Neurological Disorders

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

Cellular Reprogramming: Dissecting the Molecular Mechanism and Enhancing Efficiency

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01628
ICOC Funds Committed: 
$1 458 000
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Pluripotent stem cells have a remarkable potential to develop into virtually any cell type of the body, making them a powerful tool for the study or direct treatment of human disease. Recent demonstration that induced pluripotent stem (iPS) cells may be derived from differentiated adult cells offers unprecedented opportunities for basic biology research, regenerative medicine, disease modeling, drug discovery and toxicology. For example, using patient-derived iPS cells, one can model diseases in vitro and screen for drugs in ways never before possible, enabling the identification of promising new therapeutic candidates earlier in the drug discovery process. In addition, iPS cell derivatives represent an ideal source for autologous cell replacement therapies, as they would not be rejected upon transplantation back into the patient. While it is clear that iPS cells hold great promise for finding therapies for diseases, there are significant hurdles that need to be overcome before full clinical potential is realized. The mechanism of iPS cell derivation is largely elusive, and the process used to generate them is very inefficient and needs to be improved in significant ways. Currently, iPS cells are generated by forced expression of four molecular factors using genome-integrating viruses. This may lead to mutations and altered differentiation potential of iPS cells, as well as tumorogenesis if transplanted back into the patient. The inefficient and stochastic nature of the reprogramming process indicates that additional, as yet unidentified mechanisms may exist and contribute to iPS cell generation. Finally, increasing the efficiency of current iPS cell derivation protocols will increase the ability to generate large panels of patient-specific iPS cell lines. We propose to use a human cell-based assay to identify small molecules that can enhance the efficiency of iPS cell generation. Our strategy will allow us to identify small molecules that target events essential for derivation of iPS cells, as well as those that replace one or more of the four virally-delivered factors. We will use the identified small molecules to discover regulatory pathways and molecular targets involved in induction of pluripotency, gaining valuable insight into the mechanism of cellular reprogramming. Application of these small molecules themselves, as well as novel approaches derived from mechanism of action studies, will help overcome issues associated with viral integration and has the potential to transform personalized cell-replacement therapies as well as accelerate drug discovery based on iPS cell-derived disease models.
Statement of Benefit to California: 
California’s health care system faces significant challenges as millions of children and adults suffer from a host of incurable illnesses. It is expected that health care costs will continue to rise as California’s citizenry ages and requires treatments for age-related, chronic metabolic, cardiovascular, and neurodegenerative disorders. Both the measureable economic impact on California’s health care system and the incalculable emotional suffering of affected individuals, their families and communities, make it an imperative to develop novel therapeutic treatments to address these mounting medical and economic societal challenges. Recognizing the potential utility of novel stem cell technologies to address California’s unmet medical needs, California voters approved Proposition 71 which created the California Institute for Regenerative Medicine (CIRM), an agency that administers funds to support stem cell research that has the greatest potential for development of novel regenerative medical treatments and cures. The CIRM Basic Biology Awards II program is intended to fund studies that will lay the foundation for future stem cell-based translational and clinical advances. In keeping with this mission, our proposed research program aims to discover new methods for producing human induced pluripotent stem cells (iPS cells) on an industrial scale and in an efficient manner; and to develop a better understanding of the mechanisms underlying cellular reprogramming. As such, our research program will help accelerate the realization of the full potential of iPS cells in cell-based regenerative medicine therapies and drug discovery. Our proposed research program will benefit the State of California and its citizens in several ways. Firstly, our research program will lay the foundation for future stem cell-based clinical and translational advances to treat and manage one of California’s most pressing unmet medical needs. Secondly, execution of our research program will create new jobs in the academic, biotechnology and pharmaceutical sectors throughout California. Funding from CIRM will be expanded with additional funding from the applicant to augment achievement of the aims of this project. CIRM funding will leverage other sources of investment in this project to help ensure California’s continued future as a world leader in biomedical innovation and translational medicine for the benefit of human health. Lastly, our proposed research program will stimulate California’s economy by creating new enabling tools and technologies that can be broadly adopted across the life science industry, thus promoting development across the academic institutions and biopharmaceutical companies that create biomedical discoveries and advances. 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: 
