Neurological Disorders

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

Human iPSC modeling and therapeutics for degenerative peripheral nerve disease

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06530
ICOC Funds Committed: 
$3 206 737
Disease Focus: 
Neuropathy
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
The applicant is an MD/PhD trained physician scientist, whose clinical expertise is neuromuscular disorders including peripheral nerve disease. The proposal is aimed at providing a research proposal and career development plan that will allow the applicant to develop an independent research program, which attempts to bring stem cell based therapies to patients with peripheral nerve diseases. The proposal will use “adult stem cells” derived from patients with an inherited nerve disease, correct the genetic abnormality in those cells, and determine the feasibility of transplanting the genetically engineered cells back into peripheral nerve to slow disease progression.
Statement of Benefit to California: 
The proposed research will benefit the State of California as it will support the career development of a uniquely trained physician scientist to establish an innovative translational stem cell research program aimed toward direct clinical application to patients. The cutting edge technologies proposed are directly in line with the fundamental purpose of the California Initiative for Regenerative Medicine. If successful, both scientific and patient advocate organizations would recognize that these advances came directly from the unique efforts of CIRM and the State of California to lead the world in stem cell research. Finally, as a result of funding of this award, further financial investments from private and public funding organizations would directly benefit the State in the years to come.
Progress Report: 
  • During this award period we have made significant progress. We have established induced pluripotent stem cell (iPSC) lines from four patients with Charcot-Marie-Tooth disease type 1A (CMT1A) due to the PMP22 duplication. We have validated our strategy to genetically engineer induced pluripotent stem cells from patients with inherited neuropathy, and have genetically engineered several patient lines. We further have begun to differentiate these iPSCs into Schwann cell precursors, to begin to investigate cell type specific defects that cause peripheral neurodegeneration in patients with CMT1A. Finally we have imported and characterized a transgenic rat model of CMT1A in order to begin to investigate the ability to inject iPSC derived Schwann cell precursors into rodent nerves as a possible neuroprotective strategy.

Restoration of memory in Alzheimer’s disease: a new paradigm using neural stem cell therapy

Funding Type: 
Disease Team Therapy Development - Research
Grant Number: 
DR2A-05416
ICOC Funds Committed: 
$20 000 000
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Alzheimer’s disease (AD), the leading cause of dementia, results in profound loss of memory and cognitive function, and ultimately death. In the US, someone develops AD every 69 seconds and there are over 5 million individuals suffering from AD, including approximately 600,000 Californians. Current treatments do not alter the disease course. The absence of effective therapies coupled with the sheer number of affected patients renders AD a medical disorder of unprecedented need and a public health concern of significant magnitude. In 2010, the global economic impact of dementias was estimated at $604 billion, a figure far beyond the costs of cancer or heart disease. These numbers do not reflect the devastating social and emotional tolls that AD inflicts upon patients and their families. Efforts to discover novel and effective treatments for AD are ongoing, but unfortunately, the number of active clinical studies is low and many traditional approaches have failed in clinical testing. An urgent need to develop novel and innovative approaches to treat AD is clear. We propose to evaluate the use of human neural stem cells as a potential innovative therapy for AD. AD results in neuronal death and loss of connections between surviving neurons. The hippocampus, the part of the brain responsible for learning and memory, is particularly affected in AD, and is thought to underlie the memory problems AD patients encounter. Evidence from