Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders
Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05272
Investigator: 
ICOC Funds Committed: 
$96 448
Disease Focus: 
Parkinson's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
We proposes to use human embryonic stem cells (hESCs) differentiated into neural progenitor/stem cells (NPCs), but modified by transiently programming the cells with the transcription factor MEF2C to drive them more specifically towards dopaminergic (DA) neurons, representing the cells lost in Parkinson’s disease. We will select Parkinson’s patients that no longer respond to L-DOPA and related therapy for our study, because no alternative treatment is currently available. The transplantation of cells that become DA neurons in the brain will create a population of cells that secrete dopamine, which may stop or slow the progression of the disease. In this way, moderate to severely affected Parkinson’s patients will benefit. The impact of development of a successful cell-based therapy for late-stage Parkinson’s patients would be very significant. There are approximately one million people in the United States with Parkinson’s disease (PD) and about ten million worldwide. Though L-DOPA therapy controls symptoms in many patients for a period of time, most reach a point where they fail to respond to this treatment. This is a very devastating time for sufferers and their families as the symptoms then become much worse. A cell-based therapy that restores production of dopamine and/or the ability to effectively use L-DOPA would greatly improve the lives of these patients. Because of our extensive preclinical experience and the clinical acumen of our Disease Team, we will be able to quickly adapt our procedures to human patients and be able to seek an IND from the FDA within four years.
Statement of Benefit to California: 
It is estimated that the cost per year for a Parkinson’s patient averages over $10,000 in direct costs and over $21,000 in total cost to society (in 2007 dollars). With nearly 40 million people in California and with one in 500 estimated to have Parkinson’s (1.5-2% of the population over 60 years of age), there are approximately 80,000 people in California with Parkinson’s disease. Thus, Parkinson’s disease is a significant burden to California, not to mention the devastating effect on those who have the disease and their families. A therapy that could halt the progression or reverse Parkinson’s disease would be of great benefit to the state and its residents. It would be particularly advantageous if the disease could be halted or reversed to an early stage, since the most severe symptoms and highest costs of care are associated with the late stages of the disease. Cell-based therapies offer the hope of achieving this goal.
Progress Report: 
  • A distinguished group of scientists was assembled by Dr. Stuart Lipton to plan a strategy to develop a human embryonic stem cell line expressing a constitutively active form of the transcription factor MEF2 (MEF2CA) into a therapeutic for treatment of Parkinson’s disease (PD), as funded by this planning grant. Preliminary data presented showed directed differentiation of the stem cells into mature dopaminergic cells and a positive outcome, histologically, electrophysiologically and behaviorally, when transplanted into a rat model. The salient features of the preliminary data show that the cells showed a strong propensity to differentiate into dopaminergic neurons, remaining endogenous dopaminergic neurons were saved from death or recruited to synthesize more dopamine through trophic interactions, and the behavioral readout showed that the rats’ neuromotor deficits were improved. An additional feature of the transplanted cells produced by the presented strategy was that none of the MEF2CA-expressing cells were hyperproliferative, indicating that tumor formation will not be a problem with their use. A strategy to further develop the cells under GMP conditions, test in rat and monkey models of PD and begin regulatory compliance for FDA approval was developed. Importantly, insertion of the Mef2CA gene in the stable stem cell line was verified by sequencing to occur at non-essential site of integration.
Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05416
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$98 050
Disease Focus: 
Alzheimer's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
Alzheimer’s disease (AD) is an incurable disorder that affects memory, social interaction and the ability to perform everyday activities. In the USA alone, the number of AD patients aged 65 and older has surpassed 5 million and that number may triple by 2050. Annual health care costs have been estimated to exceed 172 billion dollars, but do not reflect loss of income and stress caused to caregivers. Therefore, there is great hope for new therapies that will both improve symptoms and alleviate suffering. There are few FDA-approved medications to treat AD and none is capable of preventing, delaying onset or curing AD. Current medications mostly tend to temporarily slow the worsening of AD-associated symptoms such as sleep disturbances, depression and memory loss/disorientation. Pharmaceutical companies continue to develop new types of drugs or combination therapies that can better treat the symptoms or improve the quality of life of AD patients. There is also an ongoing effort to discover novel drugs that may prevent, reverse, or even cure AD. Unfortunately, the number of clinical studies addressing the possible benefit of such drugs is low, and agents that have shown initial promise have failed at later stage clinical testing, despite convincing preclinical data. There are ongoing studies in AD patients using vaccines and other biological compounds but it is unclear when data from these new trials will be available and more importantly, whether they will be successful. The need for divergent and innovative approaches to AD is clearly suggested by the failure of experimental drugs. Our proposal is to use brain stem cells to treat AD. This is a completely different approach to the more standard therapies described above such as drugs, vaccines, etc., and one that we hope will be beneficial for AD patients as a one-time intervention. AD is characterized by a dysfunction and eventual loss of neurons, the specialized cells that convey information in the brain. Death or dysfunction of neurons results in the characteristic memory loss, confusion and inability to solve new problems that AD patients experience. It is our hope that stem cells transplanted into the patient’s brain may provide factors that will protect neurons and preserve their function. Even a small improvement in memory and cognitive function could significantly alter quality of life in a patient with AD.
Statement of Benefit to California: 
Of the 5.4 million Americans affected with AD, 440,000 are California residents and, according to the Alzheimer’s Association, this number is projected to increase between 49.1 - 81.0% (second highest only to Northwestern states) between 2000 and 2025. Given that California is the most populous state, AD’s impact on state finances is proportionally high and will only increase as the population ages and AD incidence increases. The dementia resulting from this devastating disease disconnects patients from their community and loved ones by eroding memory and cognitive function. Patients gradually lose their ability to drive, work, cook and even carry out simple everyday tasks, and become totally dependent on others. The quality of life of AD patients is hugely affected and the burden on their families and caregivers is very costly to the state of California. There is no cure for AD and no way to prevent it. Most approved therapies only address symptomatic aspects of AD and disease modifying drugs are currently not available. By enacting Proposition 71, California voters acknowledged and supported the need to investigate the use of novel stem cell based therapies to treat currently incurable diseases such as AD. Our goal is to leverage our proven expertise in developing neural stem cell based therapies for human neurodegenerative disorders and apply it to AD. We propose that neural stem cell transplantation into select regions of the brain will have a beneficial impact on the patient. If successful, a single intervention may be sufficient to delay or stop progression of neuronal degeneration and preserve functional levels of cognition and memory. In a disease such as AD, any therapy that can exert even a modest impact on the patient’s ability to carry out some daily activities will have an exponential positive effect not only on patients but also on families, caregivers and the health care system. The potential economic impact of such type of therapeutic intervention for California could be tremendous, not only by reducing the high costs of care but also by becoming a vital world center for stem cell interventions in AD.
Progress Report: 
  • Alzheimer's disease (AD) is an incurable disorder that affects memory, social interaction, and the ability to perform everyday activities. The number of AD patients older than 65 has surpassed 5 million in the US and 600,000 in California, numbers that may triple by 2050. Annual health care costs related to AD have been estimated to exceed $172 billion in the US, even without reflecting either the loss of income or the physical and emotional stress experienced by caregivers. 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. There is a great need for new therapies that will both improve symptoms and alleviate suffering.
  • AD is characterized by the dysfunction and eventual loss of neurons, the specialized cells that convey information in the brain. Death or dysfunction of neurons results in the characteristic memory loss, confusion, and inability to solve new problems that AD patients experience.
  • StemCells Inc. is embarking on an initiative to evaluate the use of its proprietary human neural stem cells to treat AD. We believe that neural stem cells transplanted into a patient’s brain may protect neurons and preserve their function. This represents an entirely new approach to standard therapeutic drug development for AD, which has so far resulted in drugs that only temporarily alleviate symptoms in some patients but that do not slow or change the course of the disease. We envision using neural stem cells as a one-time intervention that will improve memory and cognitive function in AD patients. Even a modest improvement in these symptoms could significantly alter the quality of life of a patient with AD.
  • StemCells Inc. received a Disease Team Planning (DTP) award from CIRM to establish a Disease Team for AD, and to begin organizing the activities required to submit a Disease Team Therapy Development (DTTD) award. We are reporting now on the successful completion of this DTP award. The main deliverables were (i) submission of a DTTD award application and (ii) development of a four year research plan that contemplates an Investigational New Drug (IND) submission to the FDA for the clinical study of neural stem cells in patients with AD, within four years.