  • iPS cell lines display high variability with respect to their growth properties, differentiation ability and disease phenotype manifestation; this is a major challenge for both in vitro disease modeling and drug screening, as well as cell replacement therapy. The cause for this variability is currently unknown and heterogeneity of the starting fibroblast population, composition of reprogramming factors, and viral integration into the genome have all been proposed to contribute to variability among different iPSC clones derived from the same starting population. In particular, the use of DNA viral vectors to deliver the reprogramming factors was suggested to be the key obstacle for eventual use of iPSCs in cell replacement therapies. Recently, several methods have been developed to derive integration-free iPS cells, but they mostly suffer from unacceptably low reprogramming efficiency or labor-intensive delivery of reprogramming factors.
  • The goal of this work is to produce integration-free iPS cells with high efficiency. To date, we developed a high-throughput platform to screen small molecules that enhance reprogramming in the presence of 3 transcription factors required for reprogramming of fibroblast cells - KLF4, SOX2, and OCT4. These efforts included assay development, characterization of our engineered stable cell lines expressing reprogramming factors under control of an inducible promoter, and optimization for high-throughput screening. We screened over 100,000 compounds from our small molecule library, comprising compounds with broad chemical diversity and covering multiple cellular target classes. We identified 130 hit candidates in our primary screen, however, in follow-up assays none of these compounds showed an effect comparable with that of a positive control compound. Moving forward, we plan on detailed characterization of other integration-free reprogramming methods to determine the differences between integrating vs. non-integrating methods for reprogramming fibroblasts. We will assess the quality and variability of iPS cell lines, as well as their potential and variability of differentiation and disease phenotype manifestation in integration-free iPSCs, and compare them to DNA virus-derived iPSCs. Our results should contribute to the understanding of the source of variability between iPS cells and bring us closer to reaching the ultimate goal: production of integration-free human iPS cells with high efficiency.
  • The use of stem cell technology to study neurodegenerative diseases has been a burgeoning area of research in recent years. Recent work by us and others have demonstrated that stem cell derived cortical neurons (CN) from Alzheimer's disease patients demonstrate key disease differentials when compared to CN’s derived from healthy controls. However, most of this work has been limited to the use of simple cultures of iPSC-derived cortical or motor neurons, or in some cases, co-culture of iPSC-derived neurons with primary human astrocytes. We have demonstrated that co-culturing of iPSC-derived neurons with primary fetal astrocytes facilitates the maturation of neurons (as assessed by electrophysiology) beyond what is observed in neuronal cultures alone. We have developed an astrocyte differentiation protocol from iPSC that allows for the generation of cells with gene expression, glutamate uptake capacity and kinetics, and neurotropic factor secretion consistent with primary fetal astrocytes. Importantly, these iPSC astrocytes can be directed to an anterior versus posterior (brain versus spinal cord) identity. The goal of this study is to compare the ability of iPSC-derived astrocytes to primary fetal astrocytes in their ability to facilitate the maturation of iPSC-derived neurons, in order to identify the appropriate co-culture conditions where iPSC-derived neurons reach functional maturity as assessed by neuronal markers and electrophysiological activity. Achievement of this goal would allow for further assessment of the contribution of neurons and astrocytes to specific disease processes in neurodegeneration and neuroinflammation through the ability to combine in co-culture either disease versus healthy iPSC-derived astrocytes and/or neurons to further elucidate the underlying mechanisms contributing to these disease phenotypes. These studies should facilitate the development of co-culture systems which drive neuronal development to a more mature phenotype, and improve the availability of stem cell-based model systems to better replicate in vivo physiological and pathological processes.
  • The use of stem cell technology to study neurodegenerative diseases has been a burgeoning area of research in recent years. Recent work by us and others have demonstrated that stem cell derived cortical neurons (CN) from Alzheimer's disease patients demonstrate key disease differentials when compared to CN's derived from healthy controls. However, most of this work has been limited to the use of simple cultures of iPSC-derived cortical or motor neurons, or in some cases, co-culture of iPSC-derived neurons with primary human astrocytes. We have demonstrated that co-culturing of iPSC-derived neurons with primary fetal astrocytes facilitates the maturation of neurons (as assessed by electrophysiology) beyond what is observed in neuronal cultures alone. We have developed an astrocyte differentiation protocol from iPSC that allows for the generation of cells with gene expression, glutumate uptake capacity and kinetics, and neurotropic factor secretion consistent with primary fetal astrocytes. The goal of this study is to compare the ability of iPSC-derived astrocytes to primary fetal astrocytes in their ability to facilitate the maturation of iPSC-derived neurons. Achievement of this goal would allow for further assessment of the contribution of neurons and astrocytes to specific diseases processes in neurodegeneration and neuroinflammation through the ability to combine co-culture either in disease vs. healthy iPSC-derived astrocytes and/or neurons to further elucidate the underlying mechanisms contributing to these disease phenotypes. These studies should facilitate the development of co-culture systems which drive neuronal development to a more mature phenotype, and improve the availability of stem-cell based model systems.

