Neurological Disorders

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

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

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

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

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

Molecular mechanisms of neural stem cell differentiation in the developing brain

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

Molecular mechanisms involved in adult neural stem cell maintenance

Funding Type: 
New Faculty I
Grant Number: 
RN1-00527
ICOC Funds Committed: 
$2 348 520
Disease Focus: 
Aging
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
The adult brain contains a pool of stem cells, termed adult neural stem cells, that could be used for regenerative purposes in diseases that affect the nervous system. The goal of this proposal is to understand the mechanisms that promote the maintenance of adult neural stem cells as an organism ages. Understanding the factors that maintain the pool of adult neural stem cells should open new avenues to prevent age-dependent decline in brain functions and to use these cells for therapeutic purposes in neurological and neurodegenerative diseases, such as Alzheimer’s or Parkinson’s diseases. Our general strategy is to use genes that play a central role in organismal aging as we have recently discovered that two of these genes, Foxo and Sirt1, have profound effects on the maintenance and self-renewal of adult neural stem cells. We propose to use these genes as a molecular handle to understand the mechanisms of maintenance of neural stem cells. Harnessing the regenerative power of stem cells by acting on genes that govern aging will provide a novel angle to identify stem cell therapeutics for neurological and neurodegenerative diseases, most of which are age-dependent.
Statement of Benefit to California: 
As the population of the State of California ages, neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease affect increasing numbers of patients. There are no efficient treatments of cures for these diseases. In addition to the devastating effects of neurodegenerative diseases on the patients and their relatives, the cost of caring for California’s Alzheimer patients—about $22.4 billion in 2000—has been estimated to triple by 2040 due to the aging of the baby-boomer’s generation. Stem cells from the brain, or neural stem cells, hold the promise of treatments and cures for these neurodegenerative diseases. One therapeutic strategy will be to replace degenerating cells in patients with stem cells. Another approach would be to identify strategy to better maintain the pool of neural stem cell with age. Both approaches will only be possible when the mechanisms controlling the maintenance of these stem cells and their capacity to produce their functional progeny are better understood in young and old individuals. We propose to study the mode of action in neural stem cells of two genes, Foxo and Sirt, that are known to play major roles to extend lifespan in a variety of species. These genes are major targets for the development of stem cell therapeutic strategies that will benefit a wide range of patients suffering from age-dependent neurodegenerative disorders. The development of effective replacement therapies in neurodegenerative diseases will be a benefit for the rapidly aging population of California; it will also alleviate the financial burden that these age-related disorders create for the State of California.
Progress Report: 
  • Aging is accompanied by a decline in the number and the function of adult stem cells in several tissues. In the brain, the depletion of adult neural stem cells (NSC) may underlie impaired cognitive performance associated with aging. Discovering the factors that govern the maintenance of adult NSC during aging should allow us to harness their regenerative potential for therapeutic purposes during normal aging and age-related neurodegenerative disorders. We have recently found that two 'longevity genes', Foxo3 and Sirt1, are critical for adult NSC function. In the past year, we have published a manuscript showing that Foxo3 is necessary for the maintenance of NSC in the adult brain. We have also started to explore the critical mechanisms by which Foxo3 maintains adult neural stem cells in the brain. We have used ultra-high throughput sequencing approach to reveal that Foxo3 is recruited to the regulatory regions of 3,000 genes in the adult neural stem cells, thereby triggering a gene expression network that regulates both the ability of neural stem cells to divide and their ability to give rise to progeny. Finally, we have obtained new results in the past year, showing that Sirt1, another 'longevity gene' is critical for the proper function of neural stem cells in the adult brain, and their ability to give rise to differentiated cells. Together, our results will help understand the regulation of neural stem cell maintenance in aging individuals and will provide new avenues to preserve the pool of these cells in the brain. Modulating longevity genes to harness the regenerative power of stem cells will provide new avenues for stem cell therapeutics for neurological and neurodegenerative diseases, most of which are age-dependent.
  • The adult brain contains pools of stem cells called neural stem cells that are critical for
  • the formation of new neurons in the adult brain. During aging, the number of neural stem
  • cells and their ability to give rise to new neurons strikingly decline. This decline could
  • underlie at least in part memory deterioration that occurs during aging and age-related
  • neurodegenerative disease such as Alzheimer’s disease. We have been interested over
  • the years in the importance of genes that regulate overall longevity in the control of the
  • pool of neural stem cells. We made the important discovery that Foxo3, a gene that has
  • been implicated in human exceptional longevity, is necessary for preserving the neural
  • stem cell pool. In the past year, we have made extensive progress in characterizing the
  • ensemble of genes regulated by Foxo3 in adult neural stem cells, a key step in
  • unraveling the mechanisms by which neural stem cells are maintained intact. In the past
  • year, we have observed that in the absence of another gene important for longevity
  • Sirt1, there is an unexpected increase in oligodendrocyte progenitors, which are cells
  • that are important for myelination of neurons, which is important for the proper
  • propagation of the neuronal information. Defects in myelination, which happen for
  • example in multiple sclerosis, have devastating consequences on the neurological
  • function. In the past year, we have made progress to understand the cellular and
  • molecular mechanism of action that enhances the production of oligodendrocytes in the
  • absence of Sirt1. Finally, we have made progress in initiating a project in human stem
  • cells that can be reprogrammed from adult cells, to extend our findings from mice to
  • humans, in particular as it relates to human diseases that have an age-dependent
  • component.
  • The number and function of adult stem cells decrease with age in a number of tissues. In the nervous system, the depletion of functional adult neural stem cells (NSC) may be responsible for impaired cognitive performance associated with normal or pathological aging. Understanding the factors that govern the maintenance of adult NSC should provide insights into their regenerative potential and open new avenues to use these cells for therapeutic purposes during normal aging and age-related neurodegenerative disorders.
  • Clues to key regulators of stem cell functions may come from studies of the genetics of aging, as genes that regulate longevity may do so by maintaining stem cells. To date, the most compelling examples for genes that control aging in a variety of organisms include the insulin-Akt-Foxo transcription factor pathway and the Sirt deacetylases. We have recently found that Foxo3 regulates a network of genes in adult NSC and interact with another transcription factor, called Ascl1, to preserve the integrity of the NSC pool and prevent the premature exhaustion of this important pool of cells. In the past year, we have also made the surprising discovery that inactivating Sirt1 in adult neural stem cells leads to the increased production of oligodendrocyte progenitors, which are cells that are crucial for myelination and could help demyelinating diseases, such as multiple sclerosis, or demyeliating injuries such as spinal cord injuries. Importantly, the enzymatic activity of Sirt1 can be targeted by small molecules, underscoring the potential for Sirt1 as a therapeutic target in stem cell and oligodendrocyte production. In the last year, we have also made significant progress in using cellular reprogramming to investigate the role of longevity genes in human cells. Our work examines the mechanisms by which ‘longevity genes’ regulate stem cell function and maintenance. Harnessing the regenerative power of stem cells by acting on longevity genes will provide a novel angle to identify stem cell therapeutics for regenerative medicine.
  • The adult brain contains reservoirs of neural stem cells that are critical for the formation of new neurons, oligodendrocytes, and astrocytes in the adult brain. During aging, the number of neural stem cells and their ability to give rise to new neurons strikingly decline. This decline could underlie at least in part the decline in memory that occurs during aging. We are interested in the importance of genes that regulate organismal longevity in the control of the reservoir of neural stem cells. We discovered that Foxo3, a transcription factor that has been implicated in human exceptional longevity, is important for regulating the neural stem cell pool pool. In the past year, we have made extensive progress in characterizing the interaction between Foxo3 and specific chromatin states at target genes in adult neural stem cells, which provides us with a mechanistic view onto how longevity genes can affect specific networks of target genes in neural stem cells in adult organisms. In the past year, we have made significant progress in testing the role of a gene involved in healthspan and longevity in a number of organisms, the deacetylase Sirt1, in adult neural stem cell function. We have observed that Sirt1 inactivation, whether genetic or pharmacological, leads to an increase in oligodendrocyte progenitors, which are cells that are important for myelination of axons. We have found that Sirt1 inactivation is beneficial for models of demyelinating injuries and diseases, which has important consequences for multiple sclerosis. Finally, we are making progress in reprogramming adult human fibroblasts into induced pluripotent stem cells and induced NSCs, with the aim to test the importance of longevity genes in this process.

