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.

A hESc-based Development Candidate for Huntington's Disease

Funding Type: 
Early Translational II
Grant Number: 
TR2-01841
ICOC Funds Committed: 
$4 045 253
Disease Focus: 
Huntington's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Huntington’s disease (HD) is a devastating degenerative brain disease with a 1 in 10,000 prevalence that inevitably leads to death. These numbers do not fully reflect the large societal and familial cost of HD, which requires extensive caregiving. HD has no effective treatment or cure and symptoms unstoppably progress for 15-20 years, with onset typically striking in midlife. Because HD is genetically dominant, the disease has a 50% chance of being inherited by the children of patients. Symptoms of the disease include uncontrolled movements, difficulties in carrying out daily tasks or continuing employment, and severe psychiatric manifestations including depression. Current treatments only address some symptoms and do not change the course of the disease, therefore a completely unmet medical need exists. Human embryonic stem cells (hESCs) offer a possible long-term treatment approach that could relieve the tremendous suffering experienced by patients and their families. HD is the 3rd most prevalent neurodegenerative disease, but because it is entirely genetic and the mutation known, a diagnosis can be made with certainty and clinical applications of hESCs may provide insights into treating brain diseases that are not caused by a single, known mutation. Trials in mice where protective factors were directly delivered to the brains of HD mice have been effective, suggesting that delivery of these factors by hESCs may help patients. Transplantation of fetal brain tissue in HD patients suggests that replacing neurons that are lost may also be effective. The ability to differentiate hESCs into neuronal populations offers a powerful and sustainable alternative for cell replacement. Further, hESCs offer an opportunity to create cell models in which to identify earlier markers of disease onset and progression and for drug development. We have assembled a multidisciplinary team of investigators and consultants who will integrate basic and translational research with the goal of generating a lead developmental candidate having disease modifying activity with sufficient promise to initiate IND-enabling activities for HD clinical trials. The collaborative research team is comprised of investigators from multiple California institutions and has been assembled to maximize leverage of existing resources and expertise within the HD and stem cell fields.
Statement of Benefit to California: 
The disability and loss of earning power and personal freedom resulting from Huntington's disease (HD) is devastating and creates a financial burden for California. Individuals are struck in the prime of life, at a point when they are their most productive and have their highest earning potential. As the disease progresses, individuals require institutional care at great financial cost. Therapies using human embryonic stem cells (hESCs) have the potential to change the lives of hundreds of individuals and their families, which brings the human cost into the thousands. For the potential of hESCs in HD to be realized, a very forward-thinking team effort will allow highly experienced investigators in HD, stem cell research and clinical trials to come together and identify a lead development candidate for treatment of HD. This early translation grant will allow for a comprehensive and systematic evaluation of hESC-derived cell lines to identify a candidate and develop a candidate line into a viable treatment option. HD is the 3rd most prevalent neurodegenerative disease, but because it is entirely genetic and the mutation known, a diagnosis can be made with certainty and clinical applications of hESCs may provide insights into treating brain diseases that are not caused by a single, known mutation. We have assembled a strong team of California-based investigators to carry out the proposed studies. Anticipated benefits to the citizens of California include: 1) development of new human stem cell-based treatments for HD with application to other neurodegenerative diseases such as Alzheimer's and Parkinson's diseases that affect thousands of individuals in California; 2) improved methods for following the course of the disease in order to treat HD as early as possible before symptoms are manifest; 3) transfer of new technologies and intellectual property to the public realm with resulting IP revenues coming into the state with possible creation of new biotechnology spin-off companies; and 4) reductions in extensive care-giving and medical costs. It is anticipated that the return to the State in terms of revenue, health benefits for its Citizens and job creation will be significant.