Using Human Embryonic Stem Cells to Understand and to Develop New Therapies for Alzheimer's Disease

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00116
ICOC Funds Committed: 
$2 512 664
Disease Focus: 
Aging
Alzheimer's Disease
Neurological Disorders
Genetic Disorder
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Alzheimer’s Disease (AD) is a progressive incurable disease that robs people of their memory and ability to think and reason. It is emotionally, and sometimes financially devastating to families that must cope when a parent or spouse develops AD. Unfortunately, however, we currently lack an understanding of Alzheimer’s Disease (AD) that is sufficient to drive the development of a broad range of therapeutic strategies. Compared to diseases such as cancer or heart disease, which are treated with a variety of therapies, AD lacks even one major effective therapeutic approach. A key problem is that there is a paucity of predictive therapeutic hypotheses driving the development of new therapies. Thus, there is tremendous need to better understand the cellular basis of AD so that effective drug and other therapies can be developed. Several key clues come from rare familial forms of AD (FAD), which identify genes that can cause disease when mutant and which have led to the leading hypotheses for AD development. Recent work on Drosophila and mouse models of Alzheimer’s Disease (AD) has led to a new suggestion that early defects in the physical transport system that is responsible for long-distance movements of vital supplies and information in neurons causes neuronal dysfunction. The type of neuronal failure caused by failures of the transport systems is predicted to initiate an autocatalytic spiral of biochemical events terminating in the classic pathologies, i.e., plaques and tangles, and the cognitive losses characteristic of AD. The problem, however, is how to test this new model and the prevailing “amyloid cascade” model, or indeed any model of human disease developed from studies in animal models, in humans. It is well known that mouse models of AD do not fully recapitulate the human disease, perhaps in part because of human-specific differences that alter the details of the biochemistry and cell biology of human neurons. One powerful approach to this problem is to use human embryonic stem cells to generate human neuronal models of hereditary AD to test rigorously the various hypotheses. These cellular models will also become crucial reagents for finding and testing new drugs for the treatment of AD. 
 