animal studies shows that transplanting human neural stem cells into the hippocampus improves memory, possibly by providing growth factors that protect neurons from degeneration. Translating this approach to humans could markedly restore memory and thus, quality of life for patients. The Disease Team has successfully initiated three clinical trials involving transplantation of human neural stem cells for neurological disorders. These trials have established that the cells proposed for this therapeutic approach are safe for transplantation into humans. The researchers in this Disease Team have shown that AD mice show a dramatic improvement in memory skills following both murine and human stem cell transplantation. With proof-of-concept established in these studies, the Disease Team intends to conduct the animal studies necessary to seek authorization by the FDA to start testing this therapeutic approach in human patients. This project will be conducted as a partnership between a biotechnology company with unique experience in clinical trials involving neural stem cell transplantation and a leading California-based academic laboratory specializing in AD research. The Disease Team also includes expert clinicians and scientists throughout California that will help guide the research project to clinical trials. The combination of all these resources will accelerate the research, and lead to a successful FDA submission to permit human testing of a novel approach for the treatment of AD; one that could enhance memory and save lives.
Statement of Benefit to California: 
The number of AD patients in the US has surpassed 5.4 million, and the incidence may triple by 2050. Roughly 1 out of every 10 patients with AD, over 550,000, is a California resident, and alarmingly, because of the large number of baby-boomers that reside in this state, the incidence is expected to more than double by 2025. Besides the personal impact of the diagnosis on the patient, the rising incidence of disease, both in the US and California, imperils the federal and state economy. The dementia induced by AD disconnects patients from their loved ones and communities by eroding memory and cognitive function. Patients gradually lose their ability to drive, work, cook, and carry out simple, everyday tasks, ultimately losing all independence. The quality of life for AD patients is hugely diminished and the burden on their families and caregivers is extremely costly to the state of California. Annual health care costs are estimated to exceed $172 billion, not including the additional costs resulting from the loss of income and physical and emotional stress experienced by caregivers of Alzheimer's patients. Given that California is the most populous state and the state with the highest number of baby-boomers, AD’s impact on California families and state finances is proportionally high and will only increase as the AD prevalence rises. Currently, there is no cure for AD and no means of prevention. Most approved therapies address only symptomatic aspects of AD and no disease-modifying approaches are currently available. By enacting Proposition 71, California voters acknowledged and supported the need to investigate the potential of novel stem cell-based therapies to treat diseases with a significant unmet medical need such as AD. In a disease like AD, any therapy that exerts even a modest impact on the patient's ability to carry out daily activities will have an exponential positive effect not only for the patients but also for their families, caregivers, and the entire health care system. We propose to evaluate the hypothesis that neural stem cell transplantation will delay the progression of AD by slowing or stabilizing loss of memory and related cognitive skills. A single, one-time intervention may be sufficient to delay progression of neuronal degeneration and preserve functional levels of memory and cognition; an approach that offers considerable cost-efficiency. The potential economic impact of this type of therapeutic research in California could be significant, and well worth the investment of this disease team proposal. Such an approach would not only reduce the high cost of care and improve the quality of life for patients, it would also make California an international leader in a pioneering approach to AD, yielding significant downstream economic benefits for the state.

