Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders
Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01021
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$918 000
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells have considerable potential as sources of differentiated cells for numerous biomedical applications. The ability to introduce targeted changes into the DNA of these cells – a process known as gene targeting – would have very broad implications. For example, mutations could readily be introduced into genes to study their roles in stem cell propagation and differentiation, to analyze mechanisms of human disease, and to develop disease models to aid in creating new therapies. Unfortunately, gene targeting efficiency in hESCs is very low. To meet this urgent need, we propose to develop new molecular tools and novel technologies for high efficiency gene targeting in hES and iPS cells. Importantly, this approach will be coupled with genome-wide identification and functional analysis of genes involved in the process in dopaminergic neuron development, work with fundamental implications for Parkinson's disease. Barriers to targeted genetic modification include the effective delivery of gene targeting constructs into cells and the introduction of defined changes into the genome. We have developed a high throughput approach to engineer novel properties into a highly promising, safe, and clinically relevant gene delivery vehicle. For example, we have engineered variants of this vehicle with highly efficient gene delivery to neural stem cells (NSCs), and the resulting vehicles can mediate efficient gene targeting. We now propose to engineer novel gene delivery and targeting vehicles optimized for use in hESCs and iPS cells. One application of such an improved vector system will be to study the mechanism of ESC differentiation into dopaminergic neurons aided by the key transcription factor Lmx1a. We propose to identify target genes that are regulated by Lmx1a during dopaminergic neuron differentiation using the newly developed technique of ChIP-seq, in combination with RNA expression and bioinformatics analysis. This work will identify essential control genes that drive dopaminergic neuron differentiation. Furthermore, our improved gene delivery and targeting system will be used for overexpressing candidate genes, knocking them down via RNA interference, and knocking in reporter genes to analyze gene expression networks during neuronal differentiation. The generation of efficient targeting technologies, in combination with genome wide analysis of gene regulation networks, will provide a general method for identifying and testing key regulatory genes for stem cell self-renewal and differentiation, as well as generating stem cell-based models of human disease. This blend of bioengineering and cell biology therefore has strong potential to create an important new capability for basic and applied stem cell research.
Statement of Benefit to California: 
This proposal will develop novel molecular tools and methodologies that will strongly enhance the scientific, technological, and economic development of stem cell therapeutics in California. The most important net benefit will be for the treatment of human diseases. Efficiently introducing specific genetic modifications into a stem cell genome is a greatly enabling technology that would impact many downstream medical applications. This capability will further enable investigations of self-renewal and differentiation, two defining properties of human stem cells. New tools to introduce targeted alterations of ES and iPS cells will also yield key model systems to elucidate mechanisms of human disease, and most importantly enable the generation of mutant cell lines to serve as models of human disease and systems for high throughput screening to develop novel therapies. Finally, the reverse process, the repair of genetic lesions responsible for disease, can in the long run enable the generation of patent-specific stem cell lines for therapeutic application. Each of these applications will directly benefit biomedical knowledge and human health. Furthermore, this proposal directly addresses several research targets of this RFA – the development and utilization of efficient homologous recombination techniques for gene targeting in human stem cells, the development of safer and more effective viral vectors for gene transduction in human stem cells, and the development and analysis of human embryonic stem cell lines with reporter genes inserted into key loci – indicating that CIRM believes that the proposed capabilities are a priority for California’s stem cell effort. While the potential applications of the proposed technology are broad, we will apply it to a specific and urgent biomedical problem: elucidating mechanisms of ES cell differentiation into dopaminergic neurons, part of a critical path towards developing therapies for Parkinson’s disease. While hESCs clearly have this capacity, the underlying mechanisms are incompletely understood, and the efficiency of this process must be improved. We will elucidate transcriptional networks that underlie this process, and utilize our novel gene targeting system to identify and analyze key components of these networks. This work will lead to a better fundamental understanding of mechanisms regulating stem cell differentiation, as well as enhance our ability to control this complex process for biomedical application. The co-investigators have a strong record of translating basic science and engineering into practice through interactions with industry, including the founding of biotech companies in California. Finally, this collaborative project will focus diverse research groups with many students on an important interdisciplinary project at the interface of science and engineering, thereby training future employees and contributing to the technological and economic development of California.
Progress Report: 
  • The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.
  • The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.
  • The central goal of this project is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. Harnessing a novel directed evolution technology we have developed to improve the properties of a promising viral vehicle, we have significantly increased its gene delivery efficiency to human embryonic and human induced pluripotent stem cells. Furthermore, this advance resulted in considerable improvements in the efficiency of gene targeting (i.e. editing) in the genomes of these cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of luripotent stem cell differentiation into neurons, with for example implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We attempted chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. Progress in this objective was ultimately hampered by the lack of a suitable antibody against Lmx1a. However, in parallel we have used an analogous approach to investigate mechanisms by which RNA transcription is regulated during the differentiation of embryonic stem cells into neurons, including motor neurons. These basic results can now be applied to enhance the efficiency of neuronal differentiation.
Funding Type: 
Shared Labs
Grant Number: 
CL1-00501-1.2
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$5 893 682
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Work in this field has been limited however, due to the limited availability of cells for transplantation. For example, cells from 6-10 human fetuses obtained 6-10 weeks post-conception are required for one patient with Parkinson’s disease to undergo transplantation. Human embryonic stem cells (hESCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body. In this proposal, we will build out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. We seek to understand how hESCs differentiate into authentic, clinically useful nerve cells and will use novel molecular tools to examine the behavior of cells transplanted in animal models of human neurological disease. We will also need to develop a noninvasive method of following cells after transplantation and we propose to develop luciferase-tagged (light-emitting) hESC lines for in vivo animal imaging. In addition, we will use hESC-derived nerve cells to screen drug and chemical libraries for compounds that protect nerve cells from toxicity, and to develop in vitro disease models. We believe that these experiments are critical to enhancing our understanding of neurological diseases and providing the tools that will be necessary to move cell therapy to the clinic. Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We propose to teach four 5-day hands-on training courses – two basic and two advanced hESC culture courses per year – to California scientists free of charge. These courses will provide scientists with an understanding of hESC biology and will enable them to set up and conduct hESC research after completion of training. In summary, the goal of this proposal is to provide over twenty investigators at the home institute and neighboring institutions with the ability to culture, differentiate, and genetically manipulate hESCs – including clinical-grade hESC lines – to develop diagnostic and therapeutic tools.