Statement of Benefit to California: 
Alzheimer’s Disease (AD) is emotionally devastating to the families it afflicts as well as causing substantial financial burdens to individuals, to families, and to society as a whole. In California, the burden of Alzheimer’s Disease is substantial, so that progress in the development of therapeutics would make a significant financial impact in the state. Although there are not a great deal of data about the burden of AD in California specifically, the population of California is 12% of that of the United States and most information suggests that California has a “typical” American burden of this disease. For example, information from the Alzheimer’s Association (http://www.alz.org/alzheimers_disease_alzheimer_statistics.asp) reveals: 1) An estimated 4.5 million Americans have Alzheimer’s disease, which has more than doubled since 1980. This creates an estimated nationwide financial burden of direct and indirect annual costs of caring for individuals with AD of at least $100 billion. Thus, a reasonable estimate is that California has more than half a million AD patients with an estimated cost to California of $12 billion per year! 2) One in 10 individuals over 65 and nearly half of those over 85 are affected, which means that as our population ages, we will be facing a tidal wave of AD. Current estimates are that with current rates of growth that the AD patient population will double or triple in the next 4 decades. 3) The potential benefit of research such as that proposed in this grant application is that finding a treatment that could delay onset by five years could reduce the number of individuals with Alzheimer’s disease by nearly 50 percent after 50 years. This would be significant since a person with Alzheimer’s disease will live an average of eight years and as many as 20 years or more from the onset of symptoms. Finding better treatments will thus have significant financial benefits to California. 4) After diagnosis, people with Alzheimer’s disease survive about half as long as those of similar age without AD or other dementia. 5) In terms of financial impact on California families, the statistics (http://www.alz.org/alzheimers_disease_alzheimer_statistics.asp) are that more than 7 out of 10 people with Alzheimer’s disease live at home. Almost 75 percent of their care is provided by family and friends. The remainder is “paid’ care costing an average of $19,000 per year. Families pay almost all of that out of pocket. The average cost for nursing home care is $42,000 per year but can exceed $70,000 per year in some areas of the country. The average lifetime cost of care for an individual with Alzheimer’s is $174,000. Thus, any progress in developing better therapy for AD will have a substantial positive impact to California. 
 
Progress Report: 
  • We have made significant progress on developing human stem cell based systems to probe the causes and features of Alzheimer's Disease. We are focusing on using human embryonic and human pluripotent stem cell lines carrying genetic changes that cause hereditary Alzheimer's Disease (AD). In one approach, we have made progress by developing iPS cells carrying small genetic changes in the presenilin 1 gene, which cause severe early onset AD. We also made substantial progress on developing methods to measure the distribution within neurons of products linked to Alzheimer's Disease. Finally, we have completed development of a cell sorting method to purify neuronal stem cells, neurons, and glia from human embryonic stem cells and human IPS cells. Together, these methods should allow us to continue making progress on using pluripotent human stem cells to probe the molecular basis for how cellular changes found in neurons in the brain of AD patients are generated. In addition, these methods we are developing are moving us closer to having sources of normal and AD human neurons generated in the laboratory for drug-testing and development.
  • We continue to make significant progress developing human stem cell based disease models to probe the causes of Alzheimer's Disease (AD) and to eventually develop drugs. In the past year we generated and analyzed several new human pluripotent stem cell lines (hIPS) carrying genetic changes that cause hereditary AD or that increase the risk of developing AD. We detected AD related characteristics in neurons with hereditary and in one case of a sporadic genetic type. While considerable confirmatory work needs to be done, our data raise the possibility that AD can be modeled in human neurons made from hIPS cells. In the coming year, we hope to continue making progress on using pluripotent human stem cells to probe the molecular basis for how cellular changes found in neurons in the brain of AD patients are generated. In addition, the methods we are developing are moving us closer to having sources of normal and AD human neurons generated in the laboratory for drug-testing and development.
  • In our final year of funding, we made significant progress developing human stem cell based disease models to probe the causes of Alzheimer's Disease (AD) and to eventually develop drugs. We generated and analyzed several new human pluripotent stem cell lines (hIPS) carrying genetic changes that cause hereditary AD or that increase the risk of developing AD. We detected AD related characteristics in neurons with hereditary and in one case of a sporadic genetic type. While considerable confirmatory work needs to be done, our data raise the possibility that AD can be modeled in human neurons made from hIPS cells. The methods we developed are moving us closer to having sources of normal and AD human neurons generated in the laboratory for drug-testing and development.

