells. We propose to leverage the power of embryonic stem (ES) cells by engineering dendritic cells that express a recombinant transgene that will specifically activate Aβ-specific T cells. We will test the effectiveness of this targeted stimulation strategy using real human T cells. If successful, this approach could provide a direct method to activate beneficial immune responses that may improve cognitive decline in Alzheimer’s disease.
Statement of Benefit to California: 
Alzheimer’s disease is the most common cause of dementia in the elderly, affecting more than 5 million people in the US. In addition to being home to more than 1 in 8 Americans, California is a retirement destination so a proportionately higher percentage of our residents are afflicted with Alzheimer’s disease. It has been estimated that the number of Alzheimer’s patients in the US will grow to 13 million by 2050, so Alzheimer’s disease is a pending health care crisis. Greater still is the emotional toll that Alzheimer’s disease takes on it’s patients, their families and loved one. Currently, there is no effective treatment or cure for Alzheimer’s disease. The research proposed here builds on more than 7 years of work showing that the body’s own immune responses keep Alzheimer’s in check in young and unaffected individuals, but deficiencies in T cell responses to beta-amyloid peptide facilitate disease progression. We have shown that boosting a very specific T cell immune response can provide cognitive and other benefits in mouse models for Alzheimer’s disease. Here we propose to use stem cell research to propel these findings into the clinical domain. This research may provide an effective therapeutic approach to treating and/or preventing Alzheimer’s disease, which will alleviate some of the financial burden caused by this disease and free those health care dollars to be spent for the well-being of all Californians.
Progress Report: 
  • We have developed new proteins that will stimulate immune responses to a major factor in Alzheimer's disease. Previous studies from our lab and others indicate that those responses can be improve memory deficits and brain pathology that occurs in Alzheimer's patients, and in Alzheimer's mice. To stimulate these immune responses the new proteins must be expressed by specific immune cells called, dendritic cells. Viruses have been made that carry the codes for these new proteins and we have confirmed that those viruses can deliver them into dendritic cells. To optimize these procedures we have made dendritic cells from human embryonic stem cells, and we developed methods to accomplish that step in our laboratory. At the end of year 2 we are nearing the completion of our preclinical studies and are poised to begin introducing the new proteins into immune cells that are derived from human blood, within the next year. The over-arching goal of this project is to develop method to trigger Alzheimer's-specific immune responses in a safe and reliable manner that could provide beneficial effects with minimal side-effects. This CIRM-funded project is on track to be completed within the 5 year time-frame.

ES-Derived Cells for the Treatment of Alzheimer's Disease

Funding Type: 
New Faculty I
Grant Number: 
RN1-00538-B
ICOC Funds Committed: 
$2 120 833
Disease Focus: 
Aging
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Alzheimer’s disease is the most common cause of dementia in the elderly, affecting over 5 million people in the US alone. Boosting immune responses to beta-Amyloid (Aβ) has proven beneficial in mouse models and Alzheimer’s disease (AD) patients. Vaccinating Alzheimer’s mice with Aβ improves cognitive performance and lessens pathological features within the brain, such as Aβ plaque loads. However, human trials with direct Aβ vaccination had to be halted to brain inflammation in some patients. We have demonstrated that T cell immunotherapy also provides cognitive benefits in a mouse model for Alzheimer’s disease, and without any detectable brain inflammation. Translating this approach into a clinical setting requires that we first develop a method to stimulate the proliferation of Aβ-specific T cells without triggering generalized inflammatory response, as happens with vaccinations. Adaptive immune responses are provided by T cells and B cells, which are regulated by the innate immune system through antigen presenting cells, such as mature dendritic cells. We propose to leverage the power of embryonic stem (ES) cells by engineering dendritic cells that express a recombinant transgene that will specifically activate Aβ-specific T cells. We will test the effectiveness of this targeted stimulation strategy using real human T cells. If successful, this approach could provide a direct method to activate beneficial immune responses that may improve cognitive decline in Alzheimer’s disease.