Statement of Benefit to California: 
We propose to build a Shared Research Laboratory and offer a Stem Cell Techniques Course for over twenty principal investigators at the home institute and neighboring institutes working collaboratively on stem-cell biology and neurological diseases of aging. We propose to: 1) Purify nerve cells at different stages of maturation from human embryonic stem cells and to develop transplantation strategies in animal models that mimic human diseases, including Parkinson’s disease, stroke and spinal cord injuries; 2) Screen drug and chemical libraries for reagents that protect nerve cells from toxicity and develop in vitro disease models using nerve cells generated from human embryonic stem cells; and 3) Assess the long-term integration and differentiation of transplanted cells using a non-invasive imaging system. We believe these experiments provide not only a blueprint for moving stem-cell transplantation for Parkinson’s disease toward the clinic, but also a generalized plan for how stem-cell therapy can be developed to treat disorders like motor neuron disease (amyotrophic lateral sclerosis, or Lou Gehrig’s disease) and spinal cord injury. As the only stem-cell research facility in California’s 10-12 most northwest counties, we are uniquely positioned to extend the promised benefits of Proposition 71 to this large part of the state. The tools and reagents we develop will be made widely available to California researchers and we will select California-based companies for commercialization of any therapies that may result. We also hope that California-based physicians will be at the forefront of translating this promising avenue of research into clinical applications. Finally, we expect that the money expended on this research will benefit the California research and business communities, and that the tools and reagents we develop will help accelerate stem-cell research in California and worldwide.
Progress Report: 
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Work in this field has been limited however, due to the limited availability of cells for transplantation. For example, cells from 6-10 human fetuses obtained 6-10 weeks post-conception are required for one patient with Parkinson’s disease to undergo transplantation. Human embryonic stem cells (hESCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. Supported by this facility, we have in the past year successfully developed a process for the production of functional dopaminergic neurons from hESCs that are suitable for potential clinical uses, e.g., in treating Parkinson’s disease (Parkinson’s disease is caused by the death of dopaminergic neurons). Our system provides a path to a scalable Good Manufacture Practice (GMP)-applicable process of generation of dopaminergic neurons from hESCs for therapeutic applications, and a ready source of large numbers of neurons for potential drug screening applications. In addition, we have developed a screening strategy that allows us to rapidly identify clinically approved drugs for use in GMP protocol that can be safely used to deplete unwanted contaminating precursor cells from dopaminergic neurons, a target for cell therapy.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 10 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 7 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 10 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 10 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Work in this field has been limited however, due to the limited availability of cells for transplantation. For example, cells from 6-10 human fetuses obtained 6-10 weeks post-conception are required for one patient with Parkinson’s disease to undergo transplantation.
  • Human embryonic stem cells (hESCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body. In this proposal, we will build out of approximately 3800 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 420 square feet for classroom facilities dedicated to training in hESC culture and manipulation. We seek to understand how hESCs differentiate into authentic, clinically useful nerve cells and will use novel molecular tools to examine the behavior of cells transplanted in rodent models of human neurological disease. We will also need to develop a noninvasive method of following cells after transplantation and we propose to develop luciferase-tagged (light-emitting) hESC lines for in vivo animal imaging. In addition, we will use hESC-derived nerve cells to screen drug and chemical libraries for compounds that protect nerve cells from toxicity, and to develop in vitro disease models. We believe that these experiments are critical to enhancing our understanding of neurological diseases and providing the tools that will be necessary to move cell therapy to the clinic.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We propose to teach four 5-day hands-on training courses: two basic and two advanced hESC culture courses per year, to California scientists free of charge. These courses will provide scientists with an understanding of hESC biology and will enable them to set up and conduct hESC research after completion of training.
  • In summary, the goal of this proposal is to provide over twenty investigators at the home institute and neighboring institutions with the ability to culture, differentiate, and genetically manipulate hESCs - including clinical-grad hESC lines—to develop diagnostic and therapeutic tools.
Funding Type: 
New Faculty II
Grant Number: 
RN2-00952
Investigator: 
Type: 
PI
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.
Funding Type: 
New Faculty II
Grant Number: 
RN2-00919
Investigator: 
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.
Funding Type: 
New Faculty II
Grant Number: 
RN2-00915
Investigator: 
Type: 
PI
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.
Funding Type: 
New Cell Lines
Grant Number: 
RL1-00682
Investigator: 
ICOC Funds Committed: 
$1 589 760
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Parkinson's disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms. The mechanisms of PD progression are currently unknown. However, genetic studies have identified that mutations (changes) in seven genes, including ?-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases. One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients). In this study, we propose to create stem cell lines that possess PD-associated mutations in two causative genes, PINK1 and parkin, using either rejected early stage embryos or cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that PD-associated abnormal parkin or PINK1 genes cause degeneration of stem cell-derived dopaminergic neurons, and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which harbor abnormal or mutant parkin or PINK1 genes; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
Statement of Benefit to California: 
Parkinson's disease (PD) is the second leading neurodegenerative disease with no current cure available. Compared to other states, California is the highest in the 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 in 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 citizens in California.
Progress Report: 
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • The mechanism of PD progression are currently unknown. However, genetic studies have identified that mutations (changes) in multiple genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that possess PD-associated mutations in two causative genes, PINK1 and parkin, using either rejected early stage embryos or cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that PD-associated abnormal parkin or PINK1 genes cause degeneration of stem cell-derived dopaminergic neurons, and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which harbor abnormal or mutant parkin or PINK1 genes; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last year, we have successfully generated primary skin fibroblast cultures from PD patients harboring mutations of parkin, PINK1, and DJ-1 genes, as well as sporadic PD patients and normal individuals. By using these cells, we have already generated four induced stem cell lines expressing multiple pluripotent markers (two from PD patients and two from normal individuals. These lines can also form teratomas with cells from three germ layers using mouse as host. These findings suggest that the induced pluripotent cell lines generated in the lab are likely PD patient specific stem cells.