Elucidating pathways from hereditary Alzheimer mutations to pathological tau phenotypes

Funding Type: 
Basic Biology V
Grant Number: 
RB5-07011
ICOC Funds Committed: 
$1 161 000
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
We propose to elucidate pathways of genes that lead from early causes to later defects in Alzheimer’s Disease (AD), which is common, fatal, and for which no effective disease-modifying drugs are available. Because no effective AD treatment is available or imminent, we propose to discover novel genetic pathways by screening purified human brain cells made from human reprogrammed stem cells (human IPS cells or hIPSC) from patients that have rare and aggressive hereditary forms of AD. We have already discovered that such human brain cells exhibit an unique biochemical behavior that indicates early development of AD in a dish. Thus, we hope to find new drug targets by using the new tools of human stem cells that were previously unavailable. We think that human brain cells in a dish will succeed where animal models and other types of cells have thus far failed.
Statement of Benefit to California: 
Alzheimer’s Disease (AD) is a fatal neurodegenerative disease that afflicts millions of Californians. The emotional and financial impact on families and on the state healthcare budget is enormous. This project seeks to find new drug targets to treat this terrible disease. If we are successful our work in the long-term may help diminish the social and familial cost of AD, and lead to establishment of new businesses in California using our approaches.

Paracrine and synaptic mechanisms underlying neural stem cell-mediated stroke recovery

Funding Type: 
Basic Biology V
Grant Number: 
RB5-07363
ICOC Funds Committed: 
$1 178 370
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Stem cell therapy holds promise for the almost million Americans yearly who suffer a stroke. Preclinical data have shown that human neural stem cells (hNSCs) aid recovery after stroke, resulting in a major effort to advance stem cell therapy to the clinic, and we are currently transitioning our hNSC product to the clinic for stroke therapy. In this proposal we will explore how these cells improve lost function. We have already shown that injected hNSCs secrete factors that promote the gross rewiring of the brain, a major component of the spontaneous recovery observed after stroke. We now intend to focus on the connections between neurons, the synapses, which are a critical part of this rewiring process. We aim to quantify the effect of hNSCs on synapse density and function, and explore whether the stem cells secrete restorative synaptogenic factors or form functional synapses with pre-existing neurons. Our pursuit is made possible by our combination of state-of-the-art imaging techniques enabling us to visualize, characterize, and quantify these tiny synaptic structures and their interaction with the hNSCs. Furthermore, by engineering the hNSCs we can identify the factors they secrete in the brain and identify those which modulate synaptic connections. Our proposed studies will provide important insight into how transplanted stem cells induce recovery after stroke, with potential applicability to other brain diseases.
Statement of Benefit to California: 
Cerebrovascular stroke is the fourth leading cause of mortality in the United States and a significant source of long-term physical and cognitive disability that has devastating consequences to patients and their families. In California alone, over 9% of adults 65 years or older have had a stroke according to a 2005 study. In the next 20 years the societal toll is projected to amount to millions of patients and 18.8 billion dollars per year in direct medical costs. To date, there is no approved therapeutic agent for the recovery phase after stroke, making the long-term care of stroke patients a tremendous socioeconomic burden that will continue to rise as our aging population increases. Our laboratory and others have demonstrated the promise of stem cell transplantation to treat stroke. We are dedicated to developing human neural stem cells (hNSCs) as a novel neuro-restorative treatment for lost motor function after stroke. The goal of our proposed work is to further understand how transplanted hNSCs improve stroke recovery, as dissecting the mechanism of action of stem cells in the stroke brain will ultimately improve the chance of clinical success. This could potentially provide significant cost savings to California, but more importantly benefit the thousands of Californians and their families who struggle with the aftermath of stroke.

Misregulated Mitophagy in Parkinsonian Neurodegeneration

Funding Type: 
Basic Biology V
Grant Number: 
RB5-06935
ICOC Funds Committed: 
$1 174 943
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Parkinson’s disease (PD), is one of the leading causes of disabilities and death and afflicting millions of people worldwide. Effective treatments are desperately needed but the underlying molecular and cellular mechanisms of Parkinson’s destructive path are poorly understood. Mitochondria are cell’s power plants that provide almost all the energy a cell needs. When these cellular power plants are damaged by stressful factors present in aging neurons, they release toxins (reactive oxygen species) to the rest of the neuron that can cause neuronal cell death (neurodegeneration). Healthy cells have an elegant mitochondrial quality control system to clear dysfunctional mitochondria and prevent their resultant devastation. Based on my work that Parkinson’s associated proteins PINK1 and Parkin control mitochondrial transport that might be essential for damaged mitochondrial clearance, I hypothesize that in Parkinson’s mutant neurons mitochondrial quality control is impaired thereby leading to neurodegeneration. I will test this hypothesis in iPSC (inducible pluripotent stem cells) from Parkinson’s patients. This work will be a major step forward in understanding the cellular dysfunctions underlying Parkinson’s etiology, and promise hopes to battle against this overwhelming health danger to our aging population.
Statement of Benefit to California: 
Parkinson's disease (PD), one of the most common neurodegenerative diseases, afflicts millions of people worldwide with tremendous global economic and societal burdens. About 500,000 people are currently living with PD in the U.S, and approximate 1/10 of them live in California. The number continues to soar as our population continues to age. An effective treatment is desperately needed but the underlying molecular and cellular mechanisms of PD’s destructive path remain poorly understood. This proposal aims to explore an innovative and critical cellular mechanism that controls mitochondrial transport and clearance via mitophagy in PD pathogenesis with elegant employment of bold and creative approaches to live image mitochondria in iPSC (inducible pluripotent stem cells)-derived dopaminergic neurons from Parkinson’s patients. This study is closely relevant to public health of the state of California and will greatly benefit its citizens, as it will illuminate the pathological causes of PD and provide novel targets for therapuetic intervention.