Defining the Isoform-Specific Effects of Apolipoprotein E on the Development of iPS Cells into Functional Neurons in Vitro and in Vivo

Funding Type: 
New Faculty II
Grant Number: 
RN2-00952
ICOC Funds Committed: 
$2 847 600
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
GOALS We propose to determine the effects of different forms of apoE on the development of induced pluripotent stem (iPS) cells into functional neurons. In Aim 1, iPS cells will be generated from skin cells of adult knock-in (KI) mice expressing different forms of human apoE and in humans with different apoE genotypes. In Aim 2, the development of the iPS cells into functional neurons in culture and in mouse brains will be compared. In Aim 3, the effects of different forms of apoE on the functional recovery of mice with acute brain injury treated with iPS cell–derived neural stem cells (NSCs) will be assessed. RATIONALE AND SIGNIFICANCE The central nervous system (CNS) has limited ability to regenerate and recover after injury. For this reason, recovery from acute and chronic neurological diseases, such as stroke and Alzheimer’s disease (AD), is often incomplete and disability results. Embryonic stem cells have great promise for treating or curing neurological diseases, but their therapeutic use is limited by ethical concerns and by rejection reactions after allogenic transplantation. The generation of iPS cells from somatic cells offers a way to potentially circumvent the ethical issues and to generate patient- and disease-specific stem cells for future therapy. In the CNS, apoE plays important roles in lipid homeostasis and in neuronal maintenance. However, apoE2, apoE3, and apoE4 differ in their ability to accomplish these tasks. ApoE4, the major genetic risk factor for AD, is associated with poor clinical outcome and more rapid progression or greater severity of head trauma, stroke, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis—all potential targets of stem cell therapy. This proposal builds on three novel findings in human apoE-KI mice. (1) NSCs express apoE. (2) ApoE plays a role in cell-fate determination (neuron vs astrocyte) of NSCs. (3) ApoE4 impairs the neuronal development of NSCs. Thus, we hypothesize that transplantation of iPS cells derived from apoE4 carriers (~20% of the general population and ~50% of AD patients) might not be beneficial or even detrimental for patients with neurological diseases. We propose in vitro and in vivo studies to assess the effects of different forms of apoE on the development of iPS cells into functional neurons and on the functional recovery of mice with acute brain injury treated with iPS cell-derived NSCs. These studies will shed light on the regulation of neuronal development of iPS cells and help to “optimize” future iPS cell therapy for neurological diseases. SPECIFIC AIMS Aim 1. To establish adult mouse and human iPS cell lines with different apoE genotypes. Aim 2. To determine the isoform-specific effects of apoE on the development of iPS cells into functional neurons in culture and in mouse brains. Aim 3. To assess the isoform-specific effects of apoE on the functional recovery of mice with acute (stroke) brain injury treated with iPS cell-derived NSCs.
Statement of Benefit to California: 
CONTRIBUTION TO THE CALFORNIA ECONOMY: A major goal of regenerative medicine is to repair damaged cells or tissue. My research focuses on (1) understanding the role of neuronal regeneration in central nervous system function and (2) developing stem cell therapy for acute and chronic neurological diseases, including stroke and Alzheimer's disease. Stroke and Alzheimer's disease are the leading causes of disability and dementia and are the fastest growing form of neurological diseases in California, in the USA, and worldwide. My research could benefit the California economy by creating jobs in the biomedical sector. Ultimately, this study could help reduce the adverse impact of neurological diseases. Thereby, I hope to increase the productivity and enhance the quality of life for Californians. The results of my studies will also help develop new technology that could contribute to the California biotechnology industry. The studies will characterize multiple lines of induced pluripotent stem (iPS) cells carrying apoE3, a protein protective to the brain, or apoE4, which is detrimental to the brain and is associated with increased risk of Alzheimer’s disease and other neurodegenerative disorders. These cell lines could be valuable for biotechnology companies and researchers who are screening for drug compounds targeting different neurological diseases. CONTRIBUTION TO THE HEALTH OF CALFORNIANS: The most important contribution of the studies will be to improve the health of Californians. Diseases that are the target of regenerative medicine, such as stroke and Alzheimer’s disease, are major causes of mortality and morbidity, resulting in billions of dollars in healthcare costs and lost productivity. As we continue our efforts in medical research, we hope to one day unlock the secrets of brain development and repair. This knowledge will help medical researchers develop beneficial therapies beyond what is currently available and potentially improve the quality of life and life expectancy of patients with neurological diseases, such as stroke and Alzheimer’s disease.