  • During the next report year, we will continue to generate more PD patient-specific induced pluripotent stem cells. We will carefully characterize all lines generated in the lab as proposed. Furthermore, we will adapt protocols to differentiate the new lines into dopaminergic neurons.
  • Public Summary of Scientific Progress
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder affecting approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately, there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • Genetic studies have identified that mutations (changes) in multiple genes cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes in PD-affected dopamine-secretion neurons may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that possess PD-associated mutations in two causative genes, PINK1 and parkin, using either rejected early stage embryos or cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that PD-associated abnormal parkin or PINK1 genes cause degeneration of stem cell-derived dopaminergic neurons, and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which harbor abnormal or mutant parkin or PINK1 genes; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last year, we have successfully obtained more primary skin fibroblast cultures from PD patients harboring mutations of parkin, PINK1, DJ-1 and PLA2G6 genes, as well as sporadic PD patients and normal control individuals. By using these cells, we have already generated 9 induced stem cell lines expressing multiple pluripotent markers (7 from PD patients and 2 from normal individuals). These lines can also form teratomas with cells from three germ layers using mouse as host. These findings suggest that the induced pluripotent cell lines generated in the lab are likely PD patient specific stem cells.
  • During the next report year, we will continue to generate more PD patient-specific induced pluripotent stem cells. We will carefully characterize all lines generated in the lab as proposed. Furthermore, we will adapt protocols to differentiate the new lines into dopaminergic neurons.
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • The mechanism of PD progression is currently unknown. However, genetic studies have identified that mutations (changes) in multiple genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that either have the genetic background of sporadic PD patients or possess PD-associated mutations using cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that the degeneration of stem cell-derived dopaminergic neurons and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which either have the genetic background of sporadic PD patients or harbor PD specific gene mutantions; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last year, we have finished to develop 15 lines of iPSCs. These include 5 lines from normal control individuals, 5 lines from sporadic Parkinson disease patients, and 5 lines from Parkinson disease patients harboring disease related mutations of PINK1, DJ-1 and PLA2G6 genes. These lines provide an unique opportunity to systematically study comparative pathophysiology of Parkinson disease using sporadic and genetic cases. Moreover, we indeed spent more than a year in optimizing the condition for differentiation of these lines. It is recognized that iPSCs are more difficult to differentiate than the hESCs. We are now able to finalize the protocols to have all lines be differentiated in vitro. Therefore, we will be able to compare differences among the controls, sporadic PD and genetic PD at the level of cell biology and molecular biology.
  • During the next report year, we will differentiate all lines into DA neurons and carefully the functional changes of these cells. We hope that the results will reveal some molecular basis of PD pathogenesis from these human neurons.
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • The mechanism of PD progression is currently unknown. However, genetic studies have identified that mutations (changes) in multiple genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that either have the genetic background of sporadic PD patients or possess PD-associated mutations using cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that the degeneration of stem cell-derived dopaminergic neurons and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which either have the genetic background of sporadic PD patients or harbor PD specific gene mutantions; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last four years, we have finished to develop 15 lines of iPSCs. These include 5 lines from normal control individuals, 5 lines from sporadic Parkinson disease patients, and 5 lines from Parkinson disease patients harboring disease related mutations of PINK1, DJ-1 and PLA2G6 genes. These iPS lines are shown to have biochemical and genomic characteristics of human ES cells. These lines provide an unique opportunity to systematically study comparative pathophysiology of Parkinson disease using sporadic and genetic cases. Using these lines, we have identified a group of genes differentially expressed and differentially methylated between iPS cells derived from PD patients and iPS cells derived from normal control individuals. However, we recognize that iPSCs are more difficult to differentiate than the hESCs. We are yet to finalize the protocols to have all lines be differentiated in vitro. Our goal is to compare differences among the controls, sporadic PD and genetic PD at the level of cell biology and molecular biology.
Funding Type: 
New Cell Lines
Grant Number: 
RL1-00681
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 382 400
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Melanoma
Cancer
Muscular Dystrophy
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
The therapeutic use of stem cells depends on the availability of pluripotent cells that are not limited by technical, ethical or immunological considerations. The goal of this proposal is to develop and bank safe and well-characterized patient-specific pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases. Several groups, including ours have recently shown that adult skin cells can be reprogrammed in the laboratory to create new cells that behave like embryonic stem cells. These new cells, known as induced pluripotent stem (iPS) cells should have the potential to develop into any cell type or tissue type in the body. Importantly, the generation of these cells does not require human embryos or human eggs. Since these cells can be derived directly from patients, they will be genetically identical to the patient, and cannot be rejected by the immune system. This concept opens the door to the generation of patient-specific stem cell lines with unlimited differentiation potential. While the current iPS cell technology enables us now to generate patient-specific stem cells, this technology has not yet been applied to derive disease-specific human stem cell lines for laboratory study. Importantly, these new cells are also not yet suitable for use in transplantation medicine. For example, the current method to make these cells uses retroviruses and genes that could generate tumors or other undesirable mutations in cells derived from iPS cells. Thus, in this proposal, we aim to improve the iPS cell reprogramming method, to make these cells safer for future use in transplant medicine. We will also generate a large number of iPS lines of different genetic or disease backgrounds, to allow us to characterize these cells for function and as targets to study new therapeutic approaches for various diseases. Lastly, we will establish protocols that would allow the preparation of these types of cells for clinical use by physicians investigating new stem cell-based therapies in a wide variety of diseases.
