Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders
Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00116
Investigator: 
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.
Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00115
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$2 879 210
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Parkinson's Disease
Genetic Disorder
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

Human embryonic stem cells (hESCs) are pluripotent entities, capable of generating a whole-body spectrum of distinct cell types. We have developmental procedures for inducing hESCs to develop into pure populations of human neural stem cells (hNS), a step required for generating authentic mature human neurons. Several protocols have currently been developed to differentiate hESCs to what appear to be differentiated dopaminergic neurons (important in Parkinson’s disease (PD) and cholinergic motor neurons (important in Amyolateral Sclerosis (ALS) in culture dishes. We have developed methods to stably insert new genes in hESC and we have demonstrated that these transgenic cells can become mature neurons in culture dishes. We plan to over express alpha synuclein and other genes associated with PD and superoxide dismutase (a gene mutated in ALS) into hESCs and then differentiate these cells to neurons, and more specifically to dopaminergic neurons and cholinergic neurons using existing protocols. These transgenic cells can be used not only for the discovery of cellular and molecular causes for dopaminergic or cholinergic cell damage and death in these devastating diseases, but also can be used as assays to screen chemical libraries to find novels drugs that may protect against the degenerative process. Until recently the investigation of the differentiation of these human cells has only been observed in culture dishes or during tumor formation. Our recent results show that hESC implanted in the brains of mice can survive and become active functional human neurons that successfully integrate into the adult mouse forebrain. This method of transplantation to generate models of human disease will permit the study of human neural development in a living environment, paving the way for the generation of new models of human neurodegenerative and psychiatric diseases. It also has the potential to speed up the screening process for therapeutic drugs.

Statement of Benefit to California: 

We plan to develop procedures to induce human ES cells into mature functioning neurons that carry genes that cause the debilitating human neurological diseases, Parkinson’s disease and Amyolateral Sclerosis (ALS). We will use the cells to reveal the genes and molecular pathways inside the cells that are responsible for how the mutant genes cause damage to specific types of brain cells. We also will make the cells available to other researchers as well as biotech companies so that other investigators can use these cells to screen small molecule and chemical libraries to discover new drugs that can interfere with the pathology caused by these mutant cells that mimic human disease, in hopes of accelerating the pace of discovery.

Progress Report: 
  • Our research is focused on studying two debilitating diseases of the nervous system: Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS) also known as Lou Gehrig’s disease. While the causes and symptoms of these two conditions are very different, they share one aspect in common: patients gradually lose specific types of nerve cells, namely the so-called dopaminergic neurons in PD, and motor neurons in ALS. If we can find ways to protect the neurons from dying, we might be able to slow or even halt disease progression in ALS and PD patients. In the past two years, our lab has developed robust procedures to generate these two classes of neurons from human embryonic stem cells and we have been studying the molecular changes that govern their specialization. Since last year, we have been using neurons to elucidate the molecular mechanisms that underlie the demise of these cells.
  • ALS is one of the most common neuromuscular diseases, afflicting more than 30,000 Americans. Patients rapidly lose their motor neurons – the nerve cells that extend from the brain through the spinal cord to the muscles, thereby controlling their movement. Therapy options are extremely limited and people with ALS usually succumb to respiratory failure or pneumonia within three to five years from the onset of symptoms. Most ALS patients have no family history of ALS and carry no known genetic defects that may help explain why they develop the disease. However, a small number of ALS patients have mutations in the superoxide dismutase 1 (SOD1) gene, which encodes an enzyme that scavenges so-called free radicals – aggressive oxidizing molecules that are by-products of the cells’ normal metabolism. Researchers therefore believe that accumulation of these free radicals may damage motor neurons in ALS and contribute to their death.
  • To test this idea, we introduced the mutated form of the SOD1 gene into astrocytes – cells that provide metabolic and structural support to neurons – and cultured our stem cell-derived motor neurons along with these SOD1-mutant astrocytes. Indeed, while motor neurons grown on ‘normal’ astrocytes were fully viable, we saw widespread death of motor neurons in cocultures with ‘mutant’ astrocytes, along with elevated levels of free radicals. We think that this is due to our mutant astrocytes being causing inflammation, and so our future efforts are focused on understanding the role of the immune system, specifically the function of microglia – the resident immune cells of the brain and spinal cord – in our co-cultures with human motor neurons. We are very excited about these results because they show that our cocultures may be a very useful tool to screen drugs that may counteract the neurotoxicity caused by inflammation and free radicals. We have already begun testing several known antioxidants, and found some of them to be very effective in improving motor neuron survival in the culture dish. Such compounds may ultimately improve the condition of ALS patients.
  • PD is the second most common neurodegenerative disease and develops when neurons in the brain, and in particular, in a part of the brain known as the substantia nigra die. These neurons are called dopaminergic because they produce dopamine, a molecule that is necessary for coordinated body movement. Many dopaminergic neurons are already lost when patients develop PD symptoms, which include trembling, stiffness, and slow movement. Around one million Americans are currently suffering from PD, and 60,000 new cases are diagnosed each year. While several surgical and pharmacological treatment options exist, they cannot slow or halt disease progression and are instead aimed at treating the symptoms. The exact causes for neuron death in PD are unknown but among others inflammation in the affected brain area may play a role in disease progression.
  • In a joint effort with the laboratories of Christopher Glass and Michael Rosenfeld at the University of California, San Diego, we showed using animal experiments that a protein called Nurr1 is crucial for the development and survival of dopaminergic neurons. We found that the Nurr1 gene is turned on by inflammatory signals and suppresses genes that encode neurotoxic factors. Microglia are the major initiators of the neurotoxic response to inflammatory stimuli, which is then amplified by astrocytes. Thus our findings reveal an important role for Nurr1 in microglia and astrocytes to protect dopaminergic neurons from exaggerated production of inflammation-induced neurotoxic mediators. We are now using human embryonic stem cell-derived dopaminergic neurons, cultured along with human atrocytes and microglia to test whether we can demonstrate this positive role of Nurr1 in a culture dish as well.
  • We are investigating the molecular mechanisms underlying two major neurological diseases: Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease. In the past year, we have taken our previously developed human embryonic stem cell (hESC)-based cell culture model for PD and ALS another step further: we have begun building an assay system that may eventually allow both the identification of biomarkers for early diagnosis and the screening of drug candidates for ALS and PD. By transplanting hESC-derived neurons into live animals and brain slices, we have also made first inroads into recapitulating the disease processes in animal model systems.
  • While the causes and symptoms of ALS and PD are very different, they share one aspect in common: in both, patients gradually lose specific types of nerve cells, namely, the so-called dopaminergic neurons in PD, and motor neurons in ALS; it this neuron death that causes both diseases. Previously, we showed with our hESC-based cell culture system that an inflammatory response in astrocytes (the brain cells that provide metabolic and structural support to neurons) is involved in loss of motor neurons. Similarly, we demonstrated that microglia (the brain’s immune cells) and astrocytes together protect dopaminergic neurons from exaggerated production of inflammation-induced neurotoxic mediators. This function of astrocytes and microglia was dependent on a protein called Nurr1: we found that the Nurr1 gene is turned on by inflammatory signals and suppresses genes that encode neurotoxic factors.
  • We have now begun to characterize in depth the specific signaling molecules that communicate the inflammation cue from the glial cells to neurons. To do this, we cultured astrocytes and microglia in the petri dish, induced inflammation and collected cell culture supernatants from the ‘inflamed’ and normal cells. We then measured the levels of specific so-called cytokines, the inflammatory signaling molecules secreted by the glial cells. Once we have obtained a characteristic cytokine ‘signature’ of disease-associated glial cells, we can begin to unravel the molecular pathways that lead to inflammation. Thus our research may lead to the discovery of early diagnostic markers and enable drug screening for compounds that suppress or prevent these neurotoxic inflammatory processes.
  • Our cell culture assays have provided a great deal of insight into the signaling cascades that eventually lead to neuron death. However, they probably cannot fully recapitulate the complex interplay between the neurons and the cellular environment in which they reside within the brain. We have therefore begun to transplant hESC-derived neurons into the brains of mice. Our results indicate that the neurons rapidly extended processes and developed dendritic branches and axons that integrated into the existing neuronal network. In the coming year, we plan to build on these results, using our hESC-derived neuronal models of PD and ALS to better understand mechanisms of dysregulation. Specifically, we will examine alterations in synapse formation, cell survival, and neuron maturation. We will also devise strategies for functional recovery and rescue in the context of the living animal.
  • We are investigating the molecular mechanisms underlying two major neurological diseases: Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease. In the past year, we took our human embryonic stem cell (hESC)-based neural cell culture model for PD and ALS another step further and built sensitive and quantitative assays that can allow for the screening of drug candidates for ALS and PD. We have also consistently improved our transplanting techniques and are now able to detect functional, electrophysiologically active, hESC-derived neurons in live animals. This experiment was crucial to show that, under our culture conditions, human neurons derived from embryonic stem cells were able to integrate and form meaningful connections with other neurons in a given adult brain environment.
  • Moreover, we are now performing an in-depth characterization of the specific signaling molecules that communicate the inflammation cues from the glial cells to neurons in the presence of ALS-causing mutations (SOD1G37R) and PD-causing mutations (recombinant alfa-synuclein). In this report we have explored another functional assay to measure glial function and inflammatory response using astrocytes that express ALS-causing mutations. In addition, we report here that adding PD-causing mutagens to mixed cultures of human neurons and astrocytes results in the death of dopaminergic neurons, the type of neurons affected in PD. We are currently testing new compounds that can decrease the neuronal toxicity observed.
  • Our research may not only lead to the discovery of early diagnostic markers but also enable drug screening for compounds that suppress or prevent these neurotoxic inflammatory processes.
Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00111
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$2 516 613
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 