Progress Report: 
  • Huntington’s disease (HD) is a devastating degenerative brain disease with a 1 in 10,000 prevalence that inevitably leads to death. Because HD is genetically dominant, the disease has a 50% chance of being inherited by the children of patients. Symptoms of the disease include uncontrolled movements, difficulties in carrying out daily tasks or continuing employment, and severe psychiatric manifestations including depression. Current treatments only address some symptoms and do not change the course of the disease, therefore a completely unmet medical need exists. Human embryonic stem cells (hESCs) offer a possible long-term treatment approach that could relieve the tremendous suffering experienced by patients and their families. Because HD is entirely genetic and the mutation known, a diagnosis can be made with certainty and clinical applications of hESCs may provide insights into treating brain diseases that are not caused by a single, known mutation. The ability to differentiate hESCs into neuronal populations offers a powerful and sustainable treatment opportunity. We have established the multidisciplinary team of investigators and consultants to integrate basic and translational research with the goal of generating a lead developmental candidate having disease modifying activity with sufficient promise to initiate IND-enabling activities for HD clinical trials.
  • In preliminary experiments, the transplantation of mouse neural stem cells, which survived in the brain for the four week period of the trial, provided protective effects in delaying disease progression in an HD mouse model and increased production of protective molecules in the brains of these mice. In the first year, the team has developed and established methods to differentiate hESCs into neural, neuronal and astrocyte precursors to be used for transplantation and has determined the correct cells to use that can be developed for future clinical development of these cells. In initial studies during this year, transplantation of neural stem cells (NSCs) provided both neurological and behavioral benefit to a HD mouse model. In addition, neuroprotective molecules were increased. Three immunosuppression regimens were tested to optimize methods for next stage preclinical trials. Finally, breeding of the three different HD mouse models has been initiated. Taken as a whole, progress supports the feasibility of the CIRM-funded studies to transplant differentiated hESCs into HD mice for preclinical development with the ultimate goal of initiating IND-enabling activities for HD clinical trials.
  • Huntington’s disease (HD) is a devastating degenerative brain disease with a 1 in 10,000 prevalence that inevitably leads to death. Because HD is genetically dominant, the disease has a 50% chance of being inherited by the children of patients. Symptoms of the disease include uncontrolled movements, difficulties in carrying out daily tasks or continuing employment, and severe psychiatric manifestations including depression. Current treatments only address some symptoms and do not change the course of the disease, therefore a completely unmet medical need exists. Human embryonic stem cells (hESCs) offer a possible long-term treatment approach that could relieve the tremendous suffering experienced by patients and their families. Because HD is entirely genetic and the mutation known, a diagnosis can be made with certainty and clinical applications of hESCs may provide insights into treating brain diseases that are not caused by a single, known mutation. The ability to differentiate hESCs into neuronal populations offers a powerful and sustainable treatment opportunity. We have established the multidisciplinary team of investigators and consultants to integrate basic and translational research with the goal of generating a lead developmental candidate having disease modifying activity with sufficient promise to initiate IND-enabling activities for HD clinical trials.
  • We previously performed transplantation of human neural stem cells into an HD mouse model and found that a subset of cells survived in the brain for the four week period of the trial, providing protective effects in delaying disease progression. In the past year, we have increased production and characterization of human neural stem cells (hNSCs) into neuronal (hNPC) and astrocyte (hAPC) precursors to be used for transplantation and optimized methods for shipping and implantation. Immunosuppression regimens were improved to optimize cell survival of implanted cells in HD mice. Transplantation of both human NSCs and NPCs are neuroprotective to HD mice and transplantation of hAPCs is in progress. Once completed, the cell giving the greatest protective benefit will be transplanted into mice that display slower progression over a longer time frame to validate and optimize approach for subsequent human application. All three HD mouse models have been bred and are ready for stem cell transplants. Taken as a whole, progress supports the feasibility of the CIRM-funded studies to transplant differentiated hESC-derived cell types into HD mice for preclinical development with the ultimate goal of identifying a lead candidate cell type and initiating IND-enabling activities for HD clinical trials.