Statement of Benefit to California: 
Several groups, including ours have recently shown that adult skin cells can be reprogrammed in the laboratory to create new cells that behave like embryonic stem cells. These new cells, known as induced pluripotent stem (iPS) cells should have, similar to embryonic stem cells, the potential to develop into any cell type or tissue type in the body. This new technology holds great promise for patient-specific stem-cell based therapies, the production of in vitro models for human disease, and is thought to provide the opportunity to perform experiments in human cells that were not previously possible, such as screening for compounds that inhibit or reverse disease progression. The advantage of using iPS cells for transplantation medicine would be that the patient’s own cells would be reprogrammed to an embryonic stem cell state and therefore, when transplanted back into the patient, the cells would not be attacked and destroyed by the body's immune system. Importantly, these new cells are not yet suitable for use in transplantation medicine or studies of human diseases, as their derivation results in permanent genetic changes, and their differentiation potential has not been fully studied. The goal of this proposal is to develop and bank genetically unmodified and well-characterized iPS cell lines of different genetic or disease backgrounds that can be used to characterize these cells for function and as targets to study new therapeutic approaches for various human diseases. We will establish protocols that would allow the preparation of these types of cells for clinical use by physicians investigating new stem cell-based therapies in a wide variety of diseases. Taken together, this would be beneficial to the people of California as tens of millions of Americans suffer from diseases and injuries that could benefit from such research. Californians will also benefit greatly as these studies should speed the transition of iPS cells to clinical use, allowing faster development of stem cell-based therapies.
Progress Report: 
  • The goal of this project is to develop and bank safe, well-characterized pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases, and that are not limited by technical, ethical or immunological considerations. To that end, we proposed to establish protocols for generation of human induced pluripotent stem cells (hiPSC) that would not involve viral vector integration, and that would be compatible with Good Manufacturing Processes (GMP) standards. To establish baseline characteristics of hiPSCs, we performed a complete molecular characterization of all existing hiPSCs in comparison to human embryonic stem cells (hESCs). We found that all hiPSC lines created to date, regardless of the method by which they were reprogrammed, shared a common gene expression signature, distinct from that of hESCs. The functional role of this gene expression signature is still unclear, but any lines that are generated under the guise of this grant will be subjected to a similar analysis to set the framework by which these new lines are functionally characterized. Our efforts to develop new strategies for the production of safe iPS cells have yielded many new cell lines generated by various techniques, all of which are safer than the standard retroviral protocol. We are currently expanding many of the hiPSCs lines generated and will soon demonstrate whether their gene expression profile, differentiation capability, and genomic stability make them suitable for banking in our iPSC core facility. Once fully characterized, these cells will be available from our bank for other investigators.
  • For hiPSC technology to be useful clinically, the procedures to derive these cells must be robust enough that iPSC can be obtained from the majority of donors. To determine the versatility of generation of iPS cells, we have now derived hiPSCs from commercially obtained fibroblasts derived from people of different ages (newborn through 66 years old) as well as from different races (Caucasian and mixed race). We are currently evaluating medium preparations that will be suitable for GMP-level use. Future work will ascertain the best current system for obtaining hiPSC, and establish GMP-compliant methodologies.
  • The goal of this project is to develop and bank safe, well-characterized pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases. To speed this process, we are taking approaches that are not limited by technical, ethical or immunological considerations. We are establishing protocols for generation of human induced pluripotent stem cells (hiPSCs) that would not involve viral vector integration, and that are compatible with Good Manufacturing Practices (GMP) standards. Our efforts to develop new strategies for the production of safe hiPSC have yielded many new cell lines generated by various techniques. We are characterizing these lines molecularly, and have found hiPSCs can be made that are nearly indistinguishable from human embryonic stem cells (hESC). We have also recently found in all the hiPSCs generated from female fibroblasts, none reactivated the X chromosome. This finding has opened a new frontier in the study and potential treatment of X-linked diseases. We are currently optimizing protocols to generate hiPSC lines that are derived, reprogrammed and differentiated in the absence of animal cell products, and preparing detailed standard operating procedures that will ready this technology for clinical utility.
  • This project was designed to generate protocols whereby human induced pluripotent stem cells could be generated in a manner consistent with use in clinical trials. This required optimization of protocols and generation of standard operating procedures such that animal products were not involved in generation and growth of the cells. We have successfully identified such a protocol as a resource to facilitate widespread adoption of these practices.
Funding Type: 
New Cell Lines
Grant Number: 
RL1-00678
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 369 800
Disease Focus: 
Huntington's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Huntington’s disease (HD) is a devastating neurodegenerative disease with a 1/10,000 disease risk that always leads to death. These numbers do not fully reflect the large societal and familial cost of HD, which requires extensive caregiving and has a 50% chance of passing the mutation to the next generation. Current treatments treat some symptoms but do not change the course of disease. Symptoms of the disease include movement abnormalities, inability to perform daily tasks and and psychiatric problems. A loss os specific regions of the brain are observed. The mutation for HD is an expansion of a region of repeated DNA in the HD gene and the longer the repeat, in general the earlier the onset of disease. While the length of this polyglutamine repeat largely determines the age-of-onset, there is variance in onset age that is not accounted for by repeat length but is determined by genetic and environmental factors. In addition, the symptoms can vary significantly among patients in a non-repeat dependent manner. To assist in preventing onset of HD, there is a great need to identify genes that are involved in why one individual with 45 repeats will manifest symptoms at age 40 while another manifests symptoms at age 70. Further, there is a lack of early readouts to determine when to begin HD treatments. Because the disease mutation is known, preimplantation genetic diagnosis (PGD) is possible and mutant Htt embryos are available. Stem cell lines can be derived from PGD embryos with varying repeat lengths and genetic backgrounds to provide new methods to identify genetic modifiers and readouts of disease progression. The development of pluripotent stem cells, termed induced pluripotent stem cells (iPS) cells, derived directly from HD patient fibroblasts, would also provide new methods for these analyses. Chemical compound screens to identify drugs that protect against the effect of mutant Htt protein expression in patient derived hESCs cells would allow evaluation of drug responses in on cells having different genetic backgrounds Ultimately, the iPS cells can provide a way to transplant neurons or neuronal support cells from affected individuals or from unaffected family members having a normal range repeat. Such cells would help reduce immune rejection effects likely to occur with transplantation, however, while patient-derived cells overcome the problems of immune rejection, the mutant protein is still expressed. To overcome this problem we will genetically modify these stem cells to reduce the mutant protein and produce a normal gene. Beyond the immediate application to HD, the development of these models is applicable to a range of neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases.