  • To begin evaluating its proprietary human neural stem cells as a potential therapy for AD, StemCells Inc. and its collaborators from UC Irvine needed to first design IND-enabling safety and efficacy studies to test these stem cells in animal models relevant for AD. The DTP funding from CIRM helped support a series of telephone, email and face-to-face meetings over the last 6 months, between investigators at UCI and StemCells Inc., to present and evaluate existing data on neural stem cells and to share information about AD in order to design pilot and definitive efficacy and safety studies. During this time, the team also discussed the logistical details required to conduct these studies.
  • After a draft research plan had been outlined, StemCells Inc. and its principal collaborator at UCI, Dr. Frank LaFerla, enlisted the help of various experts in the field of AD, including both clinicians and academic scientists, to evaluate this plan. These experts attended a meeting at UCI and provided input into the experimental design of efficacy and safety studies. Many of these experts were also recruited by StemCells Inc. to participate in preclinical and clinical working groups hosted by the Company. These working groups will ultimately evaluate the preclinical experimental results and help design the protocol for the proposed clinical trial.
  • The DTP award also allowed StemCells Inc. to establish a “Project Team” consisting of highly trained and skilled personnel at UCI, StemCells Inc., and an established Contract Research Organization. This Project Team will be responsible for the production and supply of the human neural stem cells, the execution of all efficacy and safety studies, and the preparation and submission of IND documents to the FDA within the next 4 years.
  • Finally, the DTP award allowed StemCells Inc. to timely develop and submit its DTTD application to CIRM, in which the Company requested funding in the amount of up to $20 million to facilitate execution of IND-enabling safety and efficacy studies for its proposed breakthrough neural stem cell treatment for AD.
Funding Type: 
Disease Team Therapy Planning I
Grant Number: 
DR2-05415
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$99 248
Disease Focus: 
Huntington's Disease
Neurological Disorders
oldStatus: 
Closed
Public Abstract: 
One in every ten thousand people in the USA has Huntington's disease, and it impacts many more. Multiple generations within a family can inherit the disease, resulting in escalating health care costs and draining family resources. This highly devastating and fatal disease touches all races and socioeconomic levels, and there are currently no cures. Screening for the mutant HD gene is available, but the at-risk children of an affected parent often do not wish to be tested since there are currently no early prevention strategies or effective treatments. We propose a novel therapy to treat HD; implantation of cells engineered to secrete Brain-Derived Neurotrophic factor (BDNF), a factor needed by neurons to remain alive and healthy, but which plummets to very low levels in HD patients due to interference by the mutant Huntingtin (htt) protein that is the hallmark of the disease. Intrastriatal implantation of mesenchymal stem cells (MSC) has significant neurorestorative effects and is safe in animal models. We have discovered that MSC are remarkably effective delivery vehicles, moving robustly through the tissue and infusing therapeutic molecules into each damaged cell that they contact. Thus we are utilizing nature's own paramedic system, but we are arming them with enhanced neurotrophic factor secretion to enhance the health of at-risk neurons. Our novel animal models will allow the therapy to be carefully tested in preparation for a phase 1 clinical trial of MSC/BDNF infusion into the brain tissue of HD patients, with the goal of restoring the health of neurons that have been damaged by the mutant htt protein. Delivery of BDNF by MSC into the brains of HD mice is safe and has resulted in a significant reduction in their behavioral deficits, nearly back to normal levels. We are doing further work to ensure that the proposed therapy will be safe and effective, in preparation for the phase 1 clinical trial. The significance of our studies is very high because there are currently no treatments to diminish the unrelenting decline in the numbers of medium spiny neurons in the striata of patients affected by HD. However this biological delivery system for BDNF could also be modified for other neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia (SCA1), Alzheimer's Disease, and some forms of Parkinson's Disease, where neuroregeneration is needed. Development of novel stem cell therapies is extremely important for the community of HD and neurodegenerative disease researchers, patients, and families. Since HD patients unfortunately have few other options, the benefit to risk ratio for the planned trial is very high.