Statement of Benefit to California: 
Alzheimer’s disease is the most common cause of dementia in the elderly, affecting more than 5 million people in the US. In addition to being home to more than 1 in 8 Americans, California is a retirement destination so a proportionately higher percentage of our residents are afflicted with Alzheimer’s disease. It has been estimated that the number of Alzheimer’s patients in the US will grow to 13 million by 2050, so Alzheimer’s disease is a pending health care crisis. Greater still is the emotional toll that Alzheimer’s disease takes on it’s patients, their families and loved one. Currently, there is no effective treatment or cure for Alzheimer’s disease. The research proposed here builds on more than 7 years of work showing that the body’s own immune responses keep Alzheimer’s in check in young and unaffected individuals, but deficiencies in T cell responses to beta-amyloid peptide facilitate disease progression. We have shown that boosting a very specific T cell immune response can provide cognitive and other benefits in mouse models for Alzheimer’s disease. Here we propose to use stem cell research to propel these findings into the clinical domain. This research may provide an effective therapeutic approach to treating and/or preventing Alzheimer’s disease, which will alleviate some of the financial burden caused by this disease and free those health care dollars to be spent for the well-being of all Californians.
Progress Report: 
  • Alzheimer’s disease remains the most common cause of dementia in California and the US with more than 5 million cases nationwide, a number that is expected to exceed 13 million by 2050 if treatments are not developed. We, and others, showed that T cells responses to beta-amyloid can provide beneficial effects in mouse models of this disease. However, a clinical trial of Abeta vaccination was halted due to immune cell infiltration of the meninges and consequent brain swelling. Most of the other patients seemed to benefit from the vaccination, but the uncontrolled robustness of the immune response to vaccination makes those trials unfeasible. This project aims to refine and control Abeta-specific T cell responses using antigen presenting cells derived from human embryonic stem cells (hESC). If we are successful, then we would be able to deliver only the beneficial cells responsible for the beneficial effects, and do so in a controlled manner so as to avoid encephalitogenic complications.
  • During the first 4 years of this CIRM grant, my lab developed novel methods to assess adaptive immune responses to the Alzheimer’s-linked peptide, amyloid-beta/Abeta, in human blood samples. This technique relies on the use of pluripotent stem cells to produce specific immune-modulating cells in a complicated differentiation process that takes ~50 days. Over the past year we have found that this technology can employ both human embryonic stem cells and induced-pluripotent stem cells (iPSC), the latter of which were developed in my lab through other funding sources. We have now confirmed that this method provides consistent and robust readouts. Over the past year we have moved into the clinical phase of this project and assessed these responses in over 60 human subjects. Control subjects (not affected by Alzheimer’s disease) were recruited from the university community. Initially, we looked for age-dependent changes in these responses with a cohort of >50 research subjects who ranged in age from 20-88 years. Interesting patterns emerged from that study, which are currently being prepared for publication, and will remain confidential until publication; further details are not provided in this report as it will become public record. Several Alzheimer’s patients have also been assessed. We recently entered into an agreement with a local Alzheimer’s assessment center that will allow us to expand our study by including subjects with a presumptive diagnosis of Alzheimer’s disease, as well as individuals with mild cognitive impairment (MCI) and other causes of dementia such as Fronto-temporal Dementia, Dementia with Lewy bodies and Vascular Dementia. It will be interesting to determine if the assay we have developed will be able to distinguish subjects with developing Alzheimer's pathology from those with other causes of dementia, using a small blood sample. Overall, our progress is on-track for this project to be completed at the end of year 5, with many more subject samples analyzed than were originally proposed. In the approved grant it was proposed that spleen samples from 6-8 organ donors would be assessed, but as we developed this technology it became clear that we can detect these responses using 20 mL whole blood samples from living human subjects. At present, we have used our assay to assess more than 60 human subjects – 10 times what was proposed - and by this time next year we estimate that number will double. Information gained from this research is providing exciting new insights into immune changes associated with Alzheimer’s disease. The Western University of Health Sciences is engaged in patent processes to secure intellectual property and commercialize this technology.