Statement of Benefit to California: 
The disability and loss of earning power and personal freedom resulting from Huntington's disease (HD) is devastating and creates a financial burden for California. Individuals are struck in the prime of life, at a point when they are their most productive and have their highest earning potential. Further, as the disease progressives, individuals require institutional care facilities at great financial cost. Therapies using human embryonic stem cells (hESCs) have the potential to change the lives of hundreds of individuals and their families, which brings the human cost into the thousands. Further, hESCs from HD patients will help us understand the factors that dictate the course of the disease and provide a resource for drug development. For the potential of hESCs in HD to be realized, a very forward approach such as that proposed will allow experienced investigators in HD and stem cell research and clinical trials to come together and create cell lines to more fully mimic the diseases neurons and allow for future treatment options. The federal constraints on hESCs create a critical need for the development of treatments using hESCs supported and staffed with non-federal funds. We have proposed goals and strategies for generating new stem cells derived from patient preimplantation diagnosis embryos and patient fibroblasts. We have put in place critical milestones to be met We will build on existing regional stem cell resources . Anticipated benefits to the citizens of California include: 1) development of new stem cell lines that will allow us to more closely model the disease for mechanistic studies and drug screening, 2) improved methods for following the course of the disease in order to treat HD as early as possible before symptoms are manifest; 3) development of new cell-based treatments for Huntington's disease with application to other neurodegenerative diseases such as Alzheimer's and Parkinson's diseases that affect thousands of individuals in California; 4) development of intellectual property that could form the basis of new biotech startup companies; and 5) improved methods for drug development that could directly benefit citizens of the state.
Progress Report: 
  • Huntington’s disease (HD) is a devastating neurodegenerative disease with a 1/10,000 disease risk that always leads to death. These numbers do not fully reflect the large societal and familial cost of HD, which requires extensive caregiving and has a 50% chance of passing the mutation to the next generation. Current treatments treat some symptoms but do not change the course of disease. Symptoms of the disease include movement abnormalities, inability to perform daily tasks and psychiatric problems. A loss of specific regions of the brain are observed. The mutation for HD is an expansion of a region of repeated DNA in the HD gene and the longer the repeat, in general the earlier the onset of disease. While the length of this polyglutamine repeat largely determines the age-of-onset, there is variance in onset age that is not accounted for by repeat length but is determined by genetic and environmental factors. In addition, the symptoms can vary significantly among patients in a non-repeat dependent manner. There is a lack of early readouts to determine when to begin HD treatments. Because the disease mutation is known, preimplantation genetic diagnosis (PGD) is possible and mutant Htt embryos are available. We have obtained a number of HD PGD embryos with varying repeat lengths and genetic backgrounds to derive hES cell lines and provide new methods to identify genetic modifiers and readouts of disease progression. Development of multiple lines has begun during this funding period. The development of pluripotent stem cells, termed induced pluripotent stem (iPS) cells, derived directly from HD patient fibroblasts, also provide new methods for these analyses. We have begun the establishment of a bank of HD fibroblasts and have derived three new iPS lines to date with unique CAG repeat expansions. Characterization of the lines for HD phenotypes is in progress. An additional line is being generated and additional fibroblast collection from both HD patients and individuals who do not carry the HD gene is planned for the coming year to generate other sets of iPS lines. These lines will allow mechanistic studies and chemical compound screens to identify drugs that protect against the effect of mutant Htt protein expression in patient derived stem cells to be performed. Ultimately, the iPS cells will provide a way to transplant neurons or neuronal support cells from affected individuals or from unaffected family members having a normal range repeat. Such cells would help reduce immune rejection effects likely to occur with transplantation, however, while patient-derived cells overcome the problems of immune rejection, the mutant protein is still expressed. To overcome this problem we will genetically modify these stem cells to reduce the mutant protein and produce a normal gene in the next portion of the project.
  • Huntington’s disease (HD) is a devastating neurodegenerative disease that strikes in mid-life and inevitably leads to death. As it is genetic, offspring of affected individuals are 50% at risk. Current medications treat some symptoms, which include movement abnormalities, inability to perform daily tasks and psychiatric problems, but do not change the course of disease. The mutation for HD is an expansion of a region of repeated DNA in the HD gene. In general, the longer the repeat the earlier the onset of disease. While the length of this polyglutamine repeat largely determines the age-of-onset, there is variance in onset age that is not accounted for by repeat length but is determined by genetic and environmental factors. In addition, the symptoms can vary significantly among patients in a non-repeat dependent manner. There is a lack of early readouts to determine when to begin HD treatments. Because the disease mutation is known, preimplantation genetic diagnosis (PGD) is possible and mutant Htt embryos are available. We have obtained a number of HD PGD embryos with varying repeat lengths and genetic backgrounds to derive hES cell lines and have derived a line that is now fully characterized as a stem cell line. The development of pluripotent stem cells, termed induced pluripotent stem (iPS) cells, derived directly from HD patient skin cells (fibroblasts), also provide new methods for these analyses. We have made significant progress in establishing a bank of HD fibroblasts and have derived seven new iPS lines to date with unique CAG repeat expansions. Characterization of the lines for HD phenotypes is either complete or in progress. Additional lines are being generated and additional fibroblast collection from both HD patients and individuals who do not carry the HD gene is planned for the coming year to generate other sets of iPS lines. These lines will allow mechanistic studies and chemical compound screens to identify drugs that protect against the effect of mutant Htt protein expression in patient derived stem cells to be performed.
