Neurological Disorders

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

Genetic manipulation of human embryonic stem cells and its application in studying CNS development and repair

Funding Type: 
SEED Grant
Grant Number: 
RS1-00333
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.

Modeling Parkinson's Disease Using Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00331
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.

Gene regulatory mechanisms that control spinal neuron differentiation from hES cells.

Funding Type: 
SEED Grant
Grant Number: 
RS1-00288
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.

Optimization of guidance response in human embryonic stem cell derived midbrain dopaminergic neurons in development and disease

Funding Type: 
SEED Grant
Grant Number: 
RS1-00271
ICOC Funds Committed: 
$633 170
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
A promising approach to alleviating the symptoms of Parkinson’s disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have had ongoing efforts to determine the which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and we have identified necessary cues. We now plan to extend these studies to determine how these critical guidance cues affect human stem cell derived dopaminergic neurons, the cells that will be used in transplantation. In addition, we will examine how these guidance cues affect both normal and stem cell derived dopaminergic neurons under conditions that are similar to the diseased and transplanted brain, specifically when the brain is inflamed. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.
Statement of Benefit to California: 
The goal of our work is to further optimize our ability to turn undifferentiated human stem cells into differentiated neurons that the brain can use as replacement for neurons damaged by disease. We focus onParkinson’s disease, a neurodegenerative disease that afflicts 4-6 million people worldwide in all geographical locations, but which is more common in rural farm communities compared to urban areas (Van Den Eeden et al., 2003), a criteria important for California’s large farming population. In Parkinson’s patients, a small, well-defined subset of neurons, the midbrain dopaminergic neurons have died, and one therapeutic strategy is to transplant healthy replacement neurons to the patient. Our work will further our understanding of the biology of these neurons in normal animals. This will allow us to refine the process of turning human ES cells onto biologically active dopaminergic neurons that can be used in transplantation therapy. Our work will be of benefit to all Parkinson’s patients including afflicted Californians. In addition to the direct benefit in improving PD therapies, discoveries from this work are also likely to generate substantial intellectual property and further boost clinical and biotechnical development efforts in California.
Progress Report: 
  • A promising approach to alleviating the symptoms of Parkinson's disease is to transplant healthy dopaminergic neurons into the brains of these patients. Due to the large number of transplant neurons required for each patient and the difficulty in obtaining these neurons from human tissue, the most viable transplantation strategy will utilize not fetal dopaminergic neurons but dopaminergic neurons derived from human stem cell lines. While transplantation has been promising, it has had limited success, in part due to the ability of the new neurons to find their correct targets in the brain. This incorrect targeting may be due to the lack of appropriate growth and guidance cues as well as to inflammation in the brain that occurs in response to transplantation, or to a combination of the two. Cytokines released upon inflammation can affect the ability of the new neurons to connect, and thus ultimately will affect their biological function. In out laboratory we have been examining which guidance molecules are required for proper targeting of dopaminergic neurons during normal development and have identified necessary cues. We have now extended these studies to determine that two of the molecules have dramitc effects on dopaminergic neurons made from human embryonic stem cellls and that at least in vitro, cytokines do not mask these effects. Ultimately, an understanding of how the environment of the transplanted brain influences the ability of the healthy new neurons to connect to their correct targets will lead to genetic, and/or drug-based strategies for optimizing transplantation therapy.

Development of human ES cell lines as a model system for Alzheimer disease drug discovery