  • Huntington’s disease (HD) is a devastating degenerative brain disease with a 1 in 10,000 prevalence that inevitably leads to death. Because HD is genetically dominant, the disease has a 50% chance of being inherited by the children of patients. Symptoms of the disease include uncontrolled movements, difficulties in carrying out daily tasks or continuing employment, and severe psychiatric manifestations including depression. Current treatments only address some symptoms and do not change the course of the disease, therefore a completely unmet medical need exists. Human embryonic stem cells (hESCs) offer a possible long-term treatment approach that could relieve the tremendous suffering experienced by patients and their families. Because HD is entirely genetic and the mutation known, a diagnosis can be made with certainty and clinical applications of hESCs may provide insights into treating brain diseases that are not caused by a single, known mutation. The ability to differentiate hESCs into neuronal populations offers a powerful and sustainable treatment opportunity. We have established the multidisciplinary team of investigators and consultants to integrate basic and translational research with the goal of generating a lead developmental candidate having disease modifying activity with sufficient promise to initiate Investigational New Drug (IND) enabling activities for HD clinical trials.
  • We have completed several rounds of transplantation of human neural stem cells into an HD mouse model and found that the cells survived in the brain for the four-week period of the trial, provided protective effects in delaying disease progression and increased production of protective molecules in the brains of these mice. In the last year the team differentiated hESCs into neural, neuronal and astrocyte precursors and performed transplantation studies to determine the best cell candidate to use and develop for future clinical work. We determined that the human neural stem cells produce the most robust effect. We have now selected a GMP grade hNSC line that will be carried forward for further testing in both rapidly progressing and slower progressing HD mice, as well as in mouse preclinical dosing studies. Taken as a whole, progress supports the feasibility of the CIRM-funded studies to transplant differentiated hESCs into HD mice for preclinical development with the ultimate goal on initiating IND-enabling activities for HD clinical trials.

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.

Developing a drug-screening system for Autism Spectrum Disorders using human neurons

Funding Type: 
Early Translational II
Grant Number: 
TR2-01814
ICOC Funds Committed: 
$1 491 471
Disease Focus: 
Autism
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Autism and autism spectrum disorders (ASD) are complex neurodevelopmental diseases that affect 1 in 150 children in the United States. Such diseases are mainly characterized by deficits in verbal communication, impaired social interaction, and limited and repetitive interests and behavior. Because autism is a complex spectrum of disorders, a different combination of genetic mutations is likely to play a role in each individual. One of the major impediments to ASD research is the lack of relevant human disease models. ASD animal models are limited and cannot reproduce the important language and social behavior impairment of ASD patients. Moreover, mouse models do not represent the vast human genetic variation. Reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells, iPSCs) has been accomplished using human cells. Isogenic pluripotent cells are attractive from the prospective to understanding complex diseases, such as ASD. Our preliminary data provide evidence for an unexplored developmental window in ASD wherein potential therapies could be successfully employed. The model recapitulates early stages of ASD and represents a promising cellular tool for drug screening, diagnosis and personalized treatment. By testing whether drugs have differential effects in iPSC-derived neurons from different ASD backgrounds, we can begin to unravel how genetic variation in ASD dictates responses to different drugs or modulation of different pathways. If we succeed, we may find new molecular mechanisms in ASD and new compounds that may interfere and rescue these pathways. The impact of this approach is significant, since it will help better design and anticipate results for translational medicine. Moreover, the collection and molecular/cellular characterization of these iPSCs will be an extremely valuable tool to understand the fundamental mechanism behind ASD. The current proposal uses human somatic cells converted into iPSC-derived neurons. The proposed experiments bring our analyses to real human cell models for the first time. We anticipate gaining insights into the causal molecular mechanisms of ASD and to discover potential biomarkers and specific therapeutic targets for ASD.
Statement of Benefit to California: 
Autism spectrum disorders, including Rett syndrome, Angelman syndrome, Timothy syndrome, Fragile X syndrome, Tuberous sclerosis, Asperger syndrome or childhood disintegrative disorder, affect many Californian children. In the absence of a functionally effective cure or early diagnostic tool, the cost of caring for patients with such pediatric diseases is high, in addition to a major personal and family impact since childhood. The strikingly high prevalence of ASD, dramatically increasing over the past years, has led to the emotional view that ASD can be traced to a single source, such as vaccine, preservatives or other environmental factors. Such perspective has a negative impact on science and society in general. Our major goal is to develop a drug-screening platform to rescue deficiencies showed from neurons derived from induced pluripotent stem cells generated from patients with ASD. If successful, our model will bring novel insights on the dentification of potential diagnostics for early detection of ASD risk, or ability to predict severity of particular symptoms. In addition, the development of this type of pharmacological therapeutic approach in California will serve as an important proof of principle and stimulate the formation of businesses that seek to develop these types of therapies (providing banks of inducible pluripotent stem cells) in California with consequent economic benefit.