  • Huntington’s disease (HD) is a devastating neurodegenerative disease that strikes in mid-life and inevitably leads to death. As it is genetic, offspring of affected individuals are 50% at risk. Current medications treat some symptoms, which include movement abnormalities, inability to perform daily tasks and psychiatric problems, but do not change the course of disease. The mutation for HD is an expansion of a region of repeated DNA in the HD gene. In general, the longer the repeat the earlier the onset of disease. While the length of this polyglutamine repeat largely determines the age-of-onset, there is variance in onset age that is not accounted for by repeat length but is determined by genetic and environmental factors. In addition, the symptoms can vary significantly among patients in a non-repeat dependent manner. There is a lack of early readouts to determine when to begin HD treatments. Because the disease mutation is known, preimplantation genetic diagnosis (PGD) is possible and mutant Htt embryos are available. We have obtained HD PGD embryos and have derived a line that is now fully characterized as a stem cell line that is capable of becoming brain cells. The development of pluripotent stem cells, termed induced pluripotent stem (iPS) cells, derived directly from HD patient skin cells (fibroblasts), also provide new methods for these analyses. We have established a bank of HD fibroblasts and have derived seven new iPS lines with unique CAG repeat expansions. Characterization of the lines for HD symptoms is either complete or in progress. Additional lines are being generated and additional skin cells collected from both HD patients and individuals who do not carry the HD gene. These lines are allowing mechanistic studies and chemical compound screens to identify drugs that protect against the effect of mutant Htt protein expression in patient derived stem cells to be performed. Finally, we are developing a method to reduce the level of the mutant protein to provide options for future transplantation from an individual's own skin cells to prevent immune rejection.
Funding Type: 
New Cell Lines
Grant Number: 
RL1-00650
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 708 560
Disease Focus: 
Dementia
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
We propose to generate induced pluripotent stem (iPS) cells from skin cells derived from human subjects with frontotemporal dementia (FTD). FTD accounts for 15–20% of all dementia cases and, with newly identified genetic causes, is now recognized as the most common dementia in patients under 65 years of age. FTD patients suffer progressive neurodegeneration in the frontal and temporal lobes and other brain regions, resulting in behavioral changes and memory and motor neuron deficits. The median age of onset for this devastating disease is 58 years, and disease progression is rapid, with death in 3–8 years. Compared with other age-dependent neurodegenerative diseases, the molecular, cellular, and genetic bases of FTD remain poorly understood. Genetic causes are estimated to account for ~40% of FTD. In addition to tau identified in 1998, mutations in three causative genes have been identified during the last three years. The identification of FTD mutations opens exciting new avenues for understanding the causes of FTD. Research on these mutations will help to identify effective therapies. However, the ability to study the functions of these factors is severely limited due to the lack of available human neurons from FTD patients. To address the need for disease– and patient–specific neurons, we will use the powerful new technique of iPS cells. iPS cells are derived from skin cells and can be used to generate any cell types in the body, including neurons. We will obtain human skin cells from FTD patients with disease-causing mutations and generate many FTD mutation–specific iPS cell lines. We will then use these iPS cells to generate FTD mutation–specific neurons to study disease mechanisms. The bank of iPS cell lines we generate will also enable the development of sensitive assays for drug screening and testing of therapeutic agents for treating FTD. All cell lines will be made available to the global FTD research community. The generation of human neurons from FTD patients will be a tremendous advance toward finding a cure for this disease.
Statement of Benefit to California: 
California is the U.S. leader in basic research into stem cell–based therapies for disease. To help California remain at the forefront of research on neurological disease, we propose to use induced pluripotent stem (iPS) cells—a revolutionary new technique developed recently by Dr. Shinya Yamanaka—to target frontotemporal dementia (FTD). FTD is a devastating and common form of dementia. {REDACTED} The proposed research will establish California as the leader in generating human patient–specific neurons from iPS cells. The potential long-term benefits to California include growth of the clinical enterprise in the diagnosis and treatment of FTD, the establishment of biotechnology to generate new drugs for FTD, and potential intellectual properties for driving private enterprises to develop therapies.
Progress Report: 
  • In this grant, we proposed to generate induced pluripotent stem (iPS) cells from skin cells derived from human subjects with frontotemporal dementia (FTD). FTD accounts for 15–20% of all dementia cases and, with newly identified genetic causes, is now recognized as the most common dementia in patients under 65 years of age. FTD patients suffer progressive neurodegeneration in the frontal and temporal lobes and other brain regions, resulting in behavioral changes and memory and motor neuron deficits. The median age of onset of this devastating disease is 58 years, and it progresses rapidly, causing death in 3–8 years. Compared with other age-dependent neurodegenerative diseases, the molecular, cellular, and genetic bases of FTD are poorly understood. Genetic causes are estimated to account for ~40% of FTD. In addition to tau identified in 1998, mutations in three causative genes have been identified during the last three years. The identification of FTD mutations opens exciting new avenues for understanding the causes of FTD. Research on these mutations will help to identify effective therapies. However, the ability to study the functions of these factors is severely limited due to the lack of available human neurons from FTD patients. To address the need for disease– and patient–specific neurons, we proposed to use the powerful new technique of iPS cells. iPS cells are derived from skin cells and can be used to generate any cell types in the body, including neurons. During the last 10 months, we have obtained human skin cells from more than 30 FTD patients with disease-causing mutations and unaffected family members. We have generated about 200 putative iPS cell lines from two FTD patients with defined genetic mutations, one sporadic case, and one control. We characterized some of the iPS cell lines and differentiated one patient-specific iPS cell line into human postmitotic neurons. These results represent a major advance toward finding a cure for FTD, and we will continue to pursue this line of research as proposed.
  • We have collected numerous skin samples from patients with a kind of dementia that affects the frontal lobes. We have also collected samples from unaffected family members (controls). For many of these samples we have made induced pluripotent stem cells (iPS), which can give rise to any cell type. We are in the process of generating neurons from these stem cells. Our hope and intention is to study these cells to learn about the mechanisms that give rise to this dementia and to be able to test potential therapies.
  • We have collected numerous skin samples from patients with a kind of dementia that affects the frontal lobes. We have also collected samples from unaffected family members (controls). For many of these samples we have made induced pluripotent stem cells (iPS), which can give rise to any cell type. We are in the process of generating neurons and other cell types, such as cells that mediate inflammation, from these stem cells. Our hope and intention is to study these cells to learn about the mechanisms that give rise to this dementia and to be able to test potential therapies.