Progress Report: 
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in Aims 1 and 2.
  • First, we further demonstrated that neural stem cells (NSCs) express apoE. ApoE-KO mice had significantly less hippocampal neurogenesis, but significantly more astrogenesis, than wildtype mice due to decreased Noggin expression in NSCs. In contrast, neuronal maturation in apoE4 knock-in (apoE4-KI) mice was impaired due to reduced survival and function of GABAergic interneurons in the hilus of the hippocampus, and a GABAA receptor potentiator rescued the apoE4-associated decrease in hippocampal neurogenesis. Thus, apoE plays an important role in hippocampal neurogenesis, and the apoE4 isoform impairs GABAergic input to newborn neurons, leading to decreased neurogenesis. A paper describing these data was published in Cell Stem Cell (Li G. et al. 2009, 5:634-645), which evidently is the 400th publication of CIRM-funded projects.
  • Second, we established mouse iPS cell lines from adult mouse fibroblasts of wildtype, apoE knockout (apoE-KO), human apoE2-KI, human apoE3-KI, and human apoE4-KI mice.
  • Finally, we developed NSC lines from mouse iPS cells with different apoE genotypes (wildtype mouse apoE, apoE-KO, apoE2, apoE3, and apoE4). These cell lines will be used to study the effects of apoE isoforms on neuronal development in vitro in culture and in vivo in mouse models.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in the past year, as summarized below.
  • First, We developed human iPS cells from skin fibroblasts of individuals with different apoE genotypes. We are fully characterizing these human iPS cell lines.
  • Second, We are establishing neural stem cell (NSC) lines from human iPS cells with different apoE genotypes. Some of the NSCs have been maintained in monolayer cultures for many generations. These NSCs will be used to study the effects of apoE isoforms on neuronal development in vitro in cultures and in vivo in mice.
  • Finally, we demonstrated that mouse apoE4-NSCs generated significantly fewer total neurons and fewer GABAergic interneurons than mouse apoE3-NSCs in culture. Thus, the detrimental effects of apoE4 on neurogenesis and GABAergic interneuron survival, as we observed in vivo in apoE4 knock-in mice (Li G. et al. Cell Stem Cell, 2009, 5:634-645), are recapitulated in cultures of mouse iPS cell–derived NSCs in vitro.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We have fully characterized two apoE3/3-hiPS cell lines and two apoE4/4-iPS cell lines.
  • 2) We have established NSC lines from human iPS cells with an apoE3/3 or apoE4/4 genotype. The hNSCs have been maintained in suspension or monolayer culture for multiple passages.
  • 3) We demonstrated that apoE4-hNSCs generated ~50% fewer GABAergic interneurons than apoE3-hNSCs in culture. Thus, the detrimental effects of apoE4 on GABAergic interneuron survival, as we observed in vivo in apoE4 knock-in mice (Li G. et al. Cell Stem Cell, 2009, 5:634-645), are recapitulated in cultures of human iPS cell-derived NSCs in vitro.
  • 4) We established protocols in our lab to differentiate human iPS cell-derived NSCs into different types of neurons in cultures.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We demonstrated that apoE4-miPSC-derived mNSCs had a greater “age-dependent (passage-dependent)” decrease in generation and/or survival of MAP2-positive neurons in cultures.
  • 2) We also demonstrated that apoE4-miPSC-derived mNSCs had an even greater “age-dependent (passage-dependent)” decrease in generation and/or survival of GAD67-positive GABAergic neurons, as seen in vivo in apoE4 knock-in mice (Li et al., Cell Stem Cell, 2009, 5:634–645).
  • 3) We expanded the pilot study reported last year and confirmed the detrimental effect of apoE4 on GABAergic interneuron development/survival of hiPS cell-derived hNSCs. ApoE4 also increased tau phosphorylation, one of the pathological hallmarks of Alzheimer’s disease, in neurons derived from apoE4-hiPS cells.
  • 4) We established a protocol to transplant apoE-miPS cell-derived mNSCs into mouse brains. The transplanted apoE-mNSCs developed into neurons and astrocytes and integrated into the neural circuitry.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of pluripotent stem cells into functional neurons in vitro in culture and in vivo in mice for potential cell replacement therapy. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We demonstrated that mouse GABAergic progenitors transplanted into the hilus of apoE3-KI and apoE4-KI mice developed into mature interneurons and functionally integrated into the hippocampal circuitry.
  • 2) We also demonstrated that transplantation of mouse GABAergic progenitors into the hilus of apoE4-KI mice rescued learning and memory deficits.
  • 3) Transplantation of mouse GABAergic progenitors into the hilus of hippocampus also rescued learning and memory deficits in apoE4-KI mice expressing Alzheimer’s disease-causing APP mutations.