Human embryonic stem cells (hESCs) have the potential to become all sorts of cells in human body including nerve cells. Moreover, hESCs can be expanded in culture plates into a large quantity, thus serving as an ideal source for cell transplantation in clinical use. However, the existing hESC lines are not fully characterized in terms of their potential to become specific cell types such as nerve cells. It is also unclear if the nerve cells that are derived from hESCs are totally normal when tested in cell transplantation experiments. One of the goals for our proposal is to compare the quality and the potential of eight lines of hESCs in their capacity to become nerve cells. To measure if the nerve cells that are derived from hESCs are normal when compared to the nerve cells in normal human beings, we will examine the levels of gene expression and the mechanisms that control gene expression in hESC-derived nerve cells. Specifically, we will examine the pattern of DNA modification, namely DNA methylation, in the DNA of nerve cells. This DNA modification is involved in the inhibition of gene expression. It is known that if DNA methylation pattern is abnormal, it can lead to human diseases including cancer and mental retardation disorders. We will use a DNA microarray technology to identify DNA methylation pattern in the critical regions where gene expression is controlled. Our recent results suggest that increased DNA methylation is observed in hESC-derived nerve cells. In this proposal, we will also test if we can balance the level of DNA methylation through pharmacological treatment of enzymes that are responsible for DNA methylation. Finally, we will test if hESC-derived nerve cells can repair the brain after injury . A mouse stroke model will be used for testing the mechanisms stem cell-mediated repair and recovery in the injured brain and for selecting the best nerve cells for cell transplantation. Our study will pave the way for the future use of hESC-derived nerve cells in clinical treatment of nerve injury and neurodegenerative diseases such as stroke and Parkinson’s disease.

Statement of Benefit to California: 

Neurodegenerative diseases such as stroke are the leading cause of adult disability. Stroke produces an area of damage in the brain which frequently causes the loss of crucial brain functions such as sensory and movement control, language skills, and cognition capability. Stem cell transplantation has emerged as a method that may improve recovery in these brain areas. Studies of stem cell transplantation after stroke have been limited because many of the transplanted cells do not survive, the appropriate regions for transplantation have not been identified, and the mechanisms by which transplanted stem cells improve recovery have not been determined. Also, there have been no studies of human embryonic stem cell transplantation after stroke. For the use of stem cell therapy in stroke patients, human embryonic stem cell lines have to be grown and tested for their efficacy in repairing the brain after stroke. We have recently found that the process of growing human embryonic stem cells in culture introduces genetic modifications in some of these cell lines that may decrease survival of the cells in the brain and impair their ability to repair the injured brain. The experiments in this grant will determine which human embryonic stem cell lines do not undergo this negative genetic modification. The optimum human embryonic stem cell lines will then be systematically tested for the location in the stroke brain that produces survival and integration, and the mechanisms of repair that these cells mediate in the brain after stroke. These studies will specifically test the role of human embryonic stem cells in improving sensory and movement functions after stroke. In summary, these studies will establish protocols for the proper growth of human embryonic stem cell lines, the lines that are most effective for repairing the brain after stroke, and the principles behind how human embryonic stem cells repair the brain. These results are applicable to other kinds of neurodegenerative conditions, such as Parkinsons, Alzheimer’s and Huntington’s diseases, and to the growth and culture of human embryonic stem cells in general for repair of disease of other human tissues.