Funding Type: 
New Cell Lines
Grant Number: 
RL1-00649
Investigator: 
ICOC Funds Committed: 
$1 737 720
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Autism
Blood Disorders
Rett's Syndrome
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Human embryonic stem cells (hESC) hold great promise in regenerative medicine and cell replacement therapies because of their unique ability to self-renew and their developmental potential to form all cell lineages in the body. Traditional techniques for generating hESC rely on surplus IVF embryos and are incompatible with the generation of genetically diverse, patient or disease specific stem cells. Recently, it was reported that adult human skin cells could be induced to revert back to earlier stages of development and exhibit properties of authentic hES cells. The exact method for “reprogramming” has not been optimized but currently involves putting multiple genes into skin cells and then exposing the cells to specific chemical environments tailored to hES cell growth. While these cells appear to have similar developmental potential as hES cells, they are not derived from human embryos. To distinguish these reprogrammed cells from the embryonic sourced hES cells, they are termed induced pluripotent stem (iPS) cells. Validating and optimizing the reprogramming method would prove very useful for the generation of individual cell lines from many different patients to study the nature and complexity of disease. In addition, the problems of immune rejection for future therapeutic applications of this work will be greatly relieved by being able to generate reprogrammed cells from individual patients. We have initiated a series of studies to reprogram human and mouse fibroblasts to iPS cells using the genes that have already been suggested. While induction of these genes in various combinations have been reported to reprogram human cells, we plan to optimize conditions for generating iPS cells using methods that can control the level of the “reprogramming” genes, and also can be used to excise the inducing genes once reprogramming is complete; thus avoiding unanticipated effects on the iPS cells. Once we have optimized the methods of inducing human iPS cells from human fibroblasts, we will make iPS cells from patients with 2 different neurological diseases. We will then coax these iPS cells into specific types of neurons using methods pioneered and established in our lab to explore the biological processes that lead to these neurological diseases. Once we generate these cell based models of neural diseases, we can use these cells to screen for drugs that block the progress, or reverse the detrimental effects of neural degeneration. Additionally, we will use the reprogramming technique to study models of human blood and liver disease. In these cases, genetically healthy skin cells will be reprogrammed to iPS cells, followed by introduction of the deficient gene and then coaxed to differentiate into therapeutic cell types to be used in transplantation studies in animal models of these diseases. The ability of the reprogrammed cell types to rescue the disease state will serve as a proof of principle for therapeutic grafting in
Statement of Benefit to California: 
It has been close to a decade since the culture of human embryonic stem (hES) cells was first established. To this day there are still a fairly limited number of stem cell lines that are available for study due in part to historic federal funding restrictions and the challenges associated with deriving hES cell lines from human female egg cells or discarded embryos. In this proposal we aim to advance the revolutionary new reprogramming technique for generating new stem cell lines from adult cells, thus avoiding the technical and ethical challenges associated with the use of human eggs or embryos, and creating the tools and environment to generate the much needed next generation of human stem cell lines. Stem cells offer a great potential to treat a vast array of diseases that affect the citizens of our state. The establishment of these reprogramming techniques will enable the development of cellular models of human disease via the creation of new cell lines with genetic predisposition for specific diseases. Our proposal aims to establish cellular models of two specific neurological diseases, as well as developing methods for studying blood and liver disorders that can be alleviated by stem cell therapies. California has thrived as a state with a diverse population, but the stem cell lines currently available represent a very limited genetic diversity. In order to understand the variation in response to therapeutics, we need to generate cell lines that match the rich genetic diversity of our state. The generation of disease-specific and genetically diverse stem cell lines will represent great potential not only for CA health care patients but also for our state’s pharmaceutical and biotechnology industries in terms of improved models for drug discovery and toxicological testing. California is a strong leader in clinical research developments. To maintain this position we need to be able to create stem cell lines that are specific to individual patients to overcome the challenges of immune rejection and create safe and effective transplantation therapies. Our proposal advances the very technology needed to address these issues. As a further benefit to California stem cell researchers, we will be making available the new stem cell lines created by our work.
Progress Report: 
  • Public Summary for: CIRM New Cell Line Project - Progress Report.
  • Our research team has been working over the last year on developing new human stem cell lines that are specifically useful for studying human diseases and developing new therapeutic strategies. Human embryonic stem (hES) cells were first established in 1998 and in the past decade have been shown to be capable to differentiating to a vast array of different cell types. This full developmental potential is termed pluripotency. Until recently these were the only established human cell type that could be robustly grown in the laboratory setting and still maintain full pluripotent developmental potential. In November of 1997, a new type of human pluripotent cell was created. By turning on a set of 4 genes, researchers succeeded in reprogramming human skin cells back into a cell type that appeared to have very similar properties and potential as the hES cell. These new stem cells are called induced pluripotent stem (iPS) cells in order to keep the name distinct from their embryonic derived counterpart. One of the scientific limitations of hES cells is the impracticality of generating patient or disease specific stem cell lines. This opportunity now becomes theoretically practical with the advent of human iPS cell line generation. We report here on significant progress demonstrating the practicality of generating disease-linked cellular models of human diseases.
  • We have identified 2 specific human neurological diseases that have a known, or strongly suggested genetic component, and have set about to generate disease-linked iPS cell lines. We have obtained skin cell samples from patients with these neurological diseases and have successfully reprogrammed them back to iPS cells. These disease-linked pluripotent stem cells have been carefully characterized and we have demonstrated that they do indeed behave very similar to existing hES cells and also to the genetically healthy control iPS cell lines that we have generated. Therefore the disease phenotype is not detrimental to reprogramming or proliferation as a stem cell. Furthermore, we have succeeded in coaxing these disease-linked iPS cells to turn into specific types of human neurons, the very cells that are suspected to be involved in the neurological disorders. We now have established a viable model for studying human neural disorders in the laboratory, and have already observed some potentially important functional differences between the disease-linked and control iPS generated neurons. In the coming year we will be evaluating the differences between the disease-linked and control neurons and investigating potential therapeutic approaches to stop or reverse the defects.