High throughput modeling of human neurodegenerative diseases in embryonic stem cells

Funding Type: 
New Faculty II
Grant Number: 
RN2-00919
ICOC Funds Committed: 
$2 259 092
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Neuropathy
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
An important class of neurological diseases predominantly affects spinal motor neurons, the neurons that control muscle movement. The most well known of these motor neuronopathies is Amyotrophic Lateral Sclerosis (ALS), commonly referred to as Lou Gehrig’s disease for the famous Yankee first baseman who died of the disease. The first symptoms of ALS are usually increasing difficulty walking or speaking clearly. People with ALS progressively lose their ability to initate and control movements, and may become totally paralyzed during the late stages of the disease. There are no cures or effective treatments for these diseases. Riluzole (Rilutek), the only FDA approved medication for ALS, only modestly slows disease progression. Consequently, ALS is usually fatal within one to five years from onset, with half dying within eighteen months. Although genetic studies have identified many mutations that cause these diseases, it is not understood why these mutations kill motor neurons. This lack of understanding about the root causes of motor neuron diseases currently hinders the development of effective treatments. We seek to study motor neurons carrying these mutations in cell culture dishes to understand how these diseases sicken and kill these cells. To generate these motor neurons, we will use embryonic stem cells. Embryonic stem cells can become any cell in our body, including motor neurons. We have developed a new technology that allows us to quickly replace healthy genes with mutant genes in mouse embryonic stem cells. We will use this technology to insert both normal and disease-associated versions of genes into embryonic stem cells. Study of the healthy and mutant mutant motor neurons derived from these embryonic stem cells will shed light on the ways in which the mutations cause harm. The development of cell based models of human diseases is likely to have additional benefits as well. For example, diseased motor neurons grown in cell culture dishes can be quickly and efficiently screened with potential drugs to discover agents that slow, halt or reverse the cellular damage. It is our hope that these experiments will both deepen our understanding of important neurodegenerative disorders, and lead to new directions for the development of effective therapies.
Statement of Benefit to California: 
Over 6,000 Americans are diagnosed each year with motor neuronopathies, about the same as are diagnosed with multiple sclerosis. One form of this illness, ALS, is responsible for about one in every 800 deaths, and cause many lengthy and costly hospital admissions. We propose using stem cells to model these diseases so that we can gain a deeper understanding of their root causes. It is our expectation that this deeper understanding will lead to new and better approaches to the treatment of these disorders. In addition, our technology for developing embryonic stem cell-based models of human diseases is likely to have applications in the biotechnology sector. Although our technology is most applicable for modeling simple dominant genetic diseases, it can be adapted to model recessive and complex disorders. Beyond increasing our understanding of human diseases, these cellular models represent useful screening tools for testing novel pharmacological treatments. Identification and development of these new therapies may support new companies or new products for existing companies. We hope that using stem cells to model neurodegenerative disorders will lead to progress in the fight against these diseases, as well as provide the tools and examples for those in academia and industry who hope to create stem cell models of other clinically important disorders.
Progress Report: 
  • We have been developing new tools for the genetic modification of embryonic stem cells (ESCs). Part of the potential for use of ESCs in treatments or as models of disease depends on the ability to change genes within ESCs. We have developed a novel system, which we call the Floxin system, that allows for the more efficient modification of genes within mouse ESCs than has been historically feasible. We have used this system to insert mutations that cause human diseases into mouse ESCs. Introducing human mutations into ESCs has allowed us to study the function of these mutations in the context of stem cell function and gain insight into how these mutations cause human disease.
  • We are interested in extending our findings by modeling an important class of neurological diseases that predominantly affect spinal motor neurons, the neurons that control muscle movement. The most well known of these motor neuronopathies is Amyotrophic Lateral Sclerosis (ALS), commonly referred to as Lou Gehrig’s disease, but there are a number of other motor neuronopathies including Hereditary Motor Neuronopathy and Spinal Muscular Atrophy.
  • Human genetic studies have identified many mutations that cause these diseases, but it is not understood why these mutations kill motor neurons. This lack of understanding about the root causes of motor neuron diseases currently hinders the development of effective treatments. We are currently using the Floxin system to introduce human motor neuronopathy-associated mutations into mouse ESCs. We have introduced mutations into two disease-associated genes, and are deriving motor neurons from these modified ESCs to study how the mutations kill these cells.
  • The development of cell-based models of human diseases is likely to have additional benefits as well. For example, diseased motor neurons grown in cell culture dishes can be quickly and efficiently screened with potential drugs to discover agents that slow, halt or reverse the cellular damage. It is our hope that these experiments will both deepen our understanding of important neurodegenerative disorders, and lead to new directions for the development of effective therapies.
  • We have made the resource of Floxin vectors and the greater than 24,000 characterized Floxin compatible ESC lines available to the research community. Application of the Floxin technology to this resource will allow genetic modification of more than 4,500 genes in ESCs. Furthermore, we are adapting the Floxin technology for use in human ESCs which may allow for tractable genetic engineering in these cells. We anticipate that this technology will allow many researchers to create cellular models of human disease and other genetic modifications that will facilitate the use of stem cells in fighting diverse diseases.