Multiple Sclerosis therapy: Human Pluripotent Stem Cell-Derived Neural Progenitor Cells

Funding Type: 
Early Translational III
Grant Number: 
TR3-05603
ICOC Funds Committed: 
$4 799 814
Disease Focus: 
Multiple Sclerosis
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Multiple Sclerosis (MS) is a disease of the central nervous system (CNS) caused by inflammation and loss of cells that produce myelin, which normally insulates and protects nerve cells. MS is a leading cause of neurological disability among young adults in North America. Current treatments for MS include drugs such as interferons and corticosteroids that modulate the ability of immune system cells to invade the CNS. These therapies often have unsatisfactory outcomes, with continued progression of neurologic disability over time. This is most likely due to irreversible tissue injury resulting from permanent loss of myelin and nerve destruction. The limited ability of the body to repair damaged nerve tissue highlights a critically important and unmet need for MS patients. The long-term goal of our research is to develop a stem cell-based therapy that will not only halt ongoing loss of myelin but also lead to remyelination and repair of damaged nerve tissue. Our preliminary data in animal models of human MS are very promising and suggest that this goal is possible. Research efforts will concentrate on refining techniques for production and rigorous quality control of clinically-compatible transplantable cells generated from high-quality human pluripotent stem cell lines, and to verify the therapeutic activity of these cells. We will emphasize safety and development of the most therapeutically beneficial cell type for eventual use in patients with MS.
Statement of Benefit to California: 
One in seven Americans lives in California, and these people make up the single largest health care market in the United States. The diseases and injuries that affect Californians affect the rest of the US and the world. Many of these diseases involve degeneration of healthy cells and tissues, including neuronal tissue in diseases such as Multiple Sclerosis (MS). The best estimates indicate that there are 400,000 people diagnosed with MS in the USA and 2.2 million worldwide. In California, there are approximately 160,000 people with MS – roughly half of MS patients in the US live in California. MS is a life-long, chronic disease diagnosed primarily in young adults who have a virtually normal life expectancy but suffer from progressive loss of motor and cognitive function. Consequently, the economic, social and medical costs associated with the disease are significant. Estimates place the annual cost of MS in the United States in the billions of dollars. The development of a stem cell therapy for treatment of MS patients will not only alleviate ongoing suffering but also allow people afflicted with this disease to return to work and contribute to the economic stabilization of California. Moreover, a stem cell-based therapy that will provide sustained recovery will reduce recurrence and the ever-growing cost burden to the California medical community.
Progress Report: 
  • The team has been highly productive during the first year of work on this award. A major goal of the project is to evaluate the efficacy of neural progenitor cell transplantation to promote remyelination following virus induced central nervous system damage. With intracranial infection by the virus mouse hepatitis virus (MHV), mice develop paralysis due to immune mediated destruction of cells that generate myelin. Using protocols developed in the Loring laboratory, neural precursor cells (NPC) were derived from the human embryonic stem cell line H9. Mice developing paralysis due to intracranial infection with MHV were subject to intraspinal transplantation of these NPC, resulting in significant clinical recovery beginning at 2-3 weeks following transplant. This clinical effect of NPC transplantation remained out to six months, suggesting that these NPC are effective for long-term repair following demyelination. Despite this striking recovery, these human ES cell derived NPC were rapidly rejected. Several protocols for the generation of NPC for transplantation have been characterized, with the greatest clinical impact observed for NPC cultures bearing a high level of expression of TGF beta I and TGF beta II. These findings support the hypothesis that transplanted NPC reprogram the immune system within the central nervous system (CNS), leading to the activation of endogenous NPC and other repair mechanisms. Thus, it may not be necessary to induce complete immune suppression in order to promote remyelination and CNS repair following NPC transplantation for demyelinating diseases such as multiple sclerosis.