Progress Report: 
  • Summary of Research Progress:
  • Our research aims to identify the optimal culture conditions and the best hESC lines for the derivation of nerve lineage cells in therapeutic cell transplantation. Toward this goal, we propose to compare the behavior of nerve cell differentiation in multiple lines of hESCs in one laboratory setting. We will further characterize molecular changes during directed cell differentiation and identify the cells that exhibit a pattern of DNA modification, namely DNA methylation, similar to primary neural cells in human brain. In the case of DNA hypermethylation, pharmacological treatment and genetic manipulation will be applied to correct the methylation defects by blocking enzymes involved in DNA methylation. Finally, cell transplantation in a mouse stroke model will be used to study the mechanisms and efficacy of different types of hESC-derived neural cells in neural repair.
  • In the past year, we have made progress in guiding several lines of human stem cells into nerve cells. We are now ready to compare the property of different lines of nerve cells such as the efficiency of nerve cell differentiation and the preferential production of specific nerve cells in culture. We also begin to produce and characterize a new type of human stem cells, namely induced pluripotent cells that are obtained by converting somatic cells into stem cell through reprogramming. We also test the pattern of DNA methylation in different lines of human stem cells. By engineering stem cells carrying different levels of methylation, we aim to find the optimal levels of DNA methylation for efficient nerve cell differentiation. Finally, we also made excellent progress on the procedure of cell transplantation. We have found a suitable substrate that can be used to enhance neuronal survival after cell transplantation and we expect to publish a research paper in this new method of cell transplantation.
  • Summary of Research Progress:
  • Our research aims to identify the optimal culture conditions and the best hESC lines for the derivation of nerve lineage cells in therapeutic cell transplantation. Toward this goal, we propose to compare the behavior of nerve cell differentiation in multiple lines of hESCs in one laboratory setting. We will further characterize molecular changes during directed cell differentiation and identify the cells that exhibit a pattern of DNA modification, namely DNA methylation, similar to primary neural cells in human brain. In the case of DNA hypermethylation, pharmacological treatment and genetic manipulation will be applied to correct the methylation defects by blocking enzymes involved in DNA methylation. Finally, cell transplantation in a mouse stroke model will be used to study the mechanisms and efficacy of different types of hESC-derived neural cells in neural repair.
  • In the past year, we have made great progress in converting several lines of human stem cells into nerve cells. We have compared the property of different lines of nerve cells such as the efficiency of nerve cell differentiation and the preferential production of specific nerve cells in culture. We also begin to produce and characterize a new type of human stem cells, namely induced pluripotent cells that are obtained by converting somatic cells into stem cell through reprogramming. We also test the pattern of DNA methylation in different lines of human stem cells. By engineering stem cells carrying different levels of methylation, we aim to find the optimal levels of DNA methylation for efficient nerve cell differentiation. Finally, we also made excellent progress on the procedure of cell transplantation. We have found a suitable substrate that can be used to enhance neuronal survival after cell transplantation and we expect to publish a research paper in this new method of cell transplantation.
  • Our research aims to identify the optimal culture conditions and the best hESC lines for the derivation of nerve lineage cells in therapeutic cell transplantation. Toward this goal, we propose to compare the behavior of nerve cell differentiation in multiple lines of hESCs in one laboratory setting. We will further characterize molecular changes during directed cell differentiation and identify the cells that exhibit a pattern of DNA modification, namely DNA methylation, similar to primary neural cells in human brain. In the case of DNA hypermethylation, pharmacological treatment and genetic manipulation will be applied to correct the methylation defects by blocking enzymes involved in DNA methylation. Finally, cell transplantation in a mouse stroke model will be used to study the mechanisms and efficacy of different types of hESC-derived neural cells in neural repair.
  • In the past year, we have made great progress in converting several lines of human stem cells into nerve cells. We have compared the property of different lines of nerve cells such as the efficiency of nerve cell differentiation and the preferential production of specific nerve cells in culture. We also succeeded in making a new type of human stem cells, namely induced pluripotent cells that are obtained by converting somatic cells into stem cell through reprogramming. We have tested the pattern of DNA methylation in different lines of human stem cells, including mutant cell lines from patients who exhibit defects in DNA methylaiton. Finally, we also made excellent progress on the procedure of cell transplantation and we characterized gene expression and epigenetic changes in transplanted nerve cells from human embryonic stem cells. Our studies allow us to optimize methods of neural cell differentiation and transplantation. We plan to publish additional two research papers in the near future.
Funding Type: 
SEED Grant
Grant Number: 
RS1-00462
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$791 000
Disease Focus: 
Autism
Neurological Disorders
Developmental Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

Many mental disorders are closely associated with problems that occur during brain development in early life. For instance, by 2 years of age, autistic children have larger brains than normal kids, likely due to, at least in part, excess production of neurons and support cells, the building blocks of the nervous system. In autistic brains, how neurons grow various thread-like processes also shows some abnormalities. The cause of autism is complex and likely involves many genetic factors. These developmental defects are also associated with mental disorders caused by single-gene mutations, such as Rett syndrome and fragile X syndrome, the most common form of inherited mental retardation, whose clinical features overlap with autism. However, what causes the developmental defects in brains of children with different mental disorders is largely unknown. In recent years, an exciting new regulatory pathway was discovered that may well contribute to the etiology of mental disorders. The major player in this novel pathway is a class of tiny molecules 21

Statement of Benefit to California: 

California is the most populated state in the US and has a large number of patients suffering from various mental disorders. The proposed studies in this grant application will contribute to the mission of developing novel avenues through stem cell research for the diagnosis, prevention and treatment of mental disorders

Progress Report: 
  • Human stem cells, both embryonic and induced pluripotent stem cells, offer exciting opportunities for cell-based therapies in injured or diseased human brains or spinal cords. The clinical efficacy of grafted progenitor cells critically depends on their ability to migrate to the appropriate sites in the adult central nervous system without unwanted proliferation and tumor formation. However, little is known about the cellular behavior of human neural progenitor cells derived from human stem cells or how their proliferation and migration are coordinated. During this reporting period, we continued to study human neural progenitor cells derived from human stem cells, a cell culture system established during the prior reporting period. We focused on microRNAs, a class of small, noncoding RNAs of ~21–23 nucleotides that regulate gene expression at the posttranscriptional level. These small RNAs mostly destabilize target mRNAs or suppress their translation by binding to complementary sequences in the 3' untranslated regions (3'UTRs). Our results obtained during this reporting period indicate that some microRNAs have very interesting functions in human neural progenitors, both in in vitro cell culture system and when transplanted into mouse brains. These new findings may have important implications for stem cell based therapies for neurodegenerative diseases or brain/spinal cord injuries.
Funding Type: 
SEED Grant
Grant Number: 
RS1-00413
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$625 617
Disease Focus: 
Cancer
Neurological Disorders
Skeletal Muscle
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