Stem Cell-Derived Astrocyte Precursor Transplants in Amyotrophic Lateral Sclerosis

Funding Type: 
Early Translational from Disease Team Conversion
Grant Number: 
TRX-01471
ICOC Funds Committed: 
$4 139 754
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • Project Description and Rationale:
  • Amyotrophic Lateral Sclerosis (ALS) is the most common adult motor neuron disease, affecting 30,000 people in the US and the typical age of onset is in the mid-50s or slightly younger. ALS is a degenerative neural disease in which the damage and death of neurons results in progressive loss of the body’s functions until death, which is usually in 3-5 years of diagnosis. Current ALS treatments are primarily supportive, and providing excellent clinical care is essential for patients with ALS; however, there is an urgent need for treatments that significantly change the disease course. The only Food and Drug Administration approved, disease-specific medication for treatment of ALS is Rilutek (riluzole); which demonstrated only a modest effect on survival (up to 3 months) in clinical trials.
  • The ALS Disease Team/Early Translational project is focused on developing an ALS therapy based on human embryonic stem cell (ESC) derived neural stem cells (NSC) and/or astrocyte precursor cells transplanted into the ventral horn of the spinal cord. Several lines of evidence strongly support the approach of transplanting cells that exhibit the capacity to migrate, proliferate and mature into normal healthy astrocytes which can provide a neuroprotective effect for motor neurons and reduce or prevent neural damage and disease progression in ALS. Strong evidence has been generated from extensive studies in culture dishes and in animal models to support the concept that providing normal astrocytes in the proximity of α-motor neurons can protect them from neural damage.
  • Project Plan and Progress:
  • Multiple ESC lines were acquired in 2 rounds based on early and later availability. The first round of ESCs included ESCs from City of Hope (GMP H9) and the University of California, San Francisco (UCSF4). The second round included ESCs from the University of California, San Francisco [UCSFB6 (aka UCSF4.2) and UCSFB7 (aka UCSF4.3)] and from BioTime (ESI-017). These ESC lines were tested for their ability to survive and expand under conditions required for producing a cellular therapy (FDA GMP-like and GTP compliant conditions). From these ESC lines, NSCs were generated, expanded and characterized to determine their ability to produce stable and consistent populations of NSCs under conditions required for producing a cellular therapy.
  • For the first round of cell lines, both UCSF4 and H9 were successfully induced to produce NSCs, which were mechanically enriched, expanded and implanted into immunodeficient rats and a rat model of ALS (SOD1G93A). For this small-scale in vivo screen, implanted UCSF4 and H9 NSCs survived, migrated and differentiated into neurons and astrocytic cells in 3-5 weeks, without producing tumors or other unwanted structures. NSCs from both UCSF4 and H9 performed similarly in culture and in vivo, thus the decision to use UCSF4 in the larger-scale in vivo studies for safety (implant into immunodeficient rats) and efficacy/proof of concept (SOD1G93A ALS model rats) was weighted by the difficulties obtaining H9 for future studies for a therapeutic product. These larger-scale studies began August 2013 (earlier than projected), with expected completion in February 2014.
  • For the second round of ESC lines (UCSFB6, UCSFB7 and BioTime ESI-017), UCSFB6 and UCSFB7 ESCs expanded well, while ESI-017 expansion was less robust. Because UCSFB6 and UCSFB7 ESCs are from the same blastomere, we decided to continue to NSC production with only UCSFB7, keeping UCSFB6 in reserve as a back-up. UCSFB7 ESCs were successfully induced to produce NSCs, which were mechanically enriched, expanded and implanted into immunodeficient rats and a rat model of ALS (SOD1G93A). The results from these studies are pending (some animals are still in-life), but early histology suggests the cell survival is similar to UCSF4 and H9. A second round of large-scale in vivo studies is planned to start January 2014 to evaluate this NSC line. By September 2014, the “best” NSC line will be selected as a therapeutic candidate for definitive pre-clinical studies and entry into clinical trials.
  • ESC production under GMP-like condition has been completed at the UC Davis GMP facility. UC Davis generated the first batch of NSCs, which were not sufficiently homogeneous for successful expansion beyond approximately passage 10. This prompted UCSD to investigate multiple enrichment strategies, which were tested on multiple cell lines to ensure method reproducibility. A mechanical enrichment method reproducibly resulted in more homogeneous NSC cultures, capable of expansion for 20 – 30 passages, or more. The NSC generation and enrichment methods are currently being transferred to UC Davis and the UCSD scientist who developed the methods will work side-by-side with the UC Davis GMP production team to ensure successful method transfer to the GMP facility.
  • UCSF4 NSCs are also in use in a CIRM supported early translation study for spinal cord injury.
  • Project Description and Rationale:
  • Amyotrophic Lateral Sclerosis (ALS) is the most common adult motor neuron disease, affecting 30,000 people in the US and the typical age of onset is in the mid-50s or slightly younger. ALS is a degenerative neural disease in which the damage and death of neurons results in progressive loss of the body’s functions until death, which is usually in 3-5 years of diagnosis. Current ALS treatments are primarily supportive, and providing excellent clinical care is essential for patients with ALS; however, there is an urgent need for treatments that significantly change the disease course. The only Food and Drug Administration approved, disease-specific medication for treatment of ALS is Rilutek (riluzole); which demonstrated only a modest effect on survival (up to 3 months) in clinical trials.
  • The ALS Disease Team/Early Translational project is focused on developing an ALS therapy based on human embryonic stem cell (ESC) derived neural stem cells (NSC) and/or astrocyte precursor cells transplanted into the ventral horn of the spinal cord. Several lines of evidence strongly support the approach of transplanting cells that exhibit the capacity to migrate, proliferate and mature into normal healthy astrocytes which can provide a neuroprotective effect for motor neurons and reduce or prevent neural damage and disease progression in ALS.
  • Year 2 Progress Summary:
  • The longer-term, larger-scale in vivo safety and efficacy studies using the lines that showed the most promise during previous screening studies (UCSF4 and ESI-017 NSCs) have been completed. The safety studies were performed in immunodeficient rats to evaluate the survival, migration, differentiation, function and tumorigenicity of implanted NSCs at 3 weeks, 2 months and 6 months post implant. The efficacy studies were conducted in a transgenic SOD1G93A ALS rat model to evaluate safety and cell fate in the background of disease, as well as, to evaluate disease-modifying activity (e.g. neural protection/proof-of-concept) of the implanted NSCs.
  • NOTE: A labeling error occurred during expansion and banking of the ESCs at UC Davis, and the cell line labeled as UCSFB7 (aka UCSF4.3) was determined by DNA fingerprinting to actually be ESI-017. Previous NSC generation, characterization and in vivo screening data was reported for cell line UCSFB7 (aka UCSF4.3), which was actually for ESI-017.
  • Both UCSF4 and ESI-017 NSCs were deemed acceptable in 2 out of 3 of the minimal acceptance criteria:
  • 1) Long-term survival in nude and SOD1G93A rats
  • 2) No formation of tumors or other unwanted structures when implanted into nude or SOD rats.
  • The third criterion: at least 10% greater α-motor neuron counts in cell-injected animals as compared to medium injected controls (or cell-injected side compared to the non-injected, or contralateral side) was not met due to a) variability of α-motor neuron counts and b) the aggressive nature of the current SOD1G93A rat ALS model and resulting very short 2 month treatment window which exceeds the length of time for the migration, expansion, differentiation and maturation of sufficient astrocytes to provide a neural protective effect in all implanted animals.
  • UCSF4 NSCs were originally selected as the developmental candidate, however, there are compelling reasons to reconsider ESI-017 NSCs: 1) UC Davis has found ESI-017 NSCs relatively easy to generate and is having difficulty generating UCSF4 NSCs; and 2) recent hisotological evaluations suggest that ESI-017 NSCs produce mature astrocytes earlier in vivo than UCSF4 NSCs. We are working with UC Davis on generation of UCSF4 NSCs and are quantifying astrocyte maturation histology (e.g. GFAP) to make a well-supported developmental candidate selection.
  • In parallel, mRNA sequencing has been performed 1) on cells produced in the course of this project to identify potential markers predictive of in vivo fate, 2) on naïve SOD1G93A rats to explore markers of disease onset and progression that could potentially be used as surrogate markers of disease modulation in place of motor neuron counts, and 3) on NSCs implanted into nude and SOD1G93A rats to identify potential markers of long-term post-transplant NSC cell fate and host response.