Development of Single Cell MRI Technology using Genetically-Encoded Iron-Based Reporters

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02018
ICOC Funds Committed: 
$1 930 608
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Clinical application of cell transplantation therapy requires a means of non-invasively monitoring these cells in the patient. Several imaging modalities, including MRI, bioluminescence imaging, and positron emission tomography have been used to track stem cells in vivo. For MR imaging, cells are pre-loaded with molecules or particles that substantially alter the image brightness; the most common such labelling strategy employs iron oxide particles. Several studies have shown the ability of MRI to longitudinally track transplanted iron-labeled cells in different animal models, including stroke and cancer. But there are drawbacks to this kind of labeling. Division of cells will result in the dilution of particles and loss of signal. False signal can be detected from dying cells or if the cells of interest are ingested by other cells. To overcome these roadblocks in the drive toward clinical implementation of stem cell tracking, it is now believed that a genetic labeling approach will be necessary, whereby specific protein expression causes the formation of suitable contrast agents. Such endogenous and persistent generation of cellular contrast would be particularly valuable to the field of stem cell therapy, where the homing ability of transplanted stem cells, long-term viability, and capacity for differentiation are all known to strongly influence therapeutic outcomes. However, genetic labeling or "gene reporter" strategies that permit sensitive detection of rare cells, non-invasively and deep in tissue, have not yet been developed. This is therefore the translational bottleneck that we propose to address in this grant, through the development and validation of a novel high-sensitivity MRI gene reporter technology. There have been recent reports of gene-mediated cellular production of magnetic iron-oxide nanoparticles of the same composition as the synthetic iron oxide particles used widely in exogenous labeling studies. It is an extension of this strategy, combined with our own strengths in developing high-sensitivity MRI technology, that we propose to apply to the task of single cell tracking of metastatic cancer cells and neural stem cells. If we are successful with the proposed studies, we will have substantially advanced the field of in vivo cellular imaging, by providing a stable cell tracking technology that could be used to study events occurring at arbitrary depth in tissue (unlike optical methods) and over unlimited time duration and arbitrary number of cell divisions (unlike conventional cellular MRI). With the ability to track not only the fate (migration, homing and proliferation) but also the viability and function of very small numbers of stem cells will come new knowledge of the behavior of these cells in a far more relevant micro-environment compared with current in vitro models, and yet with far better visualization and cell detection sensitivity compared with other in vivo imaging methods.
Statement of Benefit to California: 
Stem cell therapy has enormous promise to become a viable therapy for a range of illnesses, including stroke, other cardiovascular diseases, and neurological diseases. Progress in the development of these therapies depends on the ability to monitor cell delivery, migration and therapeutic action at the disease site, using imaging and other non-invasive technologies. If breakthroughs could be made along these lines, it would not only be of enormous benefit to the citizens of the state of California, but would also greatly reduce healthcare costs. From a broader research perspective, the state of California is the front-runner in stem cell research, having gathered not only private investments, as demonstrated by the numerous biotechnology companies that are developing innovative tools, but also extensive public funds that allows the state, through CIRM, to sponsor stem cell research in public and private institutions. In order to preserve the leadership position and encourage research on stem cells, CIRM is calling for research proposals to develop innovative tools and technologies that will overcome current roadblocks in translational stem cell research. This proposal will benefit the state by providing important new technology that will be valuable for both basic and translational stem cell research. A key bottleneck to the further development and translation of new stem cell therapies is the inability to track stem cells through a human body. It is possible to image stem cells using embedded optical fluorescence labels, but optical imaging does not permit tracking of cells deep in tissue. Other imaging modalities and their associated cellular labels (for example positron emission tomography) have also been used to track cells but do not have the sensitivity to detect rare or single cells. Finally, MRI has been used to track cells deep in tissue, down to the single cell level, but only by pre-loading cells with a non-renewable supply of iron oxide nanoparticles, which prevents long-term tracking and assessment of cell viability and function. We propose here to develop MRI technology and a new form of genetically-encoded, long-term cell labeling technology, to a much more advanced state than available at present. This will make it possible to use MRI to detect and follow cancer and stem cells as they migrate to and proliferate at the site of interest, even starting from the single cell stage. This will provide a technology that will help stem cell researchers, first and foremost in California, to understand stem cell behavior in a realistic in vivo environment. This technology will be translatable to future human stem cell research studies.