A variety of stem cells exist in humans throughout life and maintain their ability to divide and change into multiple cell types. Different types of adult derived stem cells occur throughout the body, and reside within specific tissues that serve as a reserve pool of cells that can replenish other cells lost due to aging, disease, trauma, chemotherapy or exposure to ionizing radiation. When conditions occur that lead to the depletion of these adult derived stem cells the recovery of normal tissue is impaired and a variety of complications result. For example, we have demonstrated that when neural stem cells are depleted after whole brain irradiation a subsequent deficit in cognition occurs, and that when muscle stem cells are depleted after leg irradiation an accelerated loss of muscle mass occurs. While an increase in stem cell numbers after depletion has been shown to lead to some functional recovery in the irradiated tissue, such recovery is usually very prolonged and generally suboptimal.Ionizing radiation is a physical agent that is effective at reducing the number of adult stem cells in nearly all tissues. Normally people are not exposed to doses of radiation that are cause for concern, however, many people are subjected to significant radiation exposures during the course of clinical radiotherapy. While radiotherapy is a front line treatment for many types of cancer, there are often unavoidable side effects associated with the irradiation of normal tissue that can be linked to the depletion of critical stem cell pools. In addition, many of these side effects pose particular threats to pediatric patients undergoing radiotherapy, since children contain more stem cells and suffer higher absolute losses of these cells after irradiation.Based on the foregoing, we will explore the potential utility and risks associated with using human embryonic stem cells (hESC) in the treatment of certain adverse effects associated with radiation-induced stem cell depletion. Our experiments will directly address whether hESCs can be used to replenish specific populations of stem cells in the brain and muscle depleted after irradiation in efforts to prevent subsequent declines in cognition and muscle mass respectively. In addition to using hESC to hasten the functional recovery of tissue after irradiation, we will also test whether implantation of such unique cells holds unforeseen risks for the development of cancer. Evidence suggests that certain types of stem cells may be prone to cancer, and since little is known regarding this issue with respect to hESC, we feel this critical issue must be addressed. Thus, we will investigate whether hESC implanted into animals develop into tumors over time. The studies proposed here comprise a first step in determining how useful hESCs will be in the treatment of humans exposed to ionizing radiation, as well as many other diseases where adult stem cell depletion might be a concern.

Statement of Benefit to California: 

Radiotherapy is a front line treatment used in California for many types of cancer, including brain, breast, prostate, bone and other cancer types presenting surgical complications. Treatment of these cancers through the use of radiation is however, often associated with side effects caused by the depletion of critical stem cell pools contained within non-cancerous normal tissue. While radiotherapy is clearly beneficial overall, many of these side effects have no viable treatment options. If we can demonstrate that human embryonic stem cells (hESC) hold promise as a safe therapeutic agent for the treatment of radiation-induced stem cell depletion, then cancer patients may have a new treatment for countering many of the debilitating side effects associated with radiotherapy. Once developed this new technology could position California to attract cancer patients throughout the United States, and the state would clearly benefit from the increased economic activity associated with a rise in patient numbers.

Progress Report: 
  • We have undertaken an extensive series of studies to delineate the radiation response of human embryonic stem cells (hESCs) and human neural stem cells (hNSCs) both in vitro and in vivo. These studies are important because radiotherapy is a frontline treatment for primary and secondary (metastatic) brain tumors. While radiotherapy is quite beneficial, it is limited by the tolerance of normal tissue to radiation injury. At clinically relevant exposures, patients often develop variable degrees of cognitive dysfunction that manifest as impaired learning and memory, and that have pronounced adverse effects on quality of life. Thus, our studies have been designed to address this serious complication of cranial irradiation.
  • We have now found that transplanted human embryonic stem cells (hESCs) can rescue radiation-induced cognitive impairment in athymic rats, providing the first evidence that such cells can ameliorate radiation-induced normal-tissue damage in the brain. Four months following head-only irradiation and hESC transplantation, the stem cells were found to have migrated toward specific regions of the brain known to support the development of new brain cells throughout life. Cells migrating toward these specialized neural regions were also found to develop into new brain cells. Cognitive analyses of these animals revealed that the rats who had received stem cells performed better in a standard test of brain function which measures the rats’ reactions to novelty. The data suggests that transplanted hESCs can rescue radiation-induced deficits in learning and memory. Additional work is underway to determine whether the rats’ improved cognitive function was due to the functional integration of transplanted stem cells or whether these cells supported and helped repair the rats’ existing brain cells.
  • The application of stem cell therapies to reduce radiation-induced normal tissue damage is still in its infancy. Our finding that transplanted hESCs can rescue radiation-induced cognitive impairment is significant in this regard, and provides evidence that similar types of approaches hold promise for ameliorating normal-tissue damage throughout other target tissues after irradiation.
  • A comprehensive series of studies was undertaken to determine if/how stem cell transplantation could ameliorate the adverse effects of cranial irradiation, both at the cellular and cognitive levels. These studies are important since radiotherapy to the head remains the only tenable option for the control of primary and metastatic brain tumors. Unfortunately, a devastating side-effect of this treatment involves cognitive decline in ~50% of those patients surviving ≥ 18 months. Pediatric patients treated for brain tumors can lose up to 3 IQ points per year, making the use of irradiation particularly problematic for this patient class. Thus, the purpose of these studies was to determine whether cranial transplantation of stem cells could afford some relief from the cognitive declines typical in patients afflicted with brain tumors, and subjected to cranial radiotherapy. Human embryonic (hESCs) and neural (hNSCs) stem cells were implanted into the brain of rats following head only irradiation. At 1 and 4 months later, rats were tested for cognitive performance using a series of specialized tests designed to determine the extent of radiation injury and the extent that transplanted cells ameliorated any radiation-induced cognitive deficits. These cognitive tasks take advantage of the innate tendency of rats to explore novelty. Successful performance of this task has been shown to rely on intact spatial memory function, a brain function known to be adversely impacted by irradiation. Our data shows that irradiation elicits significant deficits in learning and spatial task recognition 1 and 4-months following irradiation. We have now demonstrated conclusively, and for the first time, that irradiated animals receiving targeted transplantation of hESCs or hNSCs 2-days after, show significant recovery of these radiation induced cognitive decrements. In sum, our data shows the capability of 2 stem cell types (hESC and hNSC) to improve radiation-induced cognitive dysfunction at 1 and 4 months post-grafting, and demonstrates that stem cell based therapies can be used to effectively to reduce a serious complication of cranial irradiation.
Funding Type: 
SEED Grant
Grant Number: 
RS1-00409
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$425 594
Disease Focus: 
Multiple Sclerosis
Neurological Disorders
Immune Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

Multiple sclerosis (MS) is the most common neurologic disease affecting young adults under the age of 40 with the majority of MS patients diagnosed in the second or third decade of life. MS is characterized by the gradual loss of the myelin sheath that surrounds and insulates axons that allow for the conduction of nerve impulses – a process known as demyelination. For unknown reasons, the ability to remyelinate axons is impaired in MS patients making recovery of motor skills difficult. Therefore, developing novel and effective approaches to remyelinate axons in MS patients would dramatically improve the quality of life of many MS patients. The experiments described in this research proposal utilize a well-accepted model of MS to further characterize the potential clinical applicability of human embryonic stem cells (hESCs) to remyelinate axons. Such knowledge is crucial in order to increase our understanding of stem cells with regards to treatment of numerous human diseases including MS.