  • We have developed new tools for the genetic modification of embryonic stem cells (ESCs) and are using these tools to model human diseases. Part of the potential for use of ESCs in treatments or as models of disease depends on the ability to change genes within ESCs. We have developed a novel system, which we call the Floxin system, that allows for the more efficient modification of genes within mouse ESCs than has been historically feasible. We use this system to insert mutations that cause human diseases into mouse ESCs. Introducing human mutations into ESCs has allowed us to study the function of these mutations in the context of stem cell function and gain insight into how these mutations cause human disease. To date, we have investigated an inherited congenital malformation syndrome called Orofaciodigital syndrome and elucidated that the underlying birth defects are caused by misregulation of cilia and centrioles, structures within all cells. We have also used our system to investigate how genes are regulated by Polycomb-like proteins and to reveal how cilia control ESC differentiation into motor neurons, findings that shed light on the control of motor neuron production from ESCs.
  • We are extending our findings by modeling an important class of neurological diseases that predominantly affect spinal motor neurons, the neurons that control muscle movement. The most well known of these motor neuronopathies is Amyotrophic Lateral Sclerosis (ALS), commonly referred to as Lou Gehrig’s disease, but there are a number of other motor neuronopathies including Hereditary Motor Neuronopathy and Spinal Muscular Atrophy. Human genetic studies have identified many mutations that cause these diseases, but it is not understood why these mutations kill motor neurons. This lack of understanding about the root causes of motor neuron diseases currently hinders the development of effective treatments.
  • We have used the Floxin system to introduce human motor neuronopathy-associated mutations into mouse ESCs. We have introduced mutations into two disease-associated genes, and have derived motor neurons from these modified ESCs to study how the mutations kill these cells. The development of cell-based models of human diseases is likely to have additional benefits as well. For example, diseased motor neurons grown in cell culture dishes can be quickly and efficiently screened with potential drugs to discover agents that slow, halt or reverse the cellular damage. It is our hope that these experiments will both deepen our understanding of important neurodegenerative disorders, and lead to new directions for the development of effective therapies.
  • We have made the resource of Floxin vectors and the greater than 24,000 characterized Floxin compatible ESC lines available to the research community. Application of the Floxin technology to this resource will allow genetic modification of more than 4,500 genes in ESCs. Furthermore, we are hoping to adapt the Floxin technology for use in human ESCs which may allow for tractable genetic engineering in these cells. We anticipate that this technology will allow many researchers to create cellular models of human disease and other genetic modifications that will facilitate the use of stem cells in fighting diverse diseases.
  • An important class of neurological diseases predominantly affects spinal motor neurons, the neurons that control muscle movement. The most well known of these motor neuronopathies is Amyotrophic Lateral Sclerosis (ALS), commonly referred to as Lou Gehrig’s disease for the famous Yankee first baseman who died of the disease. The first symptoms of ALS are usually increasing difficulty walking or speaking clearly. People with ALS progressively lose their ability to initate and control movements, and may become totally paralyzed during the late stages of the disease. There are no cures or effective treatments for these diseases. Riluzole (Rilutek), the only FDA approved medication for ALS, only modestly slows disease progression. Consequently, ALS is usually fatal within one to five years from onset, with half dying within eighteen months.
  • Although genetic studies have identified many mutations that cause these diseases, it is not understood why these mutations kill motor neurons. This lack of understanding about the root causes of motor neuron diseases currently hinders the development of effective treatments. We seek to study motor neurons carrying these mutations in cell culture dishes to understand how these diseases sicken and kill these cells.
  • To generate these motor neurons, we are using embryonic stem cells. Embryonic stem cells can become any cell in our body, including motor neurons. We have developed a new technology that allows us to quickly replace healthy genes with mutant genes in mouse embryonic stem cells. We are using this technology to insert both normal and disease-associated versions of genes into embryonic stem cells. Study of the healthy and mutant mutant motor neurons derived from these embryonic stem cells will shed light on the ways in which the mutations cause harm.
  • We have been using the mutant embryonic stem cells to assay leading hypotheses about how diseases like ALS begin. In addition, we are using the embryonic stem cells to create new animal models of ALS. Finally, we are adapting our technology to be able to create more faithful models of disease using embryonic stem cells in order to expedite understanding into the origins of these diseases.
  • Neurodegenerative diseases, including Alzheimer disease, Parkinson disease, and Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig’s disease), affect an increasing proportion of our population as the median age increases. There are no cures for any of these disorders. One reason for the absence of cures has been the absence of good models to understand how neurodegeneration happens.
  • Genetic studies have identified many of the genes involved in neurodegeneration. To understand how these mutations lead to motor neuron degeneration in ALS, we have creased embryonic stem cells (ESCs) that contain the human ALS-associated mutations. We have also created mice that express these human ALS-associated mutations. We are studying motor neurons derived from the ESCs and the mutant mice to understand how motor neurons die in ALS. We are defining the proteins and RNAs that interact with normal and disease-associated proteins, and following the mutant neurons over time to examine how they die. Currently, we are testing the hypothesis that disease mutations alter the gene product’s normal interactions, leading to a tonic increase in cell death rate. After several decades of life, the loss of neurons surpasses compensatory mechanisms, leading to the emergence of symptoms.
  • Neurodegenerative diseases, including Alzheimer disease, Parkinson disease, and Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig’s disease), affect an increasing proportion of our population as the median age increases. There are no cures for any of these disorders. One reason for the absence of cures has been the absence of good models to understand how neurodegeneration happens.
  • Genetic studies have identified many of the genes involved in neurodegeneration. To understand how these mutations lead to motor neuron degeneration in ALS, we have creased embryonic stem cells (ESCs) that contain the human ALS-associated mutations. We have also created mice that express these human ALS-associated mutations. We studied motor neurons derived from the ESCs and the mutant mice and found that motor neurons with ALS-associated mutations die at increased rates. We identified proteins that interact with normal and disease-associated proteins. We identified that mutant proteins showed different interactions than normal proteins. After several decades of life, the loss of neurons surpasses compensatory mechanisms, leading to the emergence of symptoms.