Progress Report: 
  • We have made good progress in the first year. This project involves four separate scientific teams, brought together for the first time, representing diverse backgrounds ranging from magnetic resonance imaging (MRI) physics and cell tracking (Dr. Rutt), microbiology (Dr. Matin), nano and magnetic characterization (Dr. Moler) and stem cell imaging in stroke models (Dr. Guzman). Substantial progress has been made by all four teams, and we are starting to see important interactions between the teams. An overall summary of progress is that we have evaluated three different bacterial genes (magA, mms6, mamB) in one mammalian cell line (MDA-MB-231BR) and have shown significant iron accumulation in vitro with two of these genes, which is a very positive result implying that these genes may have the required characteristics to act as "reporter genes" for MRI-based tracking of cells labeled with these genes. MR imaging of mouse brain specimens has yielded promising results and in vivo imaging experiments are underway at medium MRI field strength (3 Tesla). At the same time, we are ramping up our higher field, higher sensitivity MR imaging methods and will be ready to evaluate the different variations of our MR reporter gene at 7 Tesla (the highest magnetic field widely available for human MRI) in the near future. Finally, methods to perform quantitative characterization of our reporter cells are being developed, with the goal of being able to characterize magnetic properties down to the single cell level, and also to be able to assess iron loading levels down to the single level in brain tissue slices.
  • We have made good progress in the second year. This project involves four separate scientific teams, brought together for the first time for this project, representing diverse backgrounds ranging from magnetic resonance imaging (MRI) physics and cell tracking (Dr. Rutt), microbiology (Dr. Matin), nano and magnetic characterization (Dr. Moler) and imaging reporter development and testing in small animal models of disease (Dr. Contag). Substantial progress has been made by all four teams, and we are starting to see important interactions between the teams.
  • An overall summary of progress is that we have been evaluating three different bacterial genes (magA, mms6, mamB) in two mammalian cell lines (MDA-MB-231BR and DAOY). In year I we had shown significant iron accumulation in vitro with two of these genes, which was a very positive result implying that these genes may have the required characteristics to act as "reporter genes" for MRI-based tracking of cells labeled with these genes. In year 2, we diversified and intensified the efforts to achieve expression of one or more of the bacterial genes in different cell lines, using different genetic constructs. We began a concerted effort to achieve optical labeling such that we could visualize the gene expression and to identify sub-cellular localization of the report gene products.
  • We obtained promising results from MR imaging of mouse brain. In vivo imaging experiments were accomplished at medium MRI field strength (3 Tesla). At the same time, we ramped up our higher field, higher sensitivity MR imaging methods and began to evaluate the sensitivity gains enabled at the higher magnetic field strength of 7 Tesla (the highest magnetic field widely available for human MRI
  • Finally, methods to perform quantitative characterization of our reporter cells were developed, with the goal of being able to characterize magnetic properties down to the single cell level, and also to be able to assess iron loading levels down to the single level in brain tissue slices.
  • We have made good progress in the third year. This project involves four separate scientific teams, brought together for the first time for this project, representing diverse backgrounds ranging from magnetic resonance imaging (MRI) physics and cell tracking (Dr. Rutt), microbiology (Dr. Matin), nano and magnetic characterization (Dr. Moler) and imaging reporter development and testing in small animal models of disease (Dr. Contag). Substantial progress has been made by all four teams, and we have benefited from important interactions between all teams in this third year.
  • An overall summary of progress is that we evaluated several iron-binding bacterial genes (magA, mamB, mms6, mms13), both singly and doubly, in two mammalian cell lines (MDA-MB-231BR and DAOY). In year 2, we diversified and intensified the efforts to achieve expression of one or more of the bacterial genes in different cell lines, using different genetic constructs. We completed an effort to achieve optical labeling such that we could visualize the gene expression and to identify sub-cellular localization of the report gene products. In year 3, while continuing to face challenges with single gene constructs, we succeeded in finding substantial iron uptake in cells containing unique double gene expression, notably magA and mms13.
  • We completed much of the development of our higher field, higher sensitivity MR imaging methods and evaluated the sensitivity gains enabled at the higher magnetic field strength of 7 Tesla (the highest magnetic field widely available for human MRI).
  • Finally, we demonstrated novel nanomagnetic methods to characterize our reporter cells, able to characterize magnetic properties down to the single cell level.