Statement of Benefit to California: 

California is the most populated state in the USA. As such, the costs of medical care for the treatment of patients with chronic diseases such as multiple sclerosis (MS) represents a significant and growing problem. MS is the most common neurologic disease affecting young adults under the age of 40 with the majority of MS patients diagnosed in the second or third decade of life. Given the population of California, there are many MS patients living in the state and the numbers will undoubtedly grow. It is unusual for MS patients to die from the disease and many will live normal life spans but will develop an increasing array of medical problems stemming from the progression of neurologic damage associated with MS. MS is characterized by the gradual loss of the myelin sheath that surrounds and insulates axons that allow for the conduction of nerve impulses – a process known as demyelination. For unknown reasons, the ability to remyelinate axons is impaired in MS patients making recovery of motor skills difficult. Therefore, developing novel and effective approaches to remyelinate axons in MS patients would dramatically alleviate some of the burden placed on the medical community by improving the quality of life of many MS patients. The experiments described in this research proposal utilize a well-accepted model of MS to further characterize the potential clinical applicability of human embryonic stem cells (hESCs) to remyelinate axons. Such knowledge is crucial in order to increase our understanding of stem cells with regards to treatment of human diseases with the ultimate goal of limiting patient suffering and reducing medical costs.

Progress Report: 
  • Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that results in demyelination and axonal loss, culminating in extensive disability through defects in neurologic function. The demyelination that defines MS pathology is progressive over time; however, studies indicate that myelin repair can occur during the course of disease in patients with MS and in animal models designed to mimic the immunopathogenesis of MS. While it is generally thought that endogenous oligodendrocyte precursor cells (OPCs) are largely responsible for spontaneous remyelination, it is unclear why these cells are only able to transiently induce myelin repair in the presence of ongoing disease. Along these lines, two therapies for demyelinating diseases look promising; implanting OPCs into sites of neuroinflammation that are directly capable of inducing remyelination of the damaged axons and/or modifying the local environment to stimulate and support remyelination by endogenous OPCs. Indeed, we have shown that human embryonic stem cell (hESC)-derived oligodendrocytes surgically implanted into the spinal cords of mice with virally induced demyelination promoted focal remyelination and axonal sparing. We are currently investigating how the implanted OPCs positionally migrate to areas of on-going demyelination and the role these cells play in repairing the damaged CNS. The purpose of this research is to identify the underlying mechanism(s) responsible for hESC-induced remyelination.
  • Oligodendrocyte progenitor cells (OPCs) are important in mediating remyelination in response to demyelinating lesions. As such, OPCs represent an attractive cell population for use in cell replacement therapies to promote remyelination for treatment of human demyelinating diseases. High-purity OPCs have been generated from hESC and have been shown to initiate remyelination associated with improved motor skills in animal models of demyelination. We have previously determined that engraftment of hESC-derived OPCs into mice with established demyelination does not significantly improve clinical recovery nor reduce the severity of demyelination. Importantly, remyelination is limited following OPC transplantation. These findings highlight that the microenvironment is critical with regards to the remyelination potential of engrafted cells. In addition, we have determined that human OPCs are capable of migrating in response to proinflammatory molecules often associated with human neuroinflammatory diseases such as multiple sclerosis. This is an important observation in that it will likely be necessary for engrafted OPCs to be able to positionally navigate within tissue in order to move from the site of surgical transplantation to areas of damage to initiate repair and tissue remodeling. Finally, we have also made a novel discovery of a unique signaling pathway that protects OPCs from damage/death in response to treatment with proinflammatory cytokines. We believe this is an important and translationally relevant observation as OPCs are critical in contributing to remyelination and remyelination failure is an important clinical feature for many human demyelinating diseases inclusing spinal cord injury and MS. We have identified a putative protective ligand/receptor interaction affords protection from cytokine-induced apoptosis. These findings may reveal novel avenues for therapeutic intervention to prevent damage/death of OPCs and enhance remyelination.
Funding Type: 
SEED Grant
Grant Number: 
RS1-00377
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$619 223
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

Our understanding of the effect of immunosuppressive agents on stem cell proliferation and differentiation in the central nervous system is limited. Indeed, even the necessity for long-term immunosuppression to promote the survival of stem cells grafted into the “immunoprivileged” central nervous system (CNS) is unknown. Grafting multipotent stem cells into the injured CNS often results in a failure of the cells to survive. If the cells survive, often they differentiate into astrocytes, a cell-type not considered beneficial. We recently grafted human stem cells (hCNS-SC) into spinal injured mice and observed behavioral improvements coupled with differentiation of these human cells into neurons and oligodendrocytes. We also observed mouse-human synapse formation and remyelination. The mice we used lacked a functional immune system, enabling us to grafting human cells into the mice without the use of immunosuppressants. When these same cells were grafted into spinal injured rats with a normal immune system, we had to immunosuppress the animals. Exposure of these human stem cells to immunosuppressive drugs resulted poor cell survival. The cells that did survive predominantly differentiated into astrocytes. Did the immunosuppressive drugs we used alter the ability of the human stem cells to differentiate into useful cells?

All cell-based therapeutic approaches are dependent upon either immunosuppression in an otherwise normal animal or testing for proof of principal in an immunodeficient animal model. This has quite significant implications for animal experiments or human trials, where continuous immunosuppression is required to obtain successful graft survival. No one knows if there are direct effects of immunosuppressant drugs on neural stem cells.

Stem cells may also respond differently to immunosuppression depending on their “ontogenetic” age (embryonic vs. fetal vs. adult). There is a common perception that “young” ES cells will have greater potential than “older” stem cells. Stem cells isolated at different ontogenetic stages might respond differently to immunosuppression.

We predict that the immunosuppressive drugs will exert direct effects on stem cell proliferation, gene expression, and fate determination, both in cell culture and when grafted into animals with spinal cord injury. We will also test if “ontogenetic” age alters the responsiveness of stem cells.