Funding Type: 
SEED Grant
Grant Number: 
RS1-00247
ICOC Funds Committed: 
$492 750
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Alzheimer disease (AD) is a progressive neurodegenerative disorder that currently affects over 4.5 million Americans. By the middle of the century, the prevalence of AD in the USA is projected to almost quadruple. As current therapies do not abate the underlying disease process, it is very likely that AD will continue to be a clinical, social, and economic burden. Progress has been made in our understanding of AD pathogenesis by studying transgenic mouse models of the disease and by utilizing primary neuronal cell cultures derived from rodents. However, key proteins that are critical to the pathogenesis of this disease exhibit many species-specific differences at both a biophysical and functional level. Additional species differences in other as yet unidentified AD-related proteins are likely to also exist. Thus, there is an urgent need to develop novel models of AD that recapitulate the complex array of human proteins involved in this disease. Cell culture-based models that allow for rapid high-throughput screening and the identification of novel compounds and drug targets are also critically needed. To that end we propose to model both sporadic and familial forms of AD by generating two novel human embryonic stem cell lines (hES cells). Differentiation of these lines along a neuronal lineage will provide researchers with an easily accessible and reproducible neuronal cell culture model of AD. These cells will also allow high-throughput screening and experimentation in neuronal cells with a species-relevant complement of human proteins. In Aim 1 we will develop and characterize hES cell lines designed to model both sporadic and familial forms of AD. To model sporadic AD we will stably transfect HUES7 hES cells (developed by Douglas Melton) with lentiviral constructs coding for human wild type amyloid precursor protein (APP-695) under control of the human APP promoter. APP is well expressed within hES cells and upregulated upon neuronal differentiation. To model familial AD and generate cells that exhibit a more aggressive formation of oligomeric A species we will also develop a second hES cell line stably transfected with human APP that includes the Arctic (E693G) mutation.In Aim 2 we will utilize our wild-type APP hES cells to perform a high-throughput siRNA screen. We will utilize AMAXA reverse-nucleofection in conjunction with a human druggable genome siRNA array (Dharmacon) that targets 7309 genes considered to be potential therapeutic targets. Following transfection conditioned media will be examined by a sensitive ELISA to identify novel targets that modulate A levels. In addition a Thioflavin S assay will determine any effects on A aggregation. Follow-up experiments will confirm promising candidates identified in the high-throughput screen. Taken together these studies aim to establish novel AD-specific hES cell lines and identify promising new therapeutic targets for this devastating disease.
Statement of Benefit to California: 
Alzheimer disease (AD) is a progressive neurodegenerative disorder that currently affects over 500 thousand Californians. As the baby-boomer generation ages the prevalence of AD in California is projected to almost quadruple such that 1 in every 45 individuals will be afflicted. As current therapies do not abate the underlying disease process, it is very likely that AD will continue to be a major clinical, social, and economic burden. Some estimates have even suggested that AD alone may bankrupt the current Californian health care system. Progress has been made in our understanding of AD by studying rodent-based models of the disease. However, key proteins that are critical to the disease exhibit many species-specific differences at both a biophysical and functional level. Thus, there is an urgent need to develop novel models of AD that exhibit the complex array of human proteins involved in this disease. Cell culture-based models that also allow for rapid high-throughput screening and the identification of novel compounds and drug targets are also in critical need. The proposed studies aim to utilize human embryonic stem (hES) cells to establish a novel cell culture based model of Alzheimer’s disease. Once developed these cells will provide Californian researchers with a unique tool to investigate genes and proteins that influence the progress of AD. In this proposal we will also utilize these hES cells to perform a high-throughput screen of over 7300 genes to identify multiple novel drug targets that may critically regulate the development of this disease. Taken together these studies aim to establish novel AD-specific hES cell lines that can be utilized by multiple Californian researchers to identify promising new therapeutic targets for this devastating disease.
Progress Report: 
  • Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. It is characterized by an irreversible loss of neurons accompanied by the accumulation of extracellular amyloid plaques and intraneuronal neurofibrillary tangles. Currently, 5.3 million Americans are afflicted with this insidious disorder, including over 588,000 in the State of California alone. Mouse models of AD have contributed significantly to our understanding of the proteins and factors involved in the pathology of AD. However, there are critical differences between mouse and human cell physiology that likely dramatically influence the development of AD-related pathologies. Hence, there is an urgent need to develop novel human neuronal cell-based models of AD.
  • To achieve this goal, we have generated stable human embryonic stem cell (hES) lines over-expressing the gene for human amyloid precursor protein (APP). We succeeded in creating several lines of hES cells that stably express either wild-type (unaltered) APP or APP that includes rare familial mutations known to cause early-onset cases of AD. In each line, transgene expression is driven under control of the human APP proximal promoter. Mutant versions of APP utilized include the “Swedish” mutation which increases production of Aß and the “Arctic” mutation which increases the assembly and accumulation of synaptotoxic Aß oligomers and protofibrils. The generation of lines that harbor familial mutations in APP both provides an aggressive model of AD, to facilitate the identification of targets that modulate not only Aß production but also the assembly of toxic oligomeric species.
  • In addition to generating stable HUES7 and H9 cell lines over-expressing mutant and wild type forms of APP, we also succeeded in establishing a neuronal differentiation protocol which results in 80% of cells adopting a mature neuronal fate. Importantly, we have also verified by biochemical measures that APP-overexpressing cells produce significantly elevated levels of Aß. As a result we are now preparing to utilize these novel cell lines to identify and examine genes that regulate Aß production and hence the development of AD.
  • Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Currently, 5.3 million individuals are afflicted with this insidious disorder, including over 588,000 in the State of California alone. Unfortunately, existing therapies provide only palliative relief. Although transgenic mouse models and cell culture experiments have contributed significantly to our understanding of the proteins and factors involved in the pathology of AD, these approaches are beset by certain critical limitations. Most notably, mouse models by definition are not based on human cells and cell culture models have been limited to non-human or non-neuronal cells. Hence, there is an urgent need to develop a human neuronal cell-based model of AD. To address this need, we have engineered human embryonic stem cell lines to overexpress mutant human genes that cause early-onset familial AD. These novel stem cell lines will provide a valuable system to test therapies and enhance our understanding of the mechanisms that mediate this devastating disease. Interestingly, we have found that overexpression of these AD-related genes can trigger the rapid differentiation of human embryonic stem cells into neuronal cells. We have examined the mechanisms involved and anticipate that our findings may provide a novel and rapid method to generate neurons from embryonic stem cells.