Use of hiPSCs to develop lead compounds for the treatment of genetic diseases

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-01920
ICOC Funds Committed: 
$1 833 054
Disease Focus: 
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
This study will use Ataxia-Telangiectasia (A-T), an early-onset inherited neurodegenerative disease of children, as a model to study the mechanisms leading to cerebellar neurodegeneration and to develop a drug that can slow or halt neurodegeneration. We will start with skin cells that were originally grown from biopsies of patients with A-T who specifically carry “nonsense” type of mutations in the ATM gene. We will convert these skin cells to stem cells capable of forming neural cells that are lacking in the brain (cerebellum) of A-T patients; presumably these neural cells need ATM protein to develop normally. We will then test the effects of our most promising new “readthrough compounds” (RTCs) on the newly-developed neural cells. Our lab has been developing the drugs over the past six years. At present, there is no other disease model (animal or in a test tube) for evaluating the effects of RTCs on the nervous system and its development. Nor is there any effective treatment for the children with A-T or other progressively-deteriorating ataxias. Success in this project would open up at least three new areas for understanding and treating neurodegenerative diseases: 1) the laboratory availability of human neural cells with specific disease-causing mutations; 2) a new approach to learning how the human brain develops and 3) a new class of drugs (RTCs) that correct nonsense mutations, even in the brain, and may correct neurodegeneration.
Statement of Benefit to California: 
This project seeks to merge the expertise of two major research cultures: one with long-standing experience in developing a treatment for a progressive childhood-onset disease called Ataxia-telangiectasia and another with recent success in converting skin cells into cells of the nervous system. California citizens will benefit by finding new ways to treat neurodegenerative diseases, like A-T, Parkinson and Alzheimer, and expanding the many possible applications of stem cell technology to medicine. More specifically, we will construct a new “disease in a dish” model for neurodegeneration, and this will enable our scientists to test the positive and negative effects of a new class of drugs for correcting inherited diseases/mutations directly on brain cells. These advances will drastically decrease drug development costs and will stimulate new biotech opportunities and increase tax revenues for California, while also training the next generation of young scientists to deliver these new medical products to physicians and patients within the next five years.
Progress Report: 
  • No effective treatments are available for most neurodegenerative diseases. This study uses Ataxia-Telangiectasia (A-T), an early-onset inherited neurodegenerative disease of children, as a model to study the mechanisms leading to cerebellar neurodegeneration and to develop a drug that can slow or halt neurodegeneration. Aim1 proposed to use “Yamanaka factors” to reprogram A-T patient-derived skin fibroblasts, which carry nonsense mutations that we have shown can be induced by RTCs to express full-length and functional ATM protein, into iPSCs. We have successfully reprogrammed A-T fibroblasts to hiPSCs and teratoma formation shows their pluripotency. Aim2 will use these established iPSCs to model neurodegeneration, focusing on differentiation to cerebellar cells, such as Purkinje cells and granule cells. We have generated the Purkinje cell promoter –driven GFP reporter system and will use this system to examine the differentiation capacity of A-T iPSCs to Purkinje cells. Aim3 will utilize the newly-developed neural cells carrying disease-causing ATM nonsense mutations as targets for evaluating the potential therapeutic effects of leading RTCs. We have already started to test the efficacy and toxicity of our lead RTC compounds on A-T iPSC-derived neural progenitor cells. The continuation of this study will help us to pick up one promising RTC compound for IND application. This project is on the right track towards its objective for the development of disease models with hiPSCs and the test of our lead small molecule compounds for the treatment of A-T or other neurodegenerative diseases.
  • No effective treatments are available for most neurodegenerative diseases. This study uses Ataxia-Telangiectasia (A-T), an early-onset inherited neurodegenerative disease of children, as a model to study the mechanisms leading to cerebellar neurodegeneration and to develop a drug that can slow or halt neurodegeneration. Aim1 proposed to use “Yamanaka factors” to reprogram A-T patient-derived skin fibroblasts, which carry nonsense mutations that we have shown can be induced by RTCs to express full-length and functional ATM protein, into iPSCs. Aim2 will use these established iPSCs to model neurodegeneration, focusing on differentiation to cerebellar cells, such as Purkinje cells and granule cells. Aim3 will utilize the newly-developed neural cells carrying disease-causing ATM nonsense mutations as targets for evaluating the potential therapeutic effects of leading RTCs.
  • During the past two years of this project, we established Ataxia-telangiectasia (A-T) patient-derived iPSC lines from two patients which contain nonsense mutations and splicing mutations. These two lines are currently used for testing the mutation-targeted therapies with small molecule readthrough (SMRT) compounds and antisense morpholino oligonucleotides (AMOs). Manuscript describing this work was recently accepted, showing that SMRT compounds can abrogate phenotypes of A-T iPSC-derived neural cells
  • This is the third year (last year) progress report. During the first two years of this project, we have already established two Ataxia-telangiectasia (A-T) patient-derived iPSC lines which contain nonsense mutations and splicing mutations, respectively. These two lines are currently used for testing the mutation-targeted therapies with small molecule readthrough (SMRT) compounds and antisense morpholino oligonucleotides (AMOs). In the third year, we have formally published our results from the first two years’ research work in Nature Communications (Lee et al., 2013). In the last year, we continue to make progresses in the characterization of A-T iPSCs and their derived neuronal cells as well as developing the mutation-targeted therapies for neurodegeneration diseases