Progress Report: 
  • During the first year of the project, we focused on creating a cell bank of reprogrammed fibroblasts derived from several autistic patients. These pluripotent stem cells were then induced to differentiate into neurons and gene expression analyses will be done at different time points along the process. We also used some of the syndromic and non-syndromic patients for neuronal phenotypic assays and found that a subset of idiopathic autism cases displayed a molecular overlap with Rett syndrome. Our plan is to use these data to test the ability of candidate drugs on reverting some of the neuronal defects observed in patient neurons.
  • The goal of this CIRM translational award is to generate a hiPSC-based drug-screening platform to identify potential therapies or biomarkers for autism spectrum disorders. In this second year we have made significant progress toward this goal by working on validating several neuronal phenotypes derived from iPSCs from idiopathic and syndromic autistic patients. We also made significant progress in order to optimize a synaptic readout for the screening platform. This step was important to speed up drug discovery. Using Rett syndrome iPSC-derived neurons as a prototype, we showed that we could rescue defect in synaptogenesis using a collection of FDA-approved drugs. Finally, we have initiated our analyses on global gene expression, from several neurons and progenitor cells derived from controls and autistic patients. We expect to find pathways that are altered in subgroups of patients, defined by specific clinical phenotypes.
  • The goal of this CIRM translational award is to generate a hiPSC-based drug-screening platform to identify potential therapies or biomarkers for ASDs. We have made significant progress toward this goal by working on validating several neuronal phenotypes derived from iPSC from Rett syndrome (RTT) and idiopathic autistic patients. We also made significant progress to optimize the readout for our screening platform. This was important to speed up drug discovery. Using RTT iPSC as a prototype, we showed that we could rescue defect in synaptogenesis using a collection of FDA-approved drugs. Finally, we initiate our analyses on gene expression, collected from several neurons and progenitor cells derived from controls and autistic patients. We expect to find pathways that are altered in subgroups of patients, defined by specific clinical phenotypes. Here, we describe the results of our drug screening, using FDA-approved drugs in a repurposing strategy. We also show for the first time that iPSC-derived human neurons are able to generate synchronized neuronal networks. RTT neurons behave differently from controls. Our focus now is on the completion of our gene expression analyses and to validate positive drugs using a battery of secondary cellular assays.

Repair of Conus Medullaris/Cauda Equina Injury using Human ES Cell-Derived Motor Neurons

Funding Type: 
Early Translational II
Grant Number: 
TR2-01785
ICOC Funds Committed: 
$1 614 441
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Injuries to the spinal cord commonly result from motor vehicle accidents, traumatic falls, diving, surfing, skiing, and snowboarding accidents, other forms of sports injuries, as well as from gunshot injuries in victims of violent crimes. Injuries to the anatomically lowest part of the spinal cord, the lumbosacral portion and its associated nerve roots commonly cause paralysis, loss of sensation, severe pain, as well as loss of bladder, bowel, and sexual function. Lumbosacral injuries represent approximately one-fifth of all traumatic lesions to the human spinal cord. As a result of the direct injury to the lumbosacral portion of the spinal cord, there is degeneration and death of spinal cord nerve cells, which control muscles in the legs as well as bladder, bowel, and sexual function. No treatments are presently available in clinical practice to reverse the effects of these devastating injuries. In order to reverse the loss of function after lumbosacral spinal cord injury, replacement of the lost nerve cells is required. Recent research studies have identified some properties that are shared by spinal cord neurons responsible for muscle and bladder control. Human embryonic stem cells can now be prepared in research laboratories to develop properties that are shared between nerve cells controlling muscle and bladder function. Such nerve cells are particularly at risk of degeneration and death as a result of injuries to the lumbosacral spinal cord. Human embryonic stem cells, which have undergone treatment to obtain properties of muscle and bladder controlling nerve cells, are now very attractive development candidates for new cell replacement therapies after lumbosacral spinal cord injuries. The proposed feasibility studies will study the properties of such cells in a clinically relevant rat model for lumbosacral spinal cord injuries. In Specific Aim 1, we will determine whether ACUTE transplantation of human embryonic stem cells, which have been treated to develop properties of specific lumbosacral spinal cord neurons, may replace lost nerve cells and result in a return of bladder function in a rat model of lumbosacral spinal cord injury and repair. In Specific Aim 2, we will determine whether DELAYED transplantation of human embryonic stem cells, which have been treated to develop properties of specific lumbosacral spinal cord neurons, may replace lost nerve cells and result in a return of bladder function in a rat model of lumbosacral spinal cord injury and repair. A variety of functional studies will determine the effect of the cell transplantation on bladder function, walking, and pain. We will also use detailed anatomical studies to determine in microscopes whether the transplanted cells have grown processes to connect with pelvic target tissues, including the lower urinary tract. If successful, the proposed experiments may lead to a new treatment strategy for patients with lumbosacral spinal cord injuries.