Mechanisms in Choroid Plexus Epithelial Development

Funding Type: 
New Faculty II
Grant Number: 
RN2-00915
ICOC Funds Committed: 
$2 994 328
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Buried deep inside the brain are cells known as choroid plexus epithelial (CPe) cells. Although not as famous as other cells in the nervous system, CPe cells perform a large number of important jobs that keep the brain and spinal cord healthy. They produce the fluid (known as cerebrospinal fluid, or CSF) that bathes the brain and spinal cord with many nourishing chemicals, which promote normal nervous system health and function, learning and memory, and neural repair following injury. In addition, CPe cells protect the brain and spinal cord from toxins – such as heavy metals and the amyloid-beta peptide associated with Alzheimer’s disease – by absorbing them or preventing them from entering the nervous system altogether by forming the so-called blood-CSF barrier. Accordingly, as CPe functions diminish during normal aging or in accelerated fashion in certain diseases, memory loss, Alzheimer’s disease, and a number of other neurologic and neuropsychiatric disorders may ensue or become worse. The ability to grow and make CPe cells should therefore enable many clinical applications, such as CPe cell replacements, transplants, and pharmaceutical studies to identify beneficial drugs that can pass through the blood-CSF barrier. However, all of these potential applications are limited by the current inability to make and expand CPe cells in culture. Our published and preliminary studies suggest that it should be feasible to generate CPe cells in culture. Our broad goals are to study how CPe cells form during normal development, then use this information to make human CPe cells for clinical applications. To achieve this goal, our approach will be to use mice to study how the CPe develops normally, then use both mouse and human stem cells to make CPe cells in culture. Our published and preliminary studies have defined one critical factor for this process (known as Bmp4) and identify candidate factors that work with Bmp4 to regulate whether or not CPe cells are formed. In Aim 1, we test whether a molecule known as Fgf8 provides CPe “competency” – i.e. whether Fgf8 allows cells to become CPe cells when exposed to Bmp4. In Aim 2, we test whether a gene known as Lhx2 prevents cortical cells from becoming CPe cells in response to Bmp4. In Aim 3, we manipulate Bmp4, Fgf8, and Lhx2 in hESC cultures to make human CPe cells. If successful, this proposal should greatly improve our understanding of normal CPe development and enable a number of CPe-based clinical applications with significant potential to improve human health.
Statement of Benefit to California: 
Our proposal to study choroid plexus epithelial (CPe) cell development and to make CPe cells in culture for clinical applications should benefit the State of California and its citizens in a number of ways. In the short term, this project will provide employment, education and training in stem cell research for a handful of California residents, and will support California-based companies that provide supplies for the stem cell and biomedical research communities. In the longer term, success in making CPe cells in culture should enable many new CPe-based clinical applications, stimulate CPe studies and applications by stem cell companies, and enable screens to identify agents that allow for passage of therapeutics across the blood-CSF barrier, which remains a significant roadblock to the development of pharmaceuticals for neurological and neuropsychiatric disorders. Such outcomes would ultimately stimulate investment in California-based companies and benefit the health of many California citizens, which may reduce the economic burden of health care in the state.
Progress Report: 
  • Our project goals are to define the factors involved in choroid plexus epithelial (CPe) cell development in mice, then apply this information to generate CPe cells from mouse and human embryonic stem cells (ESCs) for clinical applications. The first Aim is to determine whether a factor known as Fgf8 promotes CPe fate, the second Aim addresses whether the Lhx2 transcription factor inhibits CPe, and the third Aim is to generate human CPe cells in culture. Significant progress on these Aims has been made during this first year of the grant. Most importantly, multiple lines of evidence for CPe differentiation from both mouse and human ESCs have been obtained. In addition, the genetically-engineered mESC lines needed for the Lhx2 studies in Aim 2 have been successfully generated and validated. Our major goals for the next year are to further replicate, confirm, and optimize the generation of CPe cells in our mouse and human ESC cultures, and to perform the initial experiments that should determine whether manipulating Fgf8 and Lhx2 in the ESC cultures will enhance CPe generation in culture.
  • Our goal is to define the factors involved in choroid plexus epithelial (CPe) cell development in mice, then to apply this knowledge to generate CPe cells from mouse and human embryonic stem cells (ESCs) for clinical applications. The first two Aims examine Fgf8 and Lhx2 as promoter and inhibitor, respectively, of CPe fate, and the third Aim is to generate human CPe cells in culture. Unexpectedly, we obtained significant evidence for CPe differentiation from both mouse and human ESCs during year 1 of the award. Our aims for year 2 were therefore modified to accelerate the translation of our findings towards a CPe-based regenerative medicine. This year, we developed a second cell culture system for deriving mouse CPe cells, and established a functional assay for CPe cells in culture, which we used to confirm the function of our derived mouse CPe cells. To sort and purify CPe cells for clinical applications, we began characterizing CPe cell complexity, size, and mitochondrial content by flow cytometry, obtained a mouse line with fluorescent CPe cells, and identified three antibodies that may be useful for sorting human CPe cells. A stereotaxic injection system was built, and institutional approvals were obtained, to establish methods for replacing or transplanting CPe cells in the mouse brain.
  • The goal of this project is to define the factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. The first two Aims used mice to examine a potential promoter and inhibitor, respectively, of CPEC fate, and the third Aim is to generate human CPECs in culture. Unexpectedly early success in CPEC derivation from human ESCs has allowed us to accelerate Aim 3 and the pursuit of translational goals this year. We further optimized our existing human CPEC derivation method and developed a second method (a combined suspension-adherent system) that may prove to be much more efficient. Several new GMP-compliant human ESC lines were approved and obtained. To facilitate the translational efforts, we made many new mouse ESC lines that were designed to fluoresce when CPECs are produced, and this was confirmed using the first of these lines. A crude CPEC purification strategy was also developed, and using this strategy, transplantation of partially-purified CPECs into mice was established in the lab this year. Remarkably, we found that transplanted mESC-derived CPECs, on their own, can integrate into endogenous choroid plexus with relatively high efficiency. This opens up several new and exciting therapeutic possibilities. To further enhance choroid plexus engraftment, a mouse CPEC ablation approach is currently being tested. A collaboration was initiated to profile all of the genes expressed by the purified mouse ESC-derived CPECs, and to compare this profile to those expressed by the choroid plexus in developing mice and humans. Industry partnerships and non-provisional patenting were also pursued to enhance the prospects for human CPEC applications in drug screening and treating patients with a wide range of neurodegenerative and other nervous system disorders.
  • The goal of this project is to define factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. Unexpected early success in generating ESC-derived CPECs (dCPECs) allowed us to accelerate and focus on the more translational goals of the project this year. We tested two new culture systems, with promising results from a more controllable and scalable monolayer culture system that will facilitate the improvement of dCPEC generation efficiency. New transcriptome profiling studies allowed us to better define highly-expressed genes for cell surface proteins, which will be targeted to purify dCPECs for downstream applications. New double-labelling and whole mount preparations of mouse choroid plexus have been devised to facilitate ongoing efforts to improve dCPEC engraftment of host choroid plexus after injection, and a new functional assay for dCPEC barrier formation and regulation has been established to complement an already-existing functional secretion assay in the lab. Efforts are also now underway to generate fluorescent and luminescent CPEC reporter hESC lines that should greatly facilitate dCPEC process development (derivation and purification). During this past year, new industry partners were recruited, an initial paper describing the dCPEC technology was published, and an initial patent application on the dCPEC technology was filed.
  • The goal of this project is to define factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. Unexpected early success in generating ESC-derived CPECs (dCPECs) allowed us to accelerate and focus on the more translational goals of the project this year. We further developed two culture systems - a more controllable monolayer system and more scalable rotational aggregate system - that will facilitate the dCPEC work. After several disappointments, improvements in dCPEC differentiation efficiency were obtained with two pharmacologic agents. With help from transcriptome profiling studies, we identified cell surface proteins that could be utilized for dCPEC enrichment, with initial promising results for one candidate surface antigen. A robust whole mount choroid plexus culture system was newly developed to facilitate efforts to improve dCPEC engraftment of host choroid plexus, and methods surrounding the stereotactic injection of dCPECs have been improved. After some difficulties, human TTR BAC constructs that express fluorescent and luminescent reporters were created and validated; these will be used to generate new CPEC reporter mouse lines for endpoint and longitudinal studies, and for in vivo drug testing of compounds that enhance TTR production and CPEC secretion. The initial patent application on the dCPEC technology was reviewed by the US PTO, and a revision was submitted.