Banking transplant ready dopaminergic neurons using a scalable process

Funding Type: 
Early Translational II
Grant Number: 
TR2-01856
ICOC Funds Committed: 
$6 016 624
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Collaborative Funder: 
Maryland
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons (a type of nerve cells in the central nervous system) present in the midbrain. These neurons secrete dopamine (a signaling molecule) and are a critical component of the motor circuit that ensures movements are smooth and coordinated. All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy (that is, transplantation of dopaminergic neurons into the brain to replace lost cells and restore function) as proposed in this application attempts to use cells as small pumps of dopamine that will be secreted locally and in a regulated way, and will therefore avoid the complications of other modes of treatment. Indeed, cell therapy using fetal tissue-derived cells have been shown to be successful in multiple transplant studies. Work in the field has been limited however, partially due to the limited availability of cells for transplantation (e.g., 6-10 fetuses of 6-10 weeks post-conception are required for a single patient). We believe that human embryonic stem cells (hESCs) may offer a potentially unlimited source of the right kind of cell required for cell replacement therapy. Work in our laboratories and in others has allowed us to develop a process of directing hESC differentiation into dopaminergic neurons. To move forward stem cell-based therapy development it is important to develop scale-up GMP-compatible process of generating therapeutically relevant cells (dopaminergic neurons in this case). The overall goal of this proposal is to develop a hESC-based therapeutic candidate (dopaminergic neurons) by developing enabling reagents/tools/processes that will allow us to translate our efforts into clinical use. We have used PD as a model but throughout the application have focused on generalized enabling tools. The tools, reagents and processes we will develop in this project will allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects. In addition, the processes we will develop would be of benefit to the CIRM community.
Statement of Benefit to California: 
Parkinson’s disease affects more than a million patients United States with a large fraction being present in California. California, which is the home of the Parkinson’s Institute and several Parkinson’s related foundations and patient advocacy groups, has been at the forefront of this research and a large number of California based scientists supported by these foundations and CIRM have contributed to significant breakthroughs in this field. In this application we and our collaborators in California aim propose to develop a hESC-based therapeutic candidate (dopaminergic neurons) that will allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects for this currently non-curable disorder. We believe that this proposal includes the basic elements that are required for the translation of basic research to clinical research. We believe these experiments not only provide a blueprint for moving Parkinson’s disease towards the clinic for people suffering with the disorder but also a generalized blueprint for the development of stem cell therapy for multiple neurological disorders including motor neuron diseases and spinal cord injury. The tools and reagents that we develop will be made widely available to Californian researchers. We expect that the money expended on this research will benefit the Californian research community and the tools and reagents we develop will help accelerate the research of our colleagues in both California and worldwide.
Progress Report: 
  • Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons (a type of nerve cells in the central nervous system) present in the midbrain. These neurons secrete dopamine (a signaling molecule) and are a critical component of the motor circuit that ensures movements are smooth and coordinated.
  • All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy (that is, transplantation of dopaminergic neurons into the brain to replace lost cells and restore function) as proposed in this application attempts to use cells as small pumps of dopamine that will be secreted locally and in a regulated way, and will therefore avoid the complications of other modes of treatment. Indeed, cell therapy using fetal tissue-derived cells have been shown to be successful in multiple transplant studies. Work in the field has been limited however, partially due to the limited availability of cells for transplantation (e.g., 6-10 fetuses of 6-10 weeks post-conception are required for a single patient).
  • We believe that human embryonic stem cells (hESCs) may offer a potentially unlimited source of the right kind of cell required for cell replacement therapy. Work in our laboratories and in others has allowed us to develop a process of directing hESC differentiation into dopaminergic neurons. To move forward stem cell-based therapy development it is important to develop scale-up GMP-compatible process of generating therapeutically relevant cells (dopaminergic neurons in this case).
  • The overall goal of this proposal is to develop a hESC-based therapeutic candidate (dopaminergic neurons) by developing enabling reagents/tools/processes that will allow us to translate our efforts into clinical use. We have used PD as a model but throughout the application have focused on generalized enabling tools. The tools, reagents and processes we will develop in this project will allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects. In addition, the processes we will develop would be of benefit to the CIRM community.
  • Parkinson's disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons (a type of nerve cells in the central nervous system) present in the midbrain. These neurons secrete dopamine (a signaling molecule) and are a critical component of the motor circuit that ensures movements are smooth and coordinated.
  • All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy (that is, transplantation of dopaminergic neurons into the brain to replace lost cells and restore function) as proposed in this application attempts to use cells as small pumps of dopamine that will be secreted locally and in a regulated way, and will therefore avoid the complications of other modes of treatment. Indeed, cell therapy using fetal tissue-derived cells have been shown to be successful in multiple transplant studies. Work in the field has been limited however, partially due to the limited availability of cells for transplantation (e.g., 6-10 fetuses of 6-10 weeks post-conception are required for a single patient).
  • We believe that human pluripotent stem cells (PSC) may offer a potentially unlimited source of the right kind of cell required for cell replacement therapy. Work in our laboratories and in others has allowed us to develop a process of directing PSC differentiation into dopaminergic neurons. To move forward stem cell-based therapy development it is important to develop scale-up GMP-compatible process of generating therapeutically relevant cells (dopaminergic neurons in this case).
  • During this grant, we have optimized a step-wise scalable process for generating authentic dopaminergic neurons in defined media from human PSC, and have determined the time point at which dopaminergic neurons can be frozen, shipped, thawed and transplanted without compromising their ability to mature and provide therapeutic benefit in animal models. Our process has been successfully transferred to a GMP facility and we have manufactured multiple lots of GMP-equivalent cells using this process. Importantly, we have shown functional equivalency of the manufactured cells in appropriate models. The tools, reagents and processes we have developed in this project allow us to move towards translational therapy and establish processes that could be applied to future IND-enabling projects. In addition, the processes we have developed would be of benefit to the CIRM community.

Mechanisms in Choroid Plexus Epithelial Development

Funding Type: 
New Faculty II
Grant Number: 
RN2-00915
ICOC Funds Committed: 
$3 169 328
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.

Development of Induced Pluripotent Stem Cells for Modeling Human Disease

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00649
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.

Generation and characterization of high-quality, footprint-free human induced pluripotent stem cell lines from 3,000 donors to investigate multigenic diseases