Statement of Benefit to California: 
It is estimated that one in 10,000 CA residents have Huntington’s disease (HD). While the financial burden of HD is estimated to be in the billions, the emotional cost to friends, families, and those with or at risk for HD is immeasurable. Health care costs are extremely high for HD patients due to the long progression of the disease, often for two decades. The lost ability of HD patients to remain in the CA workforce, to support their families, and to pay taxes causes additional financial strain on the state’s economy. HD is inherited as an autosomal dominant trait, which means that 50% of the children of an HD patient will inherit the disease and will in turn pass it on to 50% of their children. Individuals diagnosed through genetic testing are at risk of losing insurance coverage in spite of reforms, and can be discriminated against for jobs, school, loans, or other applications. Since there are currently no cures or successful clinical trials to treat HD, many who are at risk are very reluctant to be tested. We are designing trials to treat HD through rescuing neurons in the earlier phases of the disease, before lives are devastated. Mesenchymal stem cells (MSC) have been shown to have significant effects on restoring synaptic connections between damaged neurons, promoting neurite outgrowth, secreting anti-apoptotic factors in the brain, and regulating inflammation. In addition to many trials that have assessed the safety and efficacy of human MSC delivery to tissues via systemic IV infusion, MSC are also under consideration for treatment of disorders in the CNS, although few MSC clinical trials have started so far with direct delivery to brain or spinal cord tissue. Therefore we are conducting detailed studies in support of clinical trials that will feature MSC implantation into the brain, to deliver the neurotrophic factor BDNF that is lacking in HD. MSC can be transferred from one donor to the next without tissue matching because they shelter themselves from the immune system. We have demonstrated the safe and effective production of engineered molecules from human MSC for at least 18 months, in pre-clinical animal studies, and have shown with our collaborators that delivery of BDNF can have significant effects on reducing disease progression in HD rodent models. We are developing a therapeutic strategy to treat HD, since the need is so acute. HD patient advocates are admirably among the most vocal in California about their desire for CIRM-funded cures, attending almost every public meeting of the governing board of the California Institute for Regenerative Medicine (CIRM). We are working carefully and intensely toward the first FDA-approved approved cellular therapy for HD patients which could have a major impact on those affected in California. In addition, the methods, preclinical testing models, and clincial trial design that we are developing could have far-reaching impact on the treatment of other neurodegenerative disorders.
Progress Report: 
  • A) Pre-clinical: The remainder of the IND-enabling studies were designed in consultation with Biologics Consulting Group (BCG). The project will begin with the IND-enabling phase and transition through regulatory approvals and through an observational trial and the Phase I clinical trial of stem cell therapy. The project has a Preclinical unit, under the leadership of co-PI Dr. Jan Nolta, and a Clinical unit, under the leadership of PI Dr. Vicki Wheelock. The two units are well integrated, since the team has been meeting weekly since 2009 to plan the testing of MSC trials for HD. During the planning phase we had a minimum of 4 hours of HD meetings per week, and worked continually on the project. This team is truly translational, with both PIs highly dedicated to this trial and motivated by the HD community.
  • Co-PI Jan Nolta, Ph.D. is Scientific Director of the UC Davis/CIRM GMP Facility, and will continue to direct ongoing IND-enabling studies for MSC/BDNF. The Pre-Clinical team will perform all IND-enabling studies at the level of GLP, and will manufacture and qualify the MSC and MSC/BDNF products in the GMP facility at UC Davis that is directed by Dr. Bauer (CMC lead). These studies are ongoing and we have been advised by BCG consulting lead Andra Miller, who was formerly Gene Therapy Group Leader at the FDA, CBER, Division of Cell and Gene Therapies, for almost a decade. BCG is assisting us with IND preparation.
  • Ms. Geralyn Annett is the experienced Project Manager. She is the UCD Stem Cell Program Manager and has worked in the field of academic and industry stem cell trials for 20+ years. She will oversee the regulatory team and keep the IND-enabling studies on task to meet the milestones. GMP Facility Director Gerhard Bauer will be responsible for regulatory filings with assistance from Dr. Nolta, the CMC team, and Dr. Miller. Dr. Nolta has worked on clinical trials of stem cell gene therapy, and associated translational studies with Ms. Annett and Director Bauer for over 20 years.