New Chemokine-Derived Therapeutics Targeting Stem Cell Migration

Funding Type: 
SEED Grant
Grant Number: 
RS1-00225
ICOC Funds Committed: 
$759 000
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stroke
Trauma
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
This proposal describes a sharply-focused, timely, and rigorous effort to develop new therapies for the treatment of injuries of the Central Nervous System (CNS). The underlying hypothesis for this proposal is that chemokines and their receptors (particularly those involved in inflammatory cascades) actually play important roles in mediating the directed migration of human neural stem cells (hNSCs) to, as well as engagement and interaction with, sites of CNS injury, and that understanding and manipulating the molecular mechanism of chemokine-mediated stem cell homing and engagement will lead to new, better targeted, more specific, and more efficacious chemokine-mediated stem cell-based repair strategies for CNS injury. In recent preliminary studies, we have discovered and demonstrated the important role of chemokine SDF-1-alpha and its receptor CXCR4 in mediating the directed migration of hNSCs to sites of CNS injury. To manipulate this SDF-1-alpha/CXCR4 pathway in stem cell migration, we have developed Synthetically and Modularly Modified Chemokines (SMM-chemokines) as highly potent and specific therapeutic leads. Here in this renewal application we propose to extend our research into a new area of stem cell biology and medicine involving chemokine receptors such as CXCR4 and its ligand SDF-1. Specifically, we will design more potent and specific analogs of SDF-1-alpha to direct the migration of beneficial stem cells toward the injury sites for the repair process.
Statement of Benefit to California: 
This proposal describes a sharply-focused, timely, and rigorous effort to develop new therapies for the treatment of injuries of the Central Nervous System (CNS). CNS injuries and related disorders such as stroke, traumatic brain injury and spinal cord injury are significant health issues in the nation including the state of California. The new stem cell-based therapies to be developed from this application will have important clinical application in patients with these diseases in California.
Progress Report: 
  • Human neural stem cells (hNSCs) expressing CXCR4 have been found to migrate in vivo toward an infarcted area that are representative of central nervous system (CNS) injuries, where local reactive astrocytes and vascular endothelium up-regulate the SDF-1α secretion level and generate a concentration gradient. Exposure of hNSCs to SDF-1α and the consequent induction of CXCR4-mediated signaling triggers a series of intracellular processes associated with fundamental aspects of survival, proliferation and more importantly, proper lamination and migration during the early stages of brain development [1]. To date, there is no crystal structure available for chemokine receptors [2, 3]. Structural and modeling studies of SDF-1α and D-(1~10)-L-(11~69)-vMIP-II in complexes with CXCR4 TM helical regions led us to a plausible “two-pocket” model for CXCR4 interaction with agonists or antagonists. [4-6] In this study, we extended the employment of this model into the novel design strategy for highly potent and selective CXCR4 agonist molecules, with potentials in activating CXCR4-mediated hNSC migration by mimicking a benign version of the proinflamatory signal triggered by SDF-1α. Successful verification of directed, extensive migration of hNSCs, both in vitro and in transplanted uninjured adult mouse brains, with the latter manifesting significant advantages over the natural CXCR4 agonist SDF-1α in terms of both distribution and stability in mouse brains, strongly supports the effectiveness and high potentials of these de novo designed CXCR4 agonist molecules in optimizing directed migration of transplanted human stem cells during the reparative therapeutics for a broad range of neurodegenerative diseases in a more foreseeable future.
  • Our final progress report is divided into 3 subsections, each addressing progress in the 3 fundamental areas of investigation for the successful completion of this project:
  • (1) De-novo design and synthesis of CXCR4-specific SDF-1α analogs.
  • (2) In vitro studies on validating biological potencies of molecules in (1) in activating CXCR4 down-stream signaling.
  • (3) In vivo studies on migration of transplanted neural precursor cells (NPCs) in co-administration of molecules with validated biological activities in (2).