Funding Type: 
hiPSC Derivation
Grant Number: 
ID1-06557
ICOC Funds Committed: 
$16 000 000
Disease Focus: 
Developmental Disorders
Genetic Disorder
Heart Disease
Infectious Disease
Alzheimer's Disease
Neurological Disorders
Autism
Respiratory Disorders
Vision Loss
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Induced pluripotent stem cells (iPSCs) have the potential to differentiate to nearly any cells of the body, thereby providing a new paradigm for studying normal and aberrant biological networks in nearly all stages of development. Donor-specific iPSCs and differentiated cells made from them can be used for basic and applied research, for developing better disease models, and for regenerative medicine involving novel cell therapies and tissue engineering platforms. When iPSCs are derived from a disease-carrying donor; the iPSC-derived differentiated cells may show the same disease phenotype as the donor, producing a very valuable cell type as a disease model. To facilitate wider access to large numbers of iPSCs in order to develop cures for polygenic diseases, we will use a an episomal reprogramming system to produce 3 well-characterized iPSC lines from each of 3,000 selected donors. These donors may express traits related to Alzheimer’s disease, autism spectrum disorders, autoimmune diseases, cardiovascular diseases, cerebral palsy, diabetes, or respiratory diseases. The footprint-free iPSCs will be derived from donor peripheral blood or skin biopsies. iPSCs made by this method have been thoroughly tested, routinely grown at large scale, and differentiated to produce cardiomyocytes, neurons, hepatocytes, and endothelial cells. The 9,000 iPSC lines developed in this proposal will be made widely available to stem cell researchers studying these often intractable diseases.
Statement of Benefit to California: 
Induced pluripotent stem cells (iPSCs) offer great promise to the large number of Californians suffering from often intractable polygenic diseases such as Alzheimer’s disease, autism spectrum disorders, autoimmune and cardiovascular diseases, diabetes, and respiratory disease. iPSCs can be generated from numerous adult tissues, including blood or skin, in 4–5 weeks and then differentiated to almost any desired terminal cell type. When iPSCs are derived from a disease-carrying donor, the iPSC-derived differentiated cells may show the same disease phenotype as the donor. In these cases, the cells will be useful for understanding disease biology and for screening drug candidates, and California researchers will benefit from access to a large, genetically diverse iPSC bank. The goal of this project is to reprogram 3,000 tissue samples from patients who have been diagnosed with various complex diseases and from healthy controls. These tissue samples will be used to generate fully characterized, high-quality iPSC lines that will be banked and made readily available to researchers for basic and clinical research. These efforts will ultimately lead to better medicines and/or cellular therapies to treat afflicted Californians. As iPSC research progresses to commercial development and clinical applications, more and more California patients will benefit and a substantial number of new jobs will be created in the state.

Modeling disease in human embryonic stem cells using new genetic tools

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-05855
ICOC Funds Committed: 
$1 387 800
Disease Focus: 
Neuropathy
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The use of stem cells or stem cell-derived cells to treat disease is one important goal of stem cell research. A second, important use for stem cells is the creation of cellular models of human development and disease, critical for uncovering the molecular roots of illness and testing new drugs. However, a major limitation in achieving these goals is the difficulty in manipulating human stem cells. Existing means of generating genetically modified stem cells are not ideal, as they do not preserve the normal gene regulation, are inefficient, and do not permit removal of foreign genes. We have developed a method of genetically modifying mouse embryonic stem cells that is more efficient than traditional methods. We are adapting this approach for use with human embryonic stem cells, so that these cells can be better understood and harnessed for modeling, or even treating, human diseases. We will use this approach to create a human stem cell model of Charcot-Marie-Tooth (CMT) disease, an inherited neuropathy. How gene dysfunction leads to nerve defects in CMT is not clear, and there is no cure or specific therapy for this neurological disease. Thus, we will use our genetic tools to investigate how gene function is disrupted to cause CMT. By developing these tools and using them to gain understanding of CMT, we will illustrate how this system can be used to gain insight into other important diseases.
Statement of Benefit to California: 
Although human stem cells hold the potential to generate new understanding about human biology and new approaches to important diseases, the inability to efficiently and specifically modify stem cells currently limits the pace of research. Also, there is presently no safe means of changing genes compatible with the use of the stem cells in therapies. We are developing new genetic tools to allow for the tractable manipulation of human stem cells. By accelerating diverse other stem cell research projects, these tools will enhance the scientific and economic development of California. We will use these tools to create cellular models of Charcot-Marie-Tooth (CMT), a neurological disease with no cure that affects about 15,000 Californians. This model will facilitate understanding of the etiology of CMT, and may lead to insights that can be used to develop specific therapies. Beyond gaining insight into CMT, the ability to engineer specific genetic changes in human stem cells will be useful for many applications, including the creation of replacement cells for personalized therapies, reporter lines for stem cell-based drug screens, and models of other diseases. Thus, our research will assist the endeavors of the stem cell community in both the public and private arenas, contributing to economic growth and new product development. This project will also train students and postdoctoral scholars in human stem cell biology, who will contribute to the economic capacity of California.
Progress Report: 
  • An important use for stem cells is the creation of cellular models of human development and disease, critical for uncovering the molecular roots of illness and testing new drugs. However, a major limitation in achieving these goals is the difficulty in manipulating human stem cells. We have developed a method of genetically modifying mouse embryonic stem cells that is more efficient than traditional methods. During the first year of this project, we adapted this approach for use with human embryonic stem cells. We have also created gene trap mutations in a diversity of human embryonic stem cell genes that can be used to better harness human embryonic stem cells for modeling, or even treating, human diseases.

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