  • B) Clinical. The Clinical team is led by PI Dr. Vicki Wheelock, who is Director of the HDSA Center of Excellence at UC Davis and, with nurse practitioner Terry Tempkin, follows over 250 patients with HD in the UC Davis Movement Disorders clinic. The PI has extensive experience in conducting clinical trials and has already accrued HD patients to 14 clinical trials to date. The planning grant allowed us to conduct longer weekly meetings with different team members to complete planning of the proposed clinical trial.
  • Weekly HD meetings during the planning phase included PI Dr. Wheelock, Co-PI Dr. Nolta, Nurse practitioner Terry Tempkin, Program Manager Geralyn Annett, Psychiatrist Dr. Lorin Scher, Neuropsychologist Dr. Sarah Farias, Social Worker Lisa Kjer, and members of the Imaging Unit led by Dr. Charles DeCarli. This team has worked together on multiple clinical trials for HD patients. Some meetings additionally included Dr. Kiarash Shahlaie, the UCD functional neurosurgeon who will perform the targeting and surgical implantation of the cells, Dr. Bauer who directs the GMP facility (and his team members), the translational team who is performing the IND-enabling studies in Rodents (they usually meet separately for 2 hours/week with Dr. Nolta), and Dr. Tarantal who is leading the IND-enabling studies in non-human primates.
  • We met with our CRO, Paragon, who will be responsible for regulatory and safety filings including outcomes reports, medical and safety monitoring and management including DSMB, medical writing and quality assurance, clinical events committee- adjudicate AEs, and generate clinical study reports. Paragon will also oversee the development of the electronic case report forms, site management and monitoring, biostatistical analysis, and management of the database. We had on-site meetings and conference calls with Paragon during the planning Phase.
  • Additional meetings were conducted with collaborators and consultants:
  • A) Dunbar lab and Hersch lab in the US, both leaders in the HD field – for HD trial IND-enabling study research and HD mouse and patient biomarkers, respectively.
  • B) Aylward lab in the US for detailed brain imaging analyses in HD.
  • C) Paulsen lab for interpretation of cognitive assays in HD.
  • D) Phil Starr and Dan Lim at UCSF for ClearPoint cell injection system.
  • E) Bachoud-Levi lab in France for cell implantation in HD.
  • F) Dr. Robert (Willie) Mays and Bob Deans, Athersys – for IND-enabling studies/regulatory
  • In conclusion, the planning grant helped us to finalize plans for the proposed clinical trial and to complete our detailed plans for the remainder of the IND-enabling studies required to obtain FDA approval. These goals were accomplished through frequent meetings with key consultants and collaborators during the intense planning phase, where we completed the Disease Team application to CIRM that could potentially fund our proposed Phase I clinical trial of MSC/BDNF therapy for Huntington’s disease.
Funding Type: 
Targeted Clinical Development
Grant Number: 
CT1-05168
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$24 846 856
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The proposed project is designed to assess the safety and preliminary activity of escalating doses of human embryonic stem cell (hESC) derived oligodendrocyte progenitor cells for treatment of spinal cord injury. Oligodendrocyte progenitor cells have two important functions: they produce neurotrophic factors which stimulate the survival and growth of neurons (nerve cells) after injury, and they mature in the spinal cord to produce myelin, the insulation which envelops neuronal axons (nerve cell bodies responsible for conduction) and facilitates unimpeded nerve impulse conduction. After extensive efficacy and safety testing, clinical testing of this product was initiated in 2010. Clinical testing is being initiated in paraplegic patients with neurologically complete thoracic injuries (i.e., those in which no motor or sensory function remains below the level of the injury). In the first cohort, a dose equivalent to the lowest efficacious dose observed in preclinical rodent studies is being administered. During the course of the proposed program, clinical safety studies testing increasing doses will be conducted. Upon demonstration of safety, clinical testing will be expanded to tetraplegic patients (complete cervical injuries) and to patients with incomplete thoracic injuries for additional safety testing. In each of the proposed studies, preliminary evidence of activity will be monitored using measures of improved neurological function and performance of daily living activities. The project plan also includes the manufacture of cells to be used in the clinical trials and additional supporting activities. By completion of the proposed project, we expect to have accumulated substantial safety data and preliminary efficacy data in three different patient subpopulations. This data will provide key information to inform the design and execution of advanced efficacy studies.