  • Alzheimer’s disease affects more than 5.5 million people in the USA. Problems with memory correspond with the appearance of insoluble plaques in certain brain regions, and these plaques large consist of a peptide called, amyloid-beta. For more than a decade it has known that certain immune responses to amyloid-beta improve memory in mouse models of Alzheimer’s disease, yet in humans little is known about how those responses normally occur or if they may a beneficial therapeutic strategy. In this grant we have used stem cell technology to pioneer a new method to isolate and characterize those cells using only 20 cc of whole blood from a variety of human subjects. We have found that these immune responses increase dramatically in when high-risk people are in their late 40’s and early 50’s. Those responses may provide protection against Alzheimer’s disease progression as they diminish as memory problems begin to develop. This technology will be developed as an early diagnostic test for Alzheimer's disease with private equity partners. A patent application covering this technology was submitted by the Western University of Health Sciences.
  • This CIRM grant allowed my group to translate findings from our Alzheimer’s research from mouse to man. Over several years my group, an others, showed that boosting T cell responses to a peptide found in the plaques of Alzheimer’s patients could reduce disease pathology and memory problems in mouse models of this disease. Interestingly, at least some people carry T cells in their immune system, but it was unknown who has them or if they are lost over the course of Alzheimer’s disease. In this CIRM-funded project we used stem cells to develop a new technology, called CD4see, to identify and quantify those T cells using a small sample of human blood, roughly the same amount taken for a standard blood panel. After years of development and testing of CD4see, we used it to look for and quantify those plaque-specific T cells in over 70 human subjects. We found an age-dependent decline of Aβ-specific CD4+ T cells that occurred earlier in women than in men. Men showed a 50% decline around the age of 70, but women reached the same level before the age of 60. Notably, women who carried the AD risk marker apolipoproteinE-ε4 (ApoE4) showed the earliest decline, with a precipitous drop that coincided with an age when menopause usually begins. This assay requires a sample of whole blood that is similar to standard blood panels, making it suitable as a routine test to evaluate adaptive immunity to Aβ in at-risk individuals as an early diagnostic test for Alzheimer’s disease. In future applications CD4see can be used to isolate those cells in the lab, expand them to millions of cells, and then return them back to the same person--our earlier mouse studies showed those T cells counter Alzheimer’s pathology and memory impairment, so this technology may lead to a new therapeutic approach. I am grateful to CIRM and California taxpayers for supporting young scientists and funding innovative research.

Molecular mechanisms of neural stem cell differentiation in the developing brain

Funding Type: 
New Faculty I
Grant Number: 
RN1-00530
ICOC Funds Committed: 
$2 200 715
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
One of the most exciting possibilities in stem cell biology is the potential to replace damaged or diseased neural tissues affected by neurodegenerative disorders. Stem-cell-derived neurons provide a potentially limitless supply of replacement cells to repair damaged or diseased neurons. Typically, only one or a very few types of neurons are affected in most neurodegenerative diseases, and simply transplanting stem cells directly into a degenerating or damaged brain will not guarantee that the stem cells will differentiate into the specific neurons types needed. In fact, they may instead cause tumor formation. Thus, we must learn how to guide stem cells, cultured in a laboratory, toward a specific differentiation pathway that will produce neurons of the specified type. These cells would then provide a safe, effective way to treat neurodegenerative diseases and central nervous system injuries. Since there are hundreds or thousands of types of neurons in the cerebral cortex, functionally repairing damaged neurons in the cortex will require a detailed understanding of the mechanisms controlling differentiation, survival, and connectivity of specific neuronal subtypes. In this proposal, I propose to investigate the molecular mechanisms that guide the neural stem cells in developing embryonic brains to generate two specific types of neurons – corticospinal motor neurons (CSMNs) and corticothalamic projection neurons (CTNs). Our first goal is to understand what regulates the development of CSMNs. CSMNs are clinically important neurons that degenerate in Amyotrophic Lateral Sclerosis (ALS), and are damaged in spinal cord injuries. With our current technology, replacing damaged CSMNs has been impossible, due largely to a lack of understanding of what signals regulate their development. Our second goal is to identify genes that direct the neural stem cells to generate the CTNs. Despite their essential importance in sensory processing and involvement in epilepsy, mechanisms governing the development of CTNs have not yet been revealed. CSMNs and CTNs express many identical genes, and are generated from common neural stem cells in the embryonic brains. Yet it is unclear how they are specified from common stem cells. Our third goal is to identify transcription factor codes that neural stem cells employ to specifically generate either CSMNs or CTNs. Currently, there is no cure for neurodegenerative diseases. Understanding how CSMNs and CTNs are generated during development provides the opportunity to design procedures to direct the stem cells cultured in a laboratory to specifically produce CSMNs or CTNs, which can then be used to replaced damaged or diseased neurons, such as those affected by ALS, or spinal cord injuries.