Statement of Benefit to California: 
There are presently about 250,000 patients living with neurological impairments from spinal cord injuries (SCIs) in the United States, and approximately 11,000 new cases present every year. SCIs typically result in paralysis, loss of sensation, pain as well as bladder, bowel, and sexual dysfunction. No successful treatments are available to reverse the neurological deficits that result from SCI. Common causes for SCIs include car and motorcycle accidents, skiing, diving, surfing, and snow boarding injuries, traumatic falls, sports injuries, and acts of violence. California medical centers encounter a large proportion of the overall cases in the U.S. because of our large population, extensive network of freeways, and an active life style with recreational activities taking place both along the Californian coastline and in the mountains. The proposed development candidate feasibility project will capitalize on recent progress in human stem cell science and surgical repair of conus medullaris/cauda equina (CM/CE) forms of SCI. Human embryonic stem cell-derived neurons and neuronal progenitors, which express the transcription factor Hb9, will be transplanted into the conus medullaris in attempts to replace lost motor and autonomic neurons after a lumbosacral ventral root avulsion injury in rats. Surgical replantation of avulsed lumbosacral ventral roots into the spinal cord will also be performed in this clinically relevant model for CM/CE injury and repair. If successful, our development candidate may reinnervate muscles and pelvic organs, including the lower urinary tract after CM/CE forms of SCI. Return of functional bladder control represents one of the absolute top priorities among the spinal cord injured population (Anderson, J Neurotrauma, 2004; 21, 1371-83). Successful recovery of bladder function after SCI is expected to have very significant impact on the quality of life of spinal cord injured subjects and markedly reduce health care costs. Recovery of bladder function in spinal cord injured subjects would markedly reduce or eliminate the need for intermittent bladder catheterizations and indwelling bladder catheters. The number of visits in physicians’ offices and already over-crowded California emergency rooms for bladder infections and other complications would be markedly reduced, thereby significantly reducing health care costs for both patients and our state. Improved neurological function among the SCI population is also expected to reduce care giver needs, thereby further reducing health care costs. The increased independence that will result from improved bladder control and concomitant possible recovery of other neurological functions, for instance in transfers and locomotion, will promote return to and participation in the work force for many individuals with SCI. These effects are also expected to bring a very positive effect to the California economy and increased quality of life for those living with an SCI.
Progress Report: 
  • Injuries to the lowest portion of the spine and the spinal cord commonly results in paralysis and impairment of bladder , bowel, and sexual functions. These injuries are usually referred to as conus medullaris and cauda equina forms of spinal cord injuries. Presently, no treatments are available to reverse the neurological deficits that result from these injuries.
  • In this project, we aim to reverse neurological deficits, including bladder function, in a rat model of spinal cord injury, which affects the lowermost portion of the spinal cord. This part of the spinal cord and the associated nerve roots are called the conus medullaris and cauda equina. In our experimental model, nerve roots carrying fibers that control muscle function and pelvic organs, such as the bladder and bowel, are injured at the surface of the spinal cord. This injury mimics many of the neurological deficits encountered in human cases.