Identifying small molecules that stimulate the differentiation of hESCs into dopamine-producing neurons

Funding Type: 
SEED Grant
Grant Number: 
RS1-00215
ICOC Funds Committed: 
$564 309
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
In this application, we propose to identify small molecule compounds that can stimulate human embryonic stem cells to become dopamine-producing neurons. These neurons degenerate in Parkinson’s disease, and currently have very limited availability, thus hindering the cell replacement therapy for treating Parkinson’s disease. Our proposed research, if successful, will lead to the identification of small molecule compounds that can not only stimulate cultured human embryonic stem cells to become DA neurons, but may also stimulate endogenous brain stem cells to regenerate, since the small molecule compounds can be made readily available to the brain due to their ability to cross the blood-brain barrier. In addition, these small molecule compounds may serve as important research tools, which can tell us the fundamental biology of the human embryonic stem cells.
Statement of Benefit to California: 
The proposed research will potentially lead to a cure for the devastating neurodegenerative, movement disorder, Parkinson’s disease. The proposed research will potentially provide important research tools to better understand hESCs. Such improved understanding of hESCs may lead to better treatments for a variety of diseases, in which a stem-cell based therapy could make a difference.
Progress Report: 
  • Parkinson’s disease is the most common movement disorder due to the degeneration of brain dopaminergic neurons. One strategy to combat the disease is to replenish these neurons in the patients, either through transplantation of stem cell-derived dopaminergic neurons, or through promoting endogenous dopaminergic neuronal production or survival. We have carried out a small molecule based screen to identify compounds that can affect the development and survival of dopaminergic neurons from pluripotent stem cells. The small molecules that we have identified will not only serve as important research tools for understanding dopaminergic neuron development and survival, but potentially could also lead to therapeutics in the induction of dopaminergic neurons for treating Parkinson’s disease.