Molecules to Correct Aberrant RNA Signature in Human Diseased Neurons

Funding Type: 
Early Translational III
Grant Number: 
TR3-05676
ICOC Funds Committed: 
$1 654 830
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Approximately 5,600 people in the U.S. are diagnosed with ALS each year. The incidence of ALS is two per 100,000 people, and it is estimated that as many as 30,000 Americans may have the disease at any given time. There are no effective therapies of ALS to-date. Recent genetic discoveries have pinpointed mutations that lead to the aberrant function of two proteins that bind to RNA transcripts in neurons. Misregulation of these RNA binding proteins is responsible for the aberrant levels and processing of hundreds of RNA representing genes that are important for neuronal survival and function. In this proposal, we will use neurons generated from patient cells that harbor the mutations in these RNA binding proteins to (1) prioritize a RNA “signature” unique to neurons suffering from the toxic function of these proteins and (2) as an abundant source of raw material to enable high-throughput screens of drug-like compounds that will bypass the mutations in the proteins and “correct” the RNA signature to resemble that of a healthy neuron. If successful, our unconventional approach that uses hundreds of parallel measurements of specific RNA events, will identify drugs that will treat ALS patients.
Statement of Benefit to California: 
Our research aims to develop drug-like compounds that are aimed to treat Amyotrophic Lateral Sclerosis (ALS), which may be applicable to other neurological diseases that heavily impact Californians, such as Frontotemporal Lobar Degeneration, Parkinson’s and Alzheimer’s. The cellular resources and genomic assays that we are developing in this research will have great potential for future research and can be applied to other disease areas. The cells, in particular will be beneficial to California health care patients, pharmaceutical and biotechnology industries in terms of improved human models for drug discovery and toxicology testing. Our improved knowledge base will support our efforts as well as other Californian researchers to study stem cell models of neurological disease and design new diagnostics and treatments, thereby maintaining California's position as a leader in clinical research.
Progress Report: 
  • Our research aims to develop drug-like compounds that are aimed to treat Amyotrophic Lateral Sclerosis (ALS), which may be applicable to other neurological diseases that heavily impact Californians, such as Frontotemporal Lobar Degeneration, Parkinson’s and Alzheimer’s. In the first year, we have succeeded in improving the efficiency of motor neuron differentiation to generate high-quality motor neurons from induced pluripotent stem cells. We have generated RNA signatures from motor neurons differentiated from induced pluripotent stem cells from normal, healthy individuals whereby key proteins implicated in ALS are depleted using RNAi technology. We have also generated motor neurons from induced pluripotent stem cells that contained mutations in these key proteins and are in the process of applying genomic technologies to compare these cells to ones where we have depleted the proteins themselves. In parallel, we have started to optimize conditions for a small molecule screen to identify previously FDA-approved compounds that may alter aberrant and ALS-associated phenotypes in human cell lines.