Statement of Benefit to California: 
Neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), affect tens of thousands of Californians. There are no cures for these devastating diseases, nor effective treatments that consistently slow or stop them. The research proposed in this application may provide the basis for a novel, cost-effective, cell replacement therapy for ALS, thereby benefiting the State of California and its citizens. Stem cells offer a potential renewable source of a wide range of cell types that could be used to replace damaged cells involved in neurodegenerative diseases or in spinal cord injuries. At present, transplanting stem cells directly into patients is problematic, because this approach may instead cause tumor growth. To support safe and effective cell transplants, it is important to differentiate stem cells prior to the therapy into the specific cell types affected by the diseases. Understanding how different types of neurons are generated during development provides an opportunity to develop new methods to guide the differentiation of stem cells into the proper neuron types. In this application, we propose to uncover the mechanisms that regulate the neural stem cells in developing mouse brains to generate different neuronal types in the cerebral cortex, including the corticospinal motor neurons (CSMNs) and the corticothalamic neurons (CTNs). CSMNs are the neurons that degenerate in ALS and are affected in spinal cord injuries. Dysfunction of CTNs has been implicated in epilepsy. Understanding the mechanisms regulating neural stem cells to generate CSMNs and CTNs in vivo will help scientists and physicians to direct stems cells to produce CSMNs or CTNs to replace damaged neurons in patients with neurodegenerative conditions.
Progress Report: 
  • In this reporting period, we have been continuing our work to identify genes that regulate neural stem cells to produce different types of neurons in the brain.
  • In the past grant period, we have identified Tbr1 as the major cell fate-determing gene for the corticothalamic neurons.
  • In year 4 of the grant period, we continue to explore the molecular mechanisms that regulate neural stem cells to generate various types of cortical projection neurons, in particular the corticospinal motor neurons and the corticothalamic neurons. We have identified a novel transcription factor that regulates neural stem cell differentiation.
  • During the last grant period, we continue to explore the molecular mechanisms that regulate neural stem cells to generate different types of neurons in the mammalian brains. We have identified a transcription factor that is essential for neural stem cell differentiation, neuronal migration and axon projection.
  • We have continued our study to identify the molecular mechanisms that regulate cortical neuron fate specification. We have discovered/confirmed that (1) Early cortical progenitors are multipotent, and they give rise to different types of cortical project neurons and glia based on birthdates. There is no evidence of intrinsically lineage-restricted early neural stem cells; (2) expression of Fezf2, a major cell fate determining gene for cortical neurons, is regulated by multiple enhancers and promoters. These enhancers and promotor region have distinct and sometimes overlapping activity; (3) transcription factor Nfib is essential for the differentiation of neural stem cells and required for the cortical neurons to extend corticofugal axons; and (4) splicing factor Tra2b is essential for the survival and differentiation of cortical neural progenitor cells. These results provide novel insights into the development of cortical neurons.

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