Generation of forebrain neurons from human embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00205
ICOC Funds Committed: 
$612 075
Disease Focus: 
Aging
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The goal of this proposal is to generate forebrain neurons from human embryonic stem cells. Our general strategy is to sequentially expose ES cells to signals that lead to differentiation along a neuronal lineage, and to select for cells that display characteristics of forebrain neurons. These cells would then be used in transplantation experiments to determine if they are able to make synaptic connections with host neurons. If successful these experiments would provide a therapeutic strategy for the treatment of Alzheimer’s disease and other disorders that are characterized by loss of forebrain neurons. Currently there is no effective treatments for Alzheimer’s disease, and with an aging baby-boomer population, the incidence of this disease is likely to increase sharply. One of the few promising avenues to treat Alzheimer’s is the possibility of cell replacement therapy in which the neurons lost could be replaced by transplanted neurons. Embryonic stem cells, which have the ability to differentiate into various cells of the body, could be a key component of such a therapy if we can successfully differentiate them into forebrain neurons.
Statement of Benefit to California: 
Alzheimer’s disease is a devastating sporadic neurological disorder that places all of us at risk. As the California population ages, there will be a significant increase in the incidence of Alzheimer’s disease, and the medical and financial cost on the state will be severe. There are currently no effective treatments for this disorder, and one of the few promises is the possibility of transplantation therapy to replace the neurons that are lost in the disease. Being able to generate forebrain neurons from human embryonic stem cells would provide a key tool in the fight against this disease. Needless to say, the development of an effective cell replacement therapy would not only be of immense medical significance as we care for our senior population, it will also greatly relieve the financial burden associated with the care of Alzheimer’s patients, which is often borne by the state.
Progress Report: 
  • The goal of this proposal was to generate forebrain neurons from human embryonic stem cells. Our general strategy was to sequentially expose ES cells to signals that would lead the cells to acquire characteristics typical of differentiated brain cells that are lost in disorders such as Alzheimer's Disease. The most important advance of the research was our ability to achieve this goal. We now have a well-developed protocol that can be used to generate forebrain cells in culture. We have found that these cells not only express genes typical of these cells, they extend axons and dendrites and can make synaptic connections. These cells could be very useful for transplantation studies, as well as for developing cell culture models of Alzheimer's disease. Finally, we have discovered that the same protocol is effective in generating forebrain neurons from iPS cells, attesting to the general usefulness of this strategy.

In vitro differentiation of hESCs into corticospinal motor neurons

Funding Type: 
SEED Grant
Grant Number: 
RS1-00170
ICOC Funds Committed: 
$500 000
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurological disease that leads to the degeneration of motor neurons in the brain and in the spinal cord. There are currently 20,000 ALS patients in the United States, and 5,000 new patients are diagnosed every year. Unfortunately no cure has been found for ALS. The only medication approved by the FDA to treat ALS can only slow the disease’s progression and prolong life by a few months in some patients. Thus it is critical to explore other therapeutic strategies for the treatment of ALS such as cell replacement strategy. Because of the ability to generate many different cell types, human embryonic stem cells (hESCs) may potentially serve as a renewable source of cells for replacing the damaged cells in diseases. However, transplanting ESCs directly may cause tumor growth in patients. To support cell transplants, it is important to develop methods to differentiate hESCs into the specific cell types affected by the disease. In this application, we propose to develop an effective method to differentiate