Statement of Benefit to California: 

The California Institute for Regenerative Medicine (CIRM) recognizes that the field of stem cell biology is in its infancy. CIRM has requested a broad range of research to fill in key gaps in our understanding of basic stem cell biology and the possible use of these cells as therapeutics. Grants are to be judged on impact (extent the proposed research addresses an important problem; significantly moves the field forward scientifically; moves the research closer to therapy; and changes the thinking or experimental practice in the field), quality (is proposed research planned carefully to give a meaningful result; are possible difficulties are acknowledge; does the timetable allows for achieving significant research) and innovation (to what extent the research approach is original, breaks new ground, and brings novel ideas to bear on an important problem).

We believe that the projects proposed here target several of the areas CIRM cites as beneficial to the State of California. This proposal addresses the critical area of immunosuppression and stem cell survival in animal transplantation models. Future therapies using human stem cells will have to surmount the possible rejection by the host of cells derived from another source. If traditional immunosuppressive drugs are to be used, we will need to understand whether these drugs have a direct effect on stem cell proliferation and fate determination (or differentiation). Furthermore, these projects will allow for a direct comparison of stem cells from different ontogenetic stages and the ability to improve functional outcome after spinal cord injury. Thus we may gain insight into whether embryonic derived stem cells are more useful than adult derived stem cells as a therapeutic tool.

Progress Report: 
  • We have shown that fetal human central nervous system derived stem cells (HuCNS-SC) transplanted into a mouse model of spinal cord injury (SCI) improve behavioral recovery. Transplanted human cells differentiated into myelinating oligodendrocytes and synapse forming neurons. These data suggest that efficacy is dependent upon successful cell engraftment and appropriate cell fate. The strain of mice (NOD-scid mice) are immunodeficient, which allows transplanted human cell populations to engraft and promote behavioral recovery in the absence of confounds due to a rejection response and allows us to avoid using immunosuppressant drugs. Clinically, however, it is clear that transplantation of therapeutic human cell populations will require administration of immunosuppressants (IS) such as CsA, FK506, or Rapamycin. These immunosuppressants work by altering signaling pathways which are also present within stem cells. Hence, in addition to promoting engraftment, IS have the potential to affect stem cell proliferation and/or differentiation. In Aim 1A, we tested this hypothesis in a cell culture model and found that HuCNS-SC fate and proliferation were altered by exposure to different IS. CsA and FK506 decreased the number of astrocytes in culture compared to control conditions, while Rapamyin increased the number of astrocytes. All three IS increased the number of ß-tubulin III positive neuron-like cells.
  • In Aim 1B, we tested whether cells of the inflammatory system (neutrophils and macrophages) could also directly influence stem cell proliferation and fate. To test this possibility, we exposed either fetal or embryonic neural stem cells to cell culture media from co-cultures of neutrophils or macrophages. We found that neutrophil-mediated release of inflammatory proteins promotes astrocyte differentiation of fetal derived neural stem cells but not embryonic derived neural stem cells. One way inflammatory cells might be working is via oxidative stress (e.g. hydrogen peroxide). Interestingly, excess hydrogen peroxide promoted more extensive cell death of embryonic derived versus fetal fetal derived neural stem cells, suggesting an intrinsic difference in the vulnerably of these two cell populations to oxidative stress. Conditioned media from neutrophils was found to reduce proliferation in fetal neural stem cells but not embryonic derived neural stem cells. In addition, we found neutrophil conditioned media promotes human fetal NSC astrocytic fate and migration towards sites of injury epicenter in an animal model of spinal cord injury; followup cell culture experiments enabled us to determine that neutrophil synthesized complement proteins were having a direct effect on stem cell fate and migration, resulting in a patent filing. These data demonstrate that fetal NSCs and ES-NSCs are very different by nature and nurture.
  • In Aim 2, we evaluated the hypothesis that IS could alter stem cell proliferation and/or fate in vivo, independent of rejection from the recipient’s immune system. HuCNS-SC were transplanted into NOD-scid mice, which have no immune system and hence cannot mount an immune response to the foreign cells. These animals received different immunosuppressants (CsA, FK506, Rapamycin, or vehicle) daily after transplantation until sacrifice 13 weeks later to determine if the total number of surviving human cells, or the end cell fate of the transplanted cells would be altered due to exposure to IS drugs compared to the vehicle control group. Behavioral recovery was assessed via open-field walking assessment, horizontal ladder beam testing, and video based “CatWalk” gait analysis. IS administration did not affect behavioral recovery by any of these measures compared to HuCNS-SC transplanted animals that received vehicle as an IS. Spinal cords were dissected, sectioned, and immunostained using human-specific markers in conjunction with cell lineage/fate and proliferation markers. Cell engraftment, proliferation, and fate were quantified using unbiased methods. The average number of engrafted human cells in uninjured animals was 319,700 vs 214,900 in vehicle treated injured controls. Human cell engraftment in any IS group was not significantly different than vehicle injured controls. Interestingly, 67% of human cells differentiated into Olig2+ oligodendrocyte-like cells in the uninjured controls, while 45% were Olig2 positive in vehicle treated injured controls. IS treatment did not alter Olig2 cell numbers in injured animals. 9% of human cells differentiated into GFAP positive astrocyte-like cells in the uninjured controls, compared with 9% in vehicle treated injured controls. IS treatment did not alter GFAP cell numbers in injured animals. Quantification of proliferation and other lineage markers is ongoing. The important finding thus far is that when administered to whole animals with a human stem cell transplant, a range of immunosuppressant drugs does not appear to significantly alter stem cell fate.
Funding Type: 
SEED Grant
Grant Number: 
RS1-00333
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$642 361
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