  • For treatment purposes, we transplant human derived embryonic stem cells, which have been prepared to acquire properties of motor neurons, into the lowermost portion of the rat spinal cord after injury and surgical repair of nerve roots carrying motor fibers. The studies will evaluate both acute and delayed transplantation of human embryonoic
  • During the first year of the studies, we have developed improved protocols to increase our ability to produce large number of motor neurons from human embryonic stem cells. We have also developed improved methods to detect motor neurons during the neuron production process by using fluorescent reporters inside of the cells. The latter development is of great help when sorting and preparing cells with desired properties for transplantation studies. In addition, we have refined our surgical methods to make it less invasive, using a one-sided injury model instead of lesions on both sides of the spinal cord in rats. Specifically, bladder dysfunction can be assess after a one sided injury of nerve roots and be evaluated using a combination of bladder pressure recorings and electrical recordings referred to as electromyography (EMG) from muscles along the urethra. The revised procedure is well tolerated by the rats and is a suitable approach for studies of chronic injury and cell-based long-term treatments. A research manuscript describing this improved experimental method and refinement has been submitted to a scientific journal and reviewed, and the manuscript is currently undergoing our revisions before being considered for publication. The experimental refinement will greatly assist with our long-term studies on the effects of transplanted motor neurons derived from human embryonic stem cells. We have also begun experiments using our refined model and cells, which now can be produced in high numbers and be identifiable during both the cell culture steps and during the animal studies. Initial tissues have been harvested and are being processed for morphological analyses.
  • Injuries to the lowest portion of the spine and the spinal cord commonly results in paralysis and impairment of bladder , bowel, and sexual functions. These injuries are usually referred to as conus medullaris and cauda equina forms of spinal cord injuries. Presently, no treatments are available to reverse the neurological deficits that result from these injuries.
  • In this project, we aim to reverse neurological deficits, including bladder function, in a rat model of spinal cord injury, which affects the lowermost portion of the spinal cord. This part of the spinal cord and the associated nerve roots are called the conus medullaris and cauda equina. In our experimental model, nerve roots carrying fibers that control muscle function and pelvic organs, such as the bladder and bowel, are injured at the surface of the spinal cord. This injury mimics many of the neurological deficits encountered in human cases.
  • For treatment purposes, we transplant human derived embryonic stem cells, which have been prepared to acquire properties of motor neurons, into the lowermost portion of the rat spinal cord after injury and surgical repair of nerve roots carrying motor fibers. The studies will evaluate both acute and delayed transplantation of human embryonic stem cells, which have acquired properties of motor neurons.
  • During the second year of the studies, we have developed improved protocols to increase our ability to produce large number of motor neurons from human embryonic stem cells. We have also developed improved methods to detect motor neurons during the neuron production process by using fluorescent reporters inside of the cells. The latter development is of great help when sorting and preparing cells with desired properties for transplantation studies. In addition, we have refined our surgical methods to make it less invasive, using a one-sided injury model instead of lesions on both sides of the spinal cord in rats. Specifically, bladder dysfunction can be assessed after a one sided injury of nerve roots and be evaluated using a combination of bladder pressure recordings and electrical recordings referred to as electromyography (EMG) from muscles along the urethra. The revised procedure is well tolerated by the rats and is a suitable approach for studies of chronic injury and cell-based long-term treatments. A research manuscript describing this improved experimental method and refinement has been published. The experimental refinement will greatly assist with our long-term studies on the effects of transplanted motor neurons derived from human embryonic stem cells. We have also performed transplantations of embryonic human stem cell derived motor neurons into the rat spinal cord and demonstrated surgical feasibility as well as survival of large numbers of neurons in the rat spinal cord. Some of the transplanted cells also demonstrate anatomical markers for motor neurons after transplantation.