hESCs into corticospinal motor neurons (CSMNs), the neurons in the cerebral cortex that degenerate in ALS. We will test whether these CSMNs generated from hESCs in culture conditions can form proper connections to the spinal cord when transplanted into mouse brains. To direct hESCs to become the CSMNs, it is critical to establish a reliable method to identify human CSMNs. Recent progress in developmental neuroscience have identified genes that are specifically expressed in the CSMNs in mice. However no information is available for identifying human CSMNs. We hypothesize that CSMN genes in mice will be reliable markers for human CSMNs. To test this hypothesis we will investigate whether mouse CSMN markers are specifically expressed in the human CSMNs. The therapeutic application of hESCs to replace damaged CSMNs in ALS depends on the ability to direct hESCs to develop into CSMNs. Currently a reliable condition to direct hESCs to differentiate into CSMNs has not been established. We will attempt to differentiate hESCs into CSMNs based on the knowledge gained from studying the development of nervous system. We will achieve this goal in two steps: first we will culture hESCs in a condition to make them become progenitors cells of the most anterior region of the brain; then we will culture these progenitors to become neurons of the cerebral cortex, particularly the CSMNs. We will study the identities of these neurons using the CSMN markers that we have proposed to identify. To apply the cell replacement strategy to treat ALS, it will be critical to test if human CSMNs generated from cultured hESCs can form proper connections in an animal model. We will transplant the CSMNs developed from hESCs into the brains of mice and test whether they can form connections to the spinal cord. When carried out, the proposed research will directly benefit cell replacement therapy for ALS.
Statement of Benefit to California: 
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurological disease that leads to the degeneration of motor neurons in the brain and in the spinal cord. There are currently 20,000 ALS patients in the United States, and 5,000 new patients are diagnosed every year. Unfortunately no cure has been found for ALS. The only medication approved by the FDA to treat ALS can only slow the disease’s progression and prolong life by a few months in some patients. Thus it is critical to explore other therapeutic strategies for the treatment of ALS such as cell replacement strategy. Because of the ability to generate many different types of cells, human embryonic stem cells (hESCs) may potentially serve as a renewable source of cells for replacing the damaged cells in diseases. However, transplanting ESCs directly may cause tumor growth in patients. To support cell transplants, it is important to develop methods to differentiate hESCs into the specific cell types affected by the disease. In this application, we propose to develop an effective method to differentiate hESCs into corticospinal motor neurons (CSMNs), the neurons in the cerebral cortex that degenerate in ALS. We will test whether these CSMNs generated from hESCs in culture conditions can form proper connections to the spinal cord when transplanted into mouse brains. Everyday, 15 people die from ALS. For patients diagnozied with ALS, time is running out very fast. It is critical to explore novel therapeutic strategies for this rapidly progressive and fatal disease. The research proposed in this application may provide the basis for a novel cell replacement therapy for ALS, thus it will greatly benefit the State of California and everyone in the State.
Progress Report: 
  • Corticospinal motor neurons are affected in motor neuron diseases and damaged in spinal cord injuries. In this grant application, we proposed to induce human embryonic stem cells to generate corticospinal motor neurons. In this past grant period, we have generated neurons that express the corticospinal motor neuron genes. We are currently characterizing the cell types of theses neurons in detail. In the near future we will transplant them into the brains in mice to test whether they can form functional neural circuits.
  • In the past grant period, we have been continuing to generate brain neurons from cultured human embryonic stems. We have been determining what types of neurons are generated using our protocol. We are testing the functions of these neurons.