Stem Cells Secreting GDNF for the Treatment of ALS

Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05320
ICOC Funds Committed: 
$89 834
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
This project aims to use a powerful combined stem cell and gene therapy approach to treat patients with amyotrophic lateral sclerosis (ALS or Lou Gehrig’s Disease). ALS is a devastating disease for which there is no treatment or cure. Progression from early muscle twitches to complete paralysis and death usually happens within 4 years. Every 90 minutes someone is diagnosed with ALS in the USA, and every 90 minutes someone dies from ALS. In California the death rate is one person every one and a half days. Stem cells have been shown to produce support cells for dying motor neurons called astrocytes which may slow down disease progression. Furthermore, many studies have shown that growth factors such as glial cell line-derived growth factor (or GDNF) can protect motor neurons from damage in a number of different animal models including those for ALS. However, delivering GDNF to the spinal cord has been almost impossible as it does not cross from the blood to the brain tissue. The idea behind the current proposal is to modify stem cells to produce GDNF and then transplant these cells into patients. A number of advances in human stem cell biology along with new surgical approaches has allowed us to put together this disease team approach – a first in man study to deliver cells modified to release a powerful growth factor that are expected to slow down the death of motor neurons and paralysis in patients. The focus of the proposal will be to perform essential preclinical studies in both small and large animals that will establish optimal doses and safe procedures for translating this stem cell and gene therapy into human patients. The Phase 1 clinical study will include 30 ALS patients from the state of California. This will be the first time this type of stem cell and gene therapy has been available to any ALS patients in the world.
Statement of Benefit to California: 
ALS is a devastating disease, and also puts a large burden on state resources through the need of full time care givers and hospital equipment. It is estimated that the cost of caring for an ALS patient in the late stage of disease while on a respiration is $200,00-300,000 per year. While primarily a humanitarian effort to avoid suffering, this project will also ease the cost of caring for ALS patients in California if ultimately successful. As the first trial in the world to combine stem cell and gene therapy it will make California a center of excellence for these types of studies. This in turn will attract scientists, clinicians, and companies interested in this area of medicine to the state of California thus increasing state revenue and state prestige in the rapidly growing field of Regenerative Medicine.
Progress Report: 
  • We completed the planning for submission of the CIRM Disease Team Grant on time. The series of meetings we had with leaders in the field of translational medicine as it relates to using stem cells secreting GDNF to treat ALS was extremely useful and allowed us to progress towards a very structured plan for both cell production, pre-clincial animal IND enabling studies and the final clinical trial involving 3 different institutions.

Molecular basis of plasma membrane characteristics reflecting stem cell fate potential

Funding Type: 
Basic Biology V
Grant Number: 
RB5-07254
ICOC Funds Committed: 
$1 003 590
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Closed
Public Abstract: 
Stem cells generate mature, functional cells after proteins on the cell surface interact with cues from the environment encountered during development or after transplantation. Thus, these cell surface proteins are critical for directing transplanted stem cells to form appropriate cells to treat injury or disease. A key modification regulating cell surface proteins is glycosylation, which is the addition of sugars onto proteins and has not been well studied in neural stem cells. We focus on a major unsolved problem in the neural stem cell field: do different proteins coated with sugars on the surfaces of cells in this lineage (neuron precursors, NPs and astrocyte precursors, APs) determine what types of mature cells will form? We hypothesize key players directing cellular decisions are glycosylated proteins controlling how precursors respond to extracellular cues. We will address this hypothesis with aims investigating whether (1) glycosylation pathways predicted to affect cell surface proteins differ between NPs and APs, (2) glycosylated proteins on the surface of NPs and APs serve as instructive cues governing fate or merely mark their fate potential, and (3) glycosylation pathways regulate cell surface proteins likely to affect fate choice. By answering these questions we will better understand the formation of NPs and APs, which will improve the use of these cells to treat brain and spinal cord diseases and injuries.
Statement of Benefit to California: 
The goal of this project is to determine how cell surface proteins differ between cells in the neural lineage that form two types of final, mature cells (neurons and astrocytes) in the brain and spinal cord. In the course of these studies, we will uncover specific properties of human stem cells that are used to treat neurological diseases and injuries. We expect this knowledge will improve the use of these cells in transplants by enabling more control over what type of mature cell will be formed from transplanted cells. Also, cells that specifically generate either neurons or astrocytes can be used for drug testing, which will help to predict the effects of compounds on cells in the human brain. We hope our research will greatly improve identification, isolation, and utility of specific types of human neural stem cells for treatment of human conditions. Furthermore, this project will generate new jobs for high-skilled workers and, hopefully, intellectual property that will contribute to the economic growth of California.

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