The advent of human embryonic stem cells (hESCs) has offered enormous potential for regenerative medicine and for basic understanding of human biology. On the one hand, hESCs can be turned into many different cell types in culture dish, and specific cell types derived from hESCs offer an almost infinite source for cellular replacement therapies. This is the primary reason for which hESCs have received much attention from the general public. On the other hand, scientists can study the properties of hESCs and their derivatives, and determine the effect of genes and molecules on such properties either in culture dish or with transplantation studies in live animals. This second aspect of hESC research would not only significantly enhance our understanding of the function of human genes, but will greatly augment our ability to apply hESCs in transplantation therapies and regenerative medicine. To attain the full potential of hESCs, genetic manipulation of hESCs is essential. In this proposal, we will establish the methods to genetically manipulate an increasingly used, non-federally approved hESC line, the HUES-9, and assess the feasibility to use genetically modified HUES-9 cells in cell transplantation studies to assess the integration of hESCs into the mouse central nervous system. We propose to achieve both homologous recombination (i.e. gene targeting) and transgene expression (with bacterial artificial chromosome), which have complementary utilities in assaying gene function in addition to the opportunity to label hESCs or their derivatives with fluorescent markers. Specifically, with genetic engineering of hESCs we will be able to 1) label hESCs and specific cell types derived from hESCs so that they can be readily followed in culture dish and in animals that have received cellular transplants; 2) disturb an endogenous gene or add more copies of a gene so that the effect of a gene of interest can be assessed (for this purpose, a gene involved in the development of a major motor tract, the corticospinal tract, will be studied). We will then transplant genetically engineered hESCs and their derivatives into the embryonic and adult mouse CNS to assess how well these cells integrate into the mouse CNS, and whether such transplanted animals can serve as valid models to study the effect of genes on hESC function in live animals. In transplantation studies involving adult mouse recipients, injured mouse CNS will be used in addition to intact CNS in order to evaluate the potential of hESCs to integrate into injured CNS, which has direct implications on the therapeutic potential of these cells. In summary, our proposal will establish the methods and tools to genetically manipulate HUES-9 cells, explore a paradigm to study human genes and cells in a context of neural development and cellular therapies, and will pave the way for future studies of genes and pathways in basic biology and regenerative medicine with hESCs.

Statement of Benefit to California: 

The disability, loss of earning power, and loss of personal freedom associated with spinal cord injury is devastating for the injured individual, and creates a financial burden of an estimated $400,000,000 annually for the state of California. Research is the only solution as currently there are no cures for spinal cord injury. My lab studies the underlying mechanisms for axon regeneration failure after spinal cord injury using mouse genetics and animal models of spinal cord injury. The current proposal aims to genetically manipulate human embryonic stem cells, study their potential to integrate into immature and mature central nervous system and analyze the effect of genes on such integration. Achieving genetic modification of hESCs will expedite studies with hESCs to cure a variety of human diseases and injuries including spinal cord injury. Our studies will pave the way for discoveries that might lead to novel treatment strategies for spinal cord injury and other neurological conditions. Effective treatments promoting functional repair will significantly increase personal independence for people with spinal cord injury, increase earning capacity and financial independence, and thus decrease the financial burden for the State of California. More importantly, treatments that enhance functional recovery will improve the quality of life for those who are directly or indirectly affected by spinal cord injuries.

Progress Report: 
  • A main goal of research in our laboratory is to identify strategies to promote neural repair in spinal cord injury and related neurological conditions. On the one hand, we have been using mouse models of spinal cord injury to study a long-standing puzzle in the field, namely, why axons, the fibers that connect nerve cells, do not regenerate after injury to the brain and the spinal cord. On the other hand, relevant to this CIRM SEED grant, we have started to explore the developmental and therapeutic potential of human embryonic stem cells (hESCs) for neural repair. We do this by first developing a method to genetically manipulate a HUES line of hESCs. The advent of hESCs has offered enormous potential for regenerative medicine and for basic understanding of human biology. To attain the full potential of hESCs as a tool both for therapeutic development and for basic research, we need to greatly enhance and expand our ability to genetically manipulate hESCs. A major goal for our SEED grant-sponsored research is to establish methods to genetically manipulate the HUES series of hESC lines, which are gaining wide utility in the research community due to the advantages on their growth characteristics over previously developed hESC lines. The first gene that we targeted in HUES cells, Fezf2, is critical for the development of the corticospinal tract, which plays important roles in fine motor control in humans and hence represents an important target for recovery and repair after spinal cord injury. By introducing a fluorescent reporter to the Fezf2 locus, we are now able to monitor the differentiation of hESCs into Fezf2-expressing neuronal lineages. This work has been published. A second goal is to start to explore the developmental and therapeutic potential of these cells and cells that derived from these cells in the brain and spinal cord. We are currently utilizing the cell line genetically engineered above to develop an efficient method to differentiate HUES cells into subcerebral neurons. Results so far have been encouraging. Efforts are also underway to overexpress Fezf2 as a complementary approach to drive the differentiation of HUES cells into specific neuronal types. Together, these studies will lay down the foundation for therapeutic development with HUES cells and their more differentiated derivatives for neurological disorders including spinal cord injury where neural regeneration can be beneficial. The CIRM SEED grant has allowed us to pursue a new, exciting path of research that we would have not pursued had we not been awarded the grant. Furthermore, the CIRM funded research has opened a new window of opportunity for us to explore genetic engineering of hESCs to model human neurological conditions in future.
Funding Type: 
SEED Grant
Grant Number: 
RS1-00331
Investigator: 
ICOC Funds Committed: 
$758 999
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

Parkinson’s disease (PD) is the most frequent neurodegenerative movement disorder caused by damage of dopamine-producing nerve cells (DA neuron) in patient brain. The main symptoms of PD are age-dependent tremors (shakiness). There is no cure for PD despite administration of levodopa can help to control symptoms.

Most of PD cases are sporadic in the general population. However, about 10-15% of PD cases show familial history. Genetic studies of familial cases resulted in identification of PD-linked gene changes, namely mutations, in six different genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, and DJ-1. Nevertheless, it is not known how abnormality in these genes cause PD. Our long-term research goal is to understand PD pathogenesis at cellular and molecular levels via studying functions of these PD-linked genes and dysfunction of their disease-associated genetic variants.

A proper experimental model plays critical roles in defining pathogenic mechanisms of diseases and for developing therapy. A number of cellular and animal models have been developed for PD research. Nevertheless, a model closely resembling generation processes of human DA nerve cells is not available because human neurons are unable to continuously propagate in culture. Nevertheless, human embryonic stem cells (hESCs) provide an opportunity to fulfill the task. hESCs can grow and be programmed to generate DA nerve cells. In this study, we propose to create a PD model using hESCs. The strategy is to express PD pathogenic mutants of α-synuclein or LRRK2 genes in hESCs. Mutations in α-synuclein or LRRK2 genes cause both familial and sporadic PD. α-Synuclein is a major component of Lewy body, aggregates found in the PD brain. The model will allow us to determine molecular action of PD pathogenic α-synuclein and LRRK2 mutants during generation of human DA neuron and interactions of PD related genes and environmental toxins in DA neurons derived from hESCs.

Our working hypothesis is that PD associated genes function in hESCs-derived DA neurons as in human brain DA neurons. Pathogenic mutations in combination with environmental factors (i.e. aging and oxidative stress) impair hESCs-derived DA function resulting in eventual selective neuronal death. In this study, we will firstly generate PD cellular models via expressing two PD-pathogenic genes, α-synuclein and LRRK2 in hESCs. We will next determine effects of α-synuclein and LRRK2 on hESCs and neurons derived from these cells. Finally, we will determine whether PD-causing toxins (i.e. MPP+, paraquat, and rotenone) selectively target to DA neurons derived from hESCs. Successful completion of this study will allow us to study the pathological mechanism of PD and to design strategies to treat the disease.