  • During the reporting period, we have contined to demonstrate that human embryonic stem cell derived motor neurons and motor neuron progenitors can be produced in vitro. These motor neurons and motor neuron progenitors are transplanted into the rat spinal cord after a lumbosacral ventral root avulsion injury and repair of injured roots in the form of surgical re-attachment of the roots to the spinal cord surface. The lumbosacral ventral root avulsion injury mimics cauda equina and conus medullaris forms of spinal cord injury, an underserved patient population with paralysis of the legs and loss of bladder and bowel funcion. In this clinically relevant injury and repair model in rats, we have during the past several months demonstrated that transplanted human embryonic stem cell-derived motor neurons and motor neuron progenitors are able to survive in the spinal cord of rats over extended periods of time with large numbers of neurons being detectable in the spinal cord grey matter at 1, 2, and 10 weeks after the injury, surgical root repair, and transplantation of the cells. The long term viability of translanted cells suggests integration of the transplanted cells in the host tissues. Some of the cells show expression of motor neuron markers, such as the transcription factor Hb9, as demonstrated by immunohistochemistry and light microscopy.
  • Additional studies have been performed during this reporting period to address whether the transplanted cells may extend axons into the replanted lumbosacral ventral roots. Interestingly, many human axons were detected in the replanted ventral roots using immunohistochemitry and light microscopy for the detection of human processes. Additional immunohistochemistry demonstrated that these processes contained neurofilaments, which are characteristic for axons. In control experiments, we showed that avulsed roots, which had not been replanted into the spinal cord, did not exhibit any human axons. As expected, surgical reconnection of lesioned ventral roots to the spinal cord is needed in order for the axons of the transplanted human embryonic stem cell derived motor neurons and motor neuron progenitors to be extended into avulsed ventral roots. Furthermore, in a series of sham operated animals without ventral root lesions, human motor neurons and motor neuron progenitors were also transplanted into the rat spinal cord. Interestingly, the transplanted human motor neurons and motor neuron progenitors were here also able to extend axons into ventral roots, even though the ventral roots had never been lesions. We conclude that transplanted human embryonic stem cell derived motor neurons are capable of extending axons into both intact ventrl roots and into ventral roots, which had been avulsed and surgically reattached to the spinal cord using a replantation procedure.
  • In functional studies, we have performed urodynamic studies and voiding behavioral studies in rats after the transplantation of human embryonic stem cell derived motor neurons and motor neuron progenitors. These studies are still ongoing with additional experiments being performed. However, preliminary studies suggest that the combination of acute repair of avulsed ventral roots and cell transplantation results in a gradual improvement of voiding reflexes. Ongoing studies are addressing the relative contribution that may be provided by the replantation of avulsed ventral roots and by the transplantation of human motor neurons and motor neuron progenitors into the rat spinal cord.

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.

Neural restricted, FAC-sorted, human neural stem cells to treat traumatic brain injury

Funding Type: 
Early Translational II
Grant Number: 
TR2-01767
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.

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.

Role of HLA in neural stem cell rejection using humanized mice

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01735-A
ICOC Funds Committed: 
$1 472 634
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
Public Abstract: 
One of the key issues in stem cell transplant biology is solving the problem of transplant rejection. Despite over three decades of research in human embryonic stem cells, little is known about the factors governing immune system tolerance to grafts derived from these cells. In order for the promise of embryonic stem cell transplantation for treatment of diseases to be realized, focused efforts must be made to overcome this formidable hurdle. Our proposal will directly address this critically important issue by investigating the importance of matching immune system components known as human leukocyte antigens (HLA). Because mouse and human immune systems are fundamentally different, we will establish cutting-edge mouse models that have human immune systems as suitable hosts within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human blood-derived stem cells. These mice go on to develop a human immune system, complete with HLAs, and can subsequently be used to engraft embryonic stem cell-derived brain cells that are either HLA matched or mismatched. Due to our collective expertise in the central nervous system and animal transplantation studies for Parkinson’s disease, our specific focus will be on transplanting embryonic stem cell-derived neural stem cells into brains of both healthy and Parkinson's diseased mice. We will then detect: 1) abundance of brain immune cell infiltrates, 2) production of immune molecules, and 3) numbers of brain-engrafted embryonic stem cells. Establishing this important system would allow for a predictive model of human stem cell transplant rejection based on immune system matching. We would then know how similar HLAs need to be in order to allow for acceptance stem cell grafts.