  • We have also been working on developing new methods for generating iPS cells that will make them more useful in clinical or pre-clinical settings where it is important that the original set of 4 genes used to reprogram the skin cells are removed once they have become iPS cells. Significant progress has been made in this regard and will be completed in the coming year. Looking forward we will also be applying this approach to generate human disease-linked iPS cells for specific hematological (blood) related disorders. The derivation of iPS-based models of hematological disorders will allow us develop gene therapy approaches to correct the disease causing defects and establish proof of principle for therapeutic approaches.
  • This research project is focused on developing new human stem cell lines that are specifically useful for studying human diseases and developing new therapeutic strategies. Human embryonic stem (hES) cells were first established in 1998 and in the past decade have been shown to be capable of differentiating to a vast array of different cell types. This full developmental potential is termed "pluripotency." Until recently these were the only established human cell types that could be robustly grown in the laboratory setting and still maintain full pluripotent developmental potential. In November 1997 a new type of human pluripotent cell was created. By turning on a set of 4 genes, researchers succeeded in reprogramming human skin cells back into a cell type that appeared to have very similar properties and potential as the hES cell. These new stem cells are called induced pluripotent stem (iPS) cells in order to keep the name distinct from their embryonic derived counterpart. One of the scientific limitations of hES cells is the impracticality of generating patient or disease specific stem cell lines. This opportunity now becomes theoretically practical with the advent of human iPS cell line generation. We report here on significant progress demonstrating the practicality of generating disease-linked cellular models of human diseases.
  • We have identified 2 specific human neurological diseases that have known, or strongly suggested, genetic components and have set about to generate disease-linked iPS cell lines. We have obtained skin cell samples from patients with these neurological diseases and have successfully reprogrammed them back to iPS cells. These disease-linked pluripotent stem cells have been carefully characterized and we have demonstrated that they do indeed behave very similar to existing hES cells and also to the genetically healthy control iPS cell lines that we have generated. Therefore, the disease phenotype is not detrimental to reprogramming or proliferation as a stem cell. Furthermore, we have succeeded in coaxing these disease-linked iPS cells to turn into specific types of human neurons, the very cells that are suspected to be involved in the neurological disorders. We now have established a viable model for studying human neural disorders in the laboratory, and have already observed some potentially important functional differences between the disease-linked and control iPS-generated neurons. Importantly, we have found defects in the function of disease-linked neurons that can be corrected in part following specific drug treatments. This discovery demonstrates the potential utility to use this method of modeling human diseases in the laboratory as a tool for understanding the detailed pathways, which might contribute to the development of the disease state and, importantly, as a target for screening potential therapeutic compounds that might be used to block or slow the progress of human neural disorders. In the coming year we will finalize our efforts on this project.
  • We have also succeeded in developing an improved method for the delivery of the reprogramming genes into the patient cells in order to become iPS cells. This method allows the reprogramming genes to be removed thus mitigating the potential for unwanted and potentially detrimental reactivation of these reprogramming genes subsequent to the iPS cell state. We have begun work using this new reprogramming methodology to generate iPS cell lines that are specifically linked to diseases of the blood and immune system. The new methodology appears to be working well and we anticipate completing the generation and characterization of these new disease-linked stem cell lines within the next year of this project.
  • This research project has been focused on developing new human stem cell lines that are specifically useful for studying human diseases and developing new therapeutic strategies. Human embryonic stem (hES) cells were first established in 1998 and in the past decade have been shown to be capable to differentiating of a vast array of different cell types. This full developmental potential is termed "pluripotency". Until recently these were the only established human cell type that could be robustly grown in the laboratory setting and still maintain full pluripotent developmental potential. In November of 2007, a new type of human pluripotent cell was created. By turning on a set of 4 genes, researchers succeeded in reprogramming human skin cells back into a cell type that appears to have very similar properties and potential as the hES cell. These new stem cells are called induced pluripotent stem (iPS) cells in order to keep the name distinct from their embryonic derived counterpart. One of the scientific limitations of hES cells is the impracticality of generating patient or disease specific stem cell lines. This opportunity now becomes theoretically practical with the advent of human iPS cell line generation. We report here on significant progress demonstrating the practicality of generating disease-linked cellular models of human diseases.
  • We have identified 2 specific human neurological diseases, Rett’s Syndrome and Schizophrenia that have a known, or strongly suggested genetic components, and have set about to generate disease-linked iPS cell lines. We have obtained skin cell samples from patients with these neurological diseases and have successfully reprogrammed them back to iPS cells. These disease-linked pluripotent stem cells have been carefully characterized and we have demonstrated that they do indeed behave very similar to existing hES cells and also to the healthy control iPS cell lines that we have generated. Therefore, the disease phenotype is not detrimental to reprogramming or proliferation as a stem cell. Furthermore, we have succeeded in coaxing these disease-linked iPS cells to turn into specific types of functional human neurons, the very cells that are suspected to be involved in the neurological disorders. We now have established a viable model for studying human neural disorders in the laboratory, and have already observed some potentially important functional differences between the disease-linked and control iPS generated neurons. Importantly, we have found defects in the function of disease-linked neurons that can be corrected in part following specific drug treatments. This discovery demonstrates the potential utility to use this method of modeling human diseases in the laboratory as a tool for understanding the detailed pathways that might contribute to the development of the disease state and importantly as a target for screening potential therapeutic compounds that might be used to block or slow the progress of human neural disorders.
  • We have also succeeded in developing an improved method for the delivery of the reprogramming genes into the patient cells in order to become iPS cells. This method combines all the of the reprogramming genes into a single cassette, and also allows the reprogramming genes to be removed thus mitigating the potential for unwanted and potentially detrimental reactivation of these reprogramming genes subsequent to the iPS cell state. We have demonstrated the success of this new reprogramming methodology to generate iPS cell lines that are specifically linked to a disease of the immune system. In addition to creating a panel of disease-linked iPS cell lines that are free of the externally introduced reprogramming transgenes, we have shown progress in achieving correction of the DNA mutation that leads to the disease state. Our extended research on these new disease specific iPS cell lines has shown utility for creating in vitro models of human neural disorders, and potential for genetically corrected patient specific iPS cell lines that could be used for cell based transplantation therapies.

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