Statement of Benefit to California: 

Parkinson’s disease (PD) is the second leading neurodegenerative disease with no cure currently available. Compared to other states, California is among one of the states with the highest incidence of this particular disease. First, California growers use approximately 250 million pounds of pesticides annually, about a quarter of all pesticides used in the US (Cal Pesticide use reporting system). A commonly used herbicide, paraquat, has been shown to induce parkinsonism in both animals and human. Other pesticides are also proposed as potential causative agents for PD. Studies have shown increased PD-caused mortality is agricultural pesticide-use counties in comparison to those non-use counties in California. Second, California has the largest Hispanic population. Studies suggest that incidence of PD is the highest among Hispanics (Van Den Eeden et al, American Journal of Epidemiology, Vol. 157, pages 1015-1022, 2003). Thus, finding effective treatments of PD will significantly benefit citizen in California.

Progress Report: 
  • Parkinson’s disease (PD) is the most frequent neurodegenerative movement disorder caused by damage of dopamine-producing nerve cells (DA neuron) in patient brain. The main symptoms of PD are age-dependent tremors (shakiness). There is no cure for PD despite administration of levodopa can help to control symptoms.

  • Most of PD cases are sporadic in the general population. However, about 10-15% of PD cases show familial history. Genetic studies of familial cases resulted in identification of PD-linked gene changes, namely mutations, in six different genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, and DJ-1. Nevertheless, it is not known how abnormality in these genes cause PD. Our long-term research goal is to understand PD pathogenesis at cellular and molecular levels via studying functions of these PD-linked genes and dysfunction of their disease-associated genetic variants.

  • A proper experimental model plays critical roles in defining pathogenic mechanisms of diseases and for developing therapy. A number of cellular and animal models have been developed for PD research. Nevertheless, a model closely resembling generation processes of human DA nerve cells is not available because human neurons are unable to continuously propagate in culture. Nevertheless, human embryonic stem cells (hESCs) provide an opportunity to fulfill the task. hESCs can grow and be programmed to generate DA nerve cells. In this study, we propose to create a PD model using hESCs.

  • During the funding period, we have generated a number of human ES cell lines overexpressing α-synuclein and two disease-associated α-synuclein mutants. These cells are being used to determine the cellular and molecular effects of the disease genes on human ES cells and the PD affected dopaminergic neurons made from these cells. We have found that normal and disease α-synucleins have little effect on hESC growth and differentiation. We will continue to investigate roles of this protein in modulating PD affected dopaminergic neurons. Completion of this study will allow us to study the pathological mechanism of PD and to design strategies to treat the disease.
Funding Type: 
SEED Grant
Grant Number: 
RS1-00288
Investigator: 
ICOC Funds Committed: 
$807 749
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Spinal Muscular Atrophy
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 

More than 600 disorders afflict the nervous system. Common disorders such as stroke, epilepsy, Parkinson’s disease and autism are well-known. Many other neurological disorders are rare, known only to the patients and families affected, their doctors and scientists who look to rare disorders for clues to a general understanding of the brain as well as for treatments for specific diseases. Neurological disorders strike an estimated 50 million Americans each year, exacting an incalculable personal toll and an annual economic cost of hundreds of billions of dollars in medical expenses and lost productivity. There are many potential applications for using human embryonic stem (hES) cells to treat neurological diseases and injuries; however, a critical barrier to progress in the field is the ability to efficiently and reliably control neuronal differentiation from these cells. The main goal of this proposal is to define the gene regulatory mechanisms that control the acquisition of neuronal fate from hES cells. Longer term, we plan to produce small compounds (drugs) that greatly facilitate this process. Drugs that enhance neuron formation are likely to improve scientists’ ability to manipulate hES cells and create in vitro models for studying neurological diseases. Most importantly, drugs of this type may stimulate endogenous stem cells within adults to self-repair damaged areas of the brain. Because so little is known about how hES cells differentiate into neurons at the molecular level, this grant will focus on understanding how a single neuronal subtype is generated – motor neurons. Why motor neurons? Motor neuron diseases are a group of progressive neurological disorders that destroy cells that control essential muscle activity such as speaking, walking, breathing and swallowing. Eventually, the ability to control voluntary movement can be lost. Motor neuron diseases may be inherited or acquired, and they occur in all age groups. In adults, symptoms often appear after age 40. In children, particularly in inherited or familial forms of the disease, symptoms can be present at birth or appear before the child learns to walk. Is there a treatment? There is no cure or standard treatment for motor neuron diseases. Prognosis varies depending on the type of motor neuron disease and the age of onset; however, many types such as ALS and some forms of spinal muscular atrophy are typically fatal.The experiments in this proposal seek to understand mechanisms that will be directly applicable to hES cells and their use for treating motor neuron diseases. Moreover, the mechanisms controlly motor neuron formation are also likely to be relevant to many other neuronal subtypes. Therefore, these studies should provide essential and general insight into medically deploying strategies for converting hES cells into specific neuronal subtypes and thereby serve as a platform for treating a wide range of neurological diseases.

Statement of Benefit to California: 

The long term goal of this research grant proposal is to understand and treat diseases and injuries of the nervous system using hES cells. Neurological disorders such as stroke, epilepsy, Parkinson’s disease and autism strike an estimated 5 million Californians each year, exacting an incalculable personal toll and an annual economic cost of billions of dollars in medical expenses and lost productivity. Thus, one benefit that will be derived from this area of research is the generation of specific tools and methods for reducing medical costs and increasing the quality of life and level of productivity of afflicted Californians. A second key benefit derived from this research grant proposal is the training of new scientists to serve as educators and researchers for the future, many in the burgeoning area of stem cell biology for which the State of California has emerged as a world’s leader. Finally, the discoveries derived from innovative and multidisciplinary research on hES cells described in this proposal, including the use of chemistry to create drug leads for regulating stem cell differentiation, are likely to lead to important new areas of intellectual property that are essential for creating high quality jobs in the biotechnology and pharmaceutical industries in California.

Progress Report: 
  • The differentiation of stem cells into clinically-useful cell types is directly dependent on the accurate regulation of gene activation/repression. During the last scientific period we have focused our research on aim 2 of the grant proposal -– to characterize enzymes that are recruited to DNA for the regulation of genes. This effort has employed new DNA sequencing technologies to understand how the lysine specific demethylase (KDM1, LSD1) control gene expression in embryonic stem cells.

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