Statement of Benefit to California: 
In this project, we propose to focus on the role of the human immune system in human embryonic stem cell transplant rejection. Specifically, we aim to develop cutting-edge experimental mouse models that possess human immune systems. This will allow us to determine whether immune system match versus mismatch enables embryonic stem cell brain transplant acceptance versus rejection. Further, we will explore this key problem in stem cell transplant biology both in the context of the healthy and diseased brain. Regarding neurological disease, we will focus on neural stem cell transplants for Parkinson's disease, which is one of the most common neurodegenerative diseases, second only to Alzheimer's disease. If successful, our work will pave the way toward embryonic stem cell-based treatment for this devastating neurological disorder for Californians and others. In order to accomplish these goals, we will utilize two of the most common embryonic stem cell types, known as WiCell H1 and WiCell H9 cells. It should be noted that these particular stem cells will likely not be reauthorized for funding by the federal government due to ethical considerations. This makes our research even more important to the State of California, which would not only benefit from our work but is also in a unique position to offer funding outside of the federal government to continue studies such as these on these two important types of human embryonic stem cells.
Progress Report: 
  • In order for the promise of stem cell transplantation therapy to treat or cure human disease to be realized, the key problem of stem cell transplant rejection must be solved. Yet, despite over three decades of research in human embryonic stem cells, little is known about the factors governing immune system tolerance to grafts derived from these cells.
  • The goal of our CIRM Stem Cell Transplantation Immunology Award is to overcome this formidable hurdle by generating pre-clinical mouse models that have human immune systems. This next-generation model system will provide a testing platform to evaluate the importance of matching immune system components known as human leukocyte antigens (HLAs). Because mouse and human immune systems are fundamentally different, these cutting-edge ‘humanized’ mice are currently the only animal models within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human umbilical cord blood stem cells (HSCs). These mice go on to develop a human immune system, complete with HLAs, and can subsequently be used to engraft embryonic stem cell-derived brain cells that are either HLA matched or mismatched and to monitor for graft acceptance vs. rejection.
  • During this first year of CIRM funding, we have accomplished three main goals leading to completion of Specific Aim 1: To establish mouse models with human immune systems (year 1). Firstly, we have increased purity of HSCs from 75% to 93%. This has enabled us to complete our second goal of generating 10 mice bearing 50% or more human immune cells. Thirdly, we have characterized the human adaptive immune systems of these mice and have found presence of 40-60% of human T lymphocytes in lymphoid organs of ‘humanized’ mice.
  • For the promise of stem cell transplantation therapy to treat or cure human disease to be realized, the key problem of stem cell transplant rejection must be solved. Yet, despite over three decades of research in human embryonic stem cells, little is known about the factors involved in immune system tolerance to grafts derived from embryonic stem cells.
  • The goal of our CIRM Stem Cell Transplantation Immunology Award is to overcome this formidable hurdle by generating pre-clinical mouse models that have human immune systems. This cutting-edge model system will provide a testing platform to evaluate the importance of matching immune system components, known as human leukocyte antigens (HLAs), between the human embryonic stem (hES) cell-derived neural stem cell (NSC) graft and the patient. Because mouse and human immune systems are fundamentally different, these next-generation ‘humanized’ mice are currently the only animal models within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human umbilical cord blood stem cells (HSCs). These mice go on to develop a human immune system, complete with HLAs, and can subsequently be used to engraft embryonic stem cell-derived brain cells that are either HLA matched or mismatched and to monitor for graft acceptance vs. rejection.
  • During this second year of CIRM funding, we have accomplished three main goals leading to completion of Specific Aim 2, which is designed to perform HLA haplotype ‘mix and match’ experiments using hES cell-derived NSCs as donors and ‘humanized’ mice as recipients (year 2). Firstly, we have now successfully generated ‘humanized’ mice that have 50% or more engraftment of human immune cells in lymphoid organs, defined as percentage of human immune cells within the mouse. Secondly, we have successfully HLA haplotyped these human donor CD34+ HSCs, and have additionally transplanted hES cell-derived NSCs with known HLA haplotypes. Finally, we have ‘mixed and matched’ HLA haplotypes in adoptive transfer experiments using human HSC reconstituted mice as recipients and human NSCs as donors. This critically important new tool will allow for a predictive model of human stem cell transplant acceptance vs. rejection.

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