A drug-screening platform for autism spectrum disorders using human astrocytes

Funding Type: 
Early Translational IV
Grant Number: 
TR4-06747
ICOC Funds Committed: 
$1 824 719
Disease Focus: 
Autism
Neurological Disorders
Rett's Syndrome
Pediatrics
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Autism spectrum disorders (ASD) are complex neurodevelopmental diseases that affect about 1% of 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. The causes and best treatments remain uncertain. One of the major impediments to ASD research is the lack of relevant human disease models. 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. The main goal of this project is to accelerate drug discovery to treat ASD using astrocytes generated from human iPSC. 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 astrocytes, we can begin to unravel how genetic variation in ASD dictates responses to different drugs. Insights that emerge from our studies may drive the development of new therapeutic interventions for ASD. They may also illuminate possible differences in drug responsiveness in different patients and potentially define a molecular signature resulting from ASD variants, which could predict the onset of disease before symptoms are seen.
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 brain cells 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.

Stem Cell Pathologies in Parkinson’s disease as a key to Regenerative Strategies

Funding Type: 
Research Leadership 10
Grant Number: 
LA1_C10-06535
ICOC Funds Committed: 
$6 718 471
Disease Focus: 
Parkinson's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
Protection and cell repair strategies for neurodegenerative diseases such as Parkinson’s Disease (“PD”) depend on well-characterized candidate human stem cells that are robust and show promise for generating the neurons of interest following stimulation of inherent brain stem cells or after cell transplantation. These stem cells must also be expandable in the culture dish without unwanted growth and differentiation into cancer cells, they must survive the transplantation process or, if endogenous brain stem cells are stimulated, they should insinuate themselves in established brain networks and hopefully ameliorate the disease course. The studies proposed for the CIRM Research Leadership Award have three major components that will help better understand the importance and uses of stem cells for the treatment of PD, and at the same time get a better insight into their role in disease repair and causation. First, we will characterize adult human neural stem cells from control and PD brain specimens to distinguish their genetic signatures and physiological properties of these cells. This will allow us to determine if there are stem cells that are pathological and fail in their supportive role in repairing the nervous system. Next, we will investigate a completely novel disease initiation and propagation mechanism, based on the concept that secreted vesicles from cells (also known as “exosomes”) containing a PD-associated protein, alpha-synuclein, propagate from cell-to cell. Our hypothesis is that these exosomes carry toxic forms of alpha-synuclein from cell to cell in the brain, thereby accounting disease spread. They may do the same with cells transplanted in patients with PD, thereby causing these newly transplanted cells designed to cure the disease, to be affected by the same process that causes the disease itself. This is a bottleneck that needs to be overcome for neurotransplantation to take its place as a standard treatment for PD. Our studies will address disease-associated toxicity of exosomal transmission of aggregated proteins in human neural precursor stem cells. Importantly, exosomes in spinal fluid or other peripheral tissues such as blood might represent a potentially early and reliable disease biomarker as well as a new target for molecular therapies aimed at blocking transcellular transmission of PD-associated molecules. Finally, we have chosen pre-clinical models with α-synucleinopathies to test human neural precursor stem cells as cell replacement donors for PD as well as interrogate, for the first time, their potential susceptibility to PD and contribution to disease transmission. These studies will provide a new standard of analysis of human neural precursor cells at risk for and contributing to pathology (so-called “stem cell pathologies”) in PD and other neurodegenerative diseases via transmission of altered or toxic proteins from one cell to another.
Statement of Benefit to California: 
According to the National Institute of Health, Parkinson’s disease (PD) is the second most common neurodegenerative disease in California and the United States (one in 100 people over 60 is affected) second only to Alzheimer’s Disease. Millions of Americans are challenged by PD, and according to the Parkinson’s Action Network, every 9 minutes a new case of PD is diagnosed. The cause of the majority of idiopathic PD is unknown. Identified genetic factors are responsible for less than 5% of cases and environmental factors such as pesticides and industrial toxins have been repeatedly linked to the disease. However, the vast majority of PD is thought to be etiologically multi-factorial, resulting from both genetic and environmental risk factors. Important events leading to PD probably occur in early or mid adult life. According to the Michael J. Fox Foundation, “…there is no objective test, or reliable biomarker for PD, so rate of misdiagnosis is high, and there is a seriously pressing need to develop better early detection approaches to be able to attempt disease-halting protocols at a non-symptomatic, so-called prodromal stage.” The proposed innovative and transformative research program will have a major direct impact for patients who live in California and suffer from PD and other related neurodegenerative diseases. If these high-risk high-pay-off studies are deemed successful, this new program will have tackled major culprits in the PD field. They could lead to a better understanding of the role of stem cells in health and disease. Furthermore they could greatly advance our knowledge of how the disease spreads throughout the brain which in turn could lead to entire new strategies to halt disease progression. In a similar manner these studies could lead to ways to prevent the disease from spreading to cells that have been transplanted to the brain of Parkinson’s patients in an attempt to cure their disease. This is critical for neurotransplantation to thrive as a therapeutic approach to treating PD. In addition, if we extend the cell-to-cell transmissible disease hypothesis to other neurodegenerative diseases, and cancer, the studies proposed here represent a new diagnostic approach and therapeutic targets for many diseases affecting Californians and humankind in general. This CIRM Research Leadership Award will not only have an enormous impact on understanding the cause of PD and developing new therapeutic strategies using stem cells and its technologies, this award will also be the foundation of creating a new Center for Translational Stem Cell Research within California. This could lead to further growth at the academic level and for the biotechnology industry, particularly in the area regenerative medicine.

Stem Cell Mechanisms Governing Discrete Waves of Gliogenesis in the Childhood Brain