Systemic Protein Factors as Modulators of the Aging Neurogenic Niche

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01637
ICOC Funds Committed: 
$1 522 800
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Approaches to repair the injured brain or even prevent age-related neurodegeneration are in their infancy but there is growing interest in the role of neural stem cells in these conditions. Indeed, there is hope that some day stem cells can be used for the treatment of spinal cord injury, stroke, or Parkinson’s disease and stem cells are even mentioned in the public with respect to Alzheimer’s disease. To utilize stem cells for these conditions and, equally important to avoid potential adverse events in premature clinical trials, we need to understand the environment that supports and controls neural stem cell survival, proliferation, and functional integration into the brain. This “neurogenic” environment is controlled by local cues in the neurogenic niche, by cell-intrinsic factors, and by soluble factors which can act as mitogens or inhibitory factors potentially over longer distances. While some of these factors are starting to be identified very little is known why neurogenesis decreases so dramatically with age and what factors might mediate these changes. Because exercise or diet can increase stem cell activity even in old animals and lead to the formation of new neurons there is hope that neurogenesis in the aged brain could be restored to that seen in younger brains and that stem cell transplants could survive in an old brain given the right “young” environmental factors. Indeed, our preliminary data demonstrate that systemic factors circulating in the blood are potent regulators of neurogenesis. By studying how the most promising of these factors influence key aspects of the neurogenic niche in vitro and in vivo we hope to gain an understanding about the molecular interactions that support stem cell activity and the generation of new neurons in the brain. The experiments supported under this grant will help us to identify and understand the minimal signals required to regulate adult neurogenesis. These findings could be highly significant for human health and biomedical applications if they ultimately allow us to stimulate neurogenesis in a controlled way to repair, augment, or replace neural networks that are damaged or lost due to injury and degeneration.
Statement of Benefit to California: 
In California there are hundreds of thousands of elderly individuals with age-related debilitating brain injuries, ranging from stroke to Alzheimer’s and Parkinson’s disease. Approaches to repair the injured brain or even prevent age-related neurodegeneration are in their infancy but there is growing interest in the role of neural stem cells in these conditions. However, to potentially utilize such stem cells we need to understand the basic mechanisms that control their activity in the aging brain. The proposed research will start to address this problem using a novel and innovative approach and characterize protein factors in blood that regulate stem cell activity in the old brain. Such factors could be used in the future to support stem cell transplants into the brain or to increase the activity of the brain’s own stem cells.
Progress Report: 
  • We are interested in identifying soluble protein factors in blood which can either promote or inhibit stem cell activity in the brain. Through a previous aging study and the transfer of blood from young to old mice and vice versa we had identified several proteins which correlated with reduced stem cell function and neurogenesis in young mice exposed to old blood. Over the past year we studied two factors, CCL11/eotaxin and beta2-microglobulin in more detail in tissue culture and in mice. We could demonstrate that both factors administered into the systemic environment of mice reduce neurogenesis in a brain region involved in learning and memory. We have also begun to test the effect of these factors on human neural stem cells and we started experiments to try to identify protein factors which can enhance neurogenesis.
  • While age-related cognitive dysfunction and dementia in humans are clearly distinct entities and affect different brain regions, the aging brain shows the telltale molecular and cellular changes that characterize most neurodegenerative diseases. Remarkably, the aging brain remains plastic and exercise or dietary changes can increase cognitive function in humans and animals, with animal brains showing a reversal of some of the aforementioned biological changes associated with aging. We showed recently that blood-borne factors coming outside the brain can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing an old mouse to a young systemic environment or to plasma from young mice increased neurogenesis, synaptic plasticity, and improved contextual fear conditioning and spatial learning and memory. Preliminary proteomic studies show several proteins with stem cell activity increase in old “rejuvenated” mice supporting the notion that young blood may contain increased levels of beneficial factors with regenerative capacity. We believe we have identified some of these factors now and tested them on cultured mouse and human neural stem cell derived cells. Preliminary data suggest that these factors have beneficial effects and we will test whether these effects hold true in living mice.
  • Cognitive function in humans declines in essentially all domains starting around age 50-60 and neurodegeneration and Alzheimer’s disease seems to be inevitable in all but a few who survive to very old age. Mice with a fraction of the human lifespan show similar cognitive deterioration indicating that specific biological processes rather than time alone are responsible for brain aging. While age-related cognitive dysfunction and dementia in humans are clearly distinct entities the aging brain shows the telltale molecular and cellular changes that characterize most neurodegenerative diseases including synaptic loss, dysfunctional autophagy, increased inflammation, and protein aggregation. Remarkably, the aging brain remains plastic and exercise or dietary changes can increase cognitive function in humans and animals. Using heterochronic parabiosis or systemic application of plasma we showed recently that blood-borne factors present in the systemic milieu can rejuvenate brains of old mice. Accordingly, exposing an old mouse to a young systemic environment or to plasma from young mice increased neurogenesis, synaptic plasticity, and improved contextual fear conditioning and spatial learning and memory. Unbiased genome-wide transcriptome studies from our lab show that hippocampi from old “rejuvenated” mice display increased expression of a synaptic plasticity network which includes increases in c-fos, egr-1, and several ion channels. In our most recent studies, plasma from young but not old humans reduced neuroinflammation in brains of immunodeficient mice (these mice allow us to avoid an immune response against human plasma). Together, these studies lend strong support to the existence of factors with beneficial, “rejuvenating” activity in young plasma and they offer the opportunity to try to identify such factors.
  • Cognitive function in humans declines in essentially all domains starting around age 50-60 and neurodegeneration and dementia seem to be inevitable in all but a few who survive to very old age. Mice with a fraction of the human lifespan show similar cognitive deterioration indicating that specific biological processes rather than time alone are responsible for brain aging. While age-related cognitive dysfunction and dementia in humans are clearly distinct entities and affect different brain regions the aging brain shows the telltale molecular and cellular changes that characterize most neurodegenerative diseases including synaptic loss, dysfunctional autophagy, increased inflammation, and protein aggregation. Remarkably, the aging brain remains plastic and exercise or dietary changes can increase cognitive function in humans and animals, with animal brains showing a reversal of some of the aforementioned biological changes associated with aging. Using heterochronic parabiosis we showed recently that blood-borne factors present in the systemic milieu can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing an old mouse to a young systemic environment or to plasma from young mice increased neurogenesis, synaptic plasticity, and improved contextual fear conditioning and spatial learning and memory. Over the past three years we discovered that factors in blood can actively change the number of new neurons that are being generated in the brain and that local cells in areas were neurons are generated respond to cues from the blood. We have started to identify some of these factors and hope they will allow us to regulate the activity of neural stem cells in the brain and hopefully improve cognition in diseases such as Alzheimer's.

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