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06093
ICOC Funds Committed: 
$1 264 248
Disease Focus: 
Neurological Disorders
Pediatrics
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
White matter is the infrastructure of the brain, providing conduits for communication between neural regions. White matter continues to mature from birth until early adulthood, particularly in regions of brain critical for higher cognitive functions. However, the precise timing of white matter maturation in the various neural circuits is not well described, and the mechanisms controlling white matter developmental/maturation are poorly understood. White matter is conceptually like wires and their insulating sheath is a substance called myelin. It is clear that neural stem and precursor cells contribute significantly to white matter maturation by forming the cells that generate myelin. In the proposed experiments, we will map the precise timing of myelination in the human brain and changes in the populations of neural precursor cells that generate myelin from birth to adulthood and define mechanisms that govern the process of white matter maturation. The resulting findings about how white matter develops may provide insights for white matter regeneration to aid in therapy for diseases such as cerebral palsy, multiple sclerosis and chemotherapy-induced cognitive dysfunction.
Statement of Benefit to California: 
Diseases of white matter account for significant neurological morbidity in both children and adults in California. Understanding the cellular and molecular mechanisms that govern white matter development the may unlock clues to the regenerative potential of endogeneous stem and precursor cells in the childhood and adult brain. Although the brain continues robust white matter development throughout childhood, adolescence and young adulthood, relatively little is known about the mechanisms that orchestrate proliferation, differentiation and functional maturation of neural stem and precursor cells to generate myelin-forming oligodendrocytes during postnatal brain development. In the present proposal, we will define how white matter precursor cell populations vary with age throughout the brain and determine if and how neuronal activity instructs neural stem and precursor cell contributions to human white matter myelin maturation. Disruption of white matter myelination is implicated in a range of neurological diseases, including cerebral palsy, multiple sclerosis, cognitive dysfunction from chemotherapy exposure, attention deficit and hyperactivity disorder (ADHD) and even psychiatric diseases such as schizophrenia. The results of these studies have the potential to elucidate clues to white matter regeneration that may benefit hundreds of thousands of Californians.
Progress Report: 
  • Formation of the insulated fiber infrastructure of the human brain (called "myelin") depends upon the function of a precursor cell type called "oligodendrocyte precursor cells (OPC)". The first Aim of this study seeks to determine how OPCs differ from each other in different regions of the brain, and over different ages. Understanding this heterogeneity is important as we explore the regenerative capacity of this class of precursor cells. We have, in the past year, isolated OPCs from various regions of the human brain from individuals at various ages and are studying the molecular characteristics of these precursor cells at the single cell level in order to define distinct OPC subpopulations. We have also begun to study the functional capabilities of OPCs isolated from different brain regions. The second Aim of this study seeks to understand how interactions between electrically active neurons and OPCs affect OPC function and myelin formation. We have found that when mouse motor cortex neurons "fire" signals in such a way as to elicit a complex motor behavior, much as would happen when one practices a motor task, OPCs within that circuit respond and myelination increases. This affects the function of that circuit in a lasting way. These results indicate that neurons and OPCs interact in important ways to modulate myelination and supports the hypothesis that OPC function may play a role in learning.

Stem cell models to analyze the role of mutated C9ORF72 in neurodegeneration

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06045
ICOC Funds Committed: 
$1 393 200
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Dementia
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Amyotrophic lateral sclerosis (ALS) is an idiopathic adult-onset degenerative disease characterized by progressive weakness from loss of upper and lower motor neurons. Onset is insidious, progression is essentially linear, and death occurs within 3-5 years in 90% of patients. In the US, 5,000 deaths occur per year and in the world, 100,000. In October, 2011, the causative gene defect in a long sought after locus on chromosome 9 for ALS, frontotemporal dementia (FTD) and overlap ALS-FTD was identified to be a expansion of a hexanucleotide repeat in the uncharacterized C9ORF72 gene. The goal of the proposed research is to generate human stem cell models from cells derived from ALS patients with the C9ORF72 expanded repeats and relevant control cells using genome-editing technology. We will also generate a stem cell model expressing the repeat independent of the C9ORF72 gene to study if the repeat alone is causing neural defects. Using advanced genome technologies, biochemical and cellular approaches, we will study the molecular pathways affected in motor neurons derived from these stem cell models. Finally, we will use innovative technologies to rescue the abnormal phenotypes that arise from the expanded repeat in human motor neurons. Completion of the proposed research is expected to transform our understanding of the regulatory and pathogenetic mechanisms underlying ALS and FTD, and establish therapeutic options for these debilitating diseases.
Statement of Benefit to California: 
Our research provides the foundation for decoding the mechanisms that underlie the single most frequent genetic mutation found to contribute to both ALS and FTD, debilitating neurological diseases that impact many Californians. In California, the expected prevalence of ALS (the number of total existing cases) is 2,200 to 3,000 cases at any one time, and the incidence is 750-1,100 new cases each year. The number of FTD cases is five times as many. Our research has and will continue to serve as a basis for understanding deviations from normal and disease patient neuronal cells, enabling us to make inroards to understanding neurological disease modeling using neurons differentiated from reprogammed patient-specific lines. Such disease modeling will have great potential for California health care patients, pharmaceutical and biotechnology industries in terms of improved human models for drug discovery and toxicology testing. Our improved knowledge base will support our efforts as well as other Californian researchers to study stem cell models of neurological disease and design new diagnostics and treatments, thereby maintaining California's position as a leader in clinical research.
Progress Report: 
  • Expanded hexanucleotide repeats in the C9ORF72 gene were identified in Oct 2011 as a cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), thus identifying the single most frequent genetic cause of each and connecting them to repeat expansion disease. We are developing stem cell disease models to enable key hypotheses of pathogenesis and new interventions to be tested. We have successfully engineered stem cell models to analyze the effects of C9ORF72 mutations, and have differentiated these stem cell models into motor neurons which enabled us to conduct transcriptomic and biochemical studies. In addition, we have utilized antisense-oligonucleotides (ASOs) from ISIS Pharmaceuticals to deplete mutant C9ORF72 in motor neurons. We expect our efforts to provide mechanistic insights and a potential therapy in human cells.

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