Statement of Benefit to California: 
The proposed project has the potential to benefit the state of California through 1) providing improved medical outcomes for patients with spinal cord injury and their families, 2) increasing California’s leadership in the emerging field of stem cell research, and 3) preserving and creating high quality, high paying jobs for Californians. Over 12,000 Americans suffer spinal cord injuries each year, and approximately 1.3 million people in the US are estimated to be living with spinal cord injuries. Although specific estimates for the state of California are not available, it is known that the majority of spinal cord injuries result from motor vehicle accidents, falls, acts of violence and recreational sporting activities, all of which are prevalent in California. Spinal cord injury affects not only the patient but family members, friends, healthcare workers and employers. It is estimated that one year after injury, only 11.6% of spinal cord injury patients are employed, and that spinal cord injuries cost $40.5 billion annually in the US. As the most populous state, California is disproportionately affected, negatively impacting our productivity, healthcare system and public finances. There are currently no approved therapies for the treatment of spinal cord injury. The product described in this application has initiated phase 1 clinical testing in patients with complete thoracic spinal cord injury. Even partial correction of any of the debilitating consequences of spinal cord injury could potentially enhance activities of daily living and increase employment while decreasing reliance on attendant care and subsequent medical interventions. California has a history of leadership in biotechnology, and is emerging as a leader in the development of stem cell therapeutics. Cutting edge stem cell research, in many cases funded by CIRM, is already underway in academic research laboratories and biotechnology companies throughout the state. The proposed project has the potential to further increase California’s leadership in the field of stem cell therapeutics through the performance of the first clinical testing of an hESC-derived therapy. The applicant has been located in California since its inception, and currently employs 182 full-time employees at its California headquarters with more than 50% of employees holding an advanced degree. These positions are highly skilled positions, offering competitive salaries and comprehensive benefits. The successful performance of the proposed project would enable significant additional jobs creation in preparation for pivotal trials and product registration.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-02221
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 482 822
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
The goal of this research is to utilize novel research tools to investigate the molecular mechanisms that cause Parkinson’s disease (PD). The proposed work builds on previous funding from CIRM that directed the developed patient derived models of PD. The majority of PD patients suffer from sporadic disease with no clear etiology. However some PD patients harbor specific inherited mutations have been shown to cause PD. The most frequently observed form of genetic parkinsonism is caused by the LRRK2 G2019S mutation it the most common. This mutation accounts for approximately 1.5-2% of patients with apparently sporadic PD, increasing to 4-6% of patients with a family history of PD, and even higher in isolated populations. Importantly, LRRK2 induced PD is clinically and pathologically largely indistinguishable from sporadic PD. This proposal focuses on studying the most frequent cause of familial PD and induces disease that is clinically and pathologically identical to sporadic PD cases. It is likely that LRRK2 regulates a pathway(s) that is important in the more common sporadic form of PD as well. Therefore by employing relevant models of PD, we hope to drive the biological understanding of LRRK2 in a direction that facilitates the development of disease therapeutics in the future. We ascertained patients harboring mutations in LRRK2 [heterozygous (+/G2019S) and homozygous (G2019S/G2019S)] as well as sporadic cases and age matched controls. We have successfully derived iPSCs from each genotype and differentiated these to DA neurons. We will use these as a model system to investigate these LRRK2 based models of PD. We will adapt current biochemical assays of LRRK2, which are source material intensive, to the small culture volumes required for the differentiation of iPSCs to DA neurons. This is a crucial necessity for development for utilizing iPSC derived DA neurons as tractable models of LRRK2 based PD. We will then probe the roles of LRRK2 in neuronal cell differentiation and survival. We will also ask whether the mutant LRRK2 induces changes in autophagy, as this has been postulated as a mechanism of LRRK2 induced pathogenesis. By studying wild-type and disease mutant LRRK2, in DA models of PD we hope to provide crucial understanding of the role mutant LRRK2 has in disease.
Statement of Benefit to California: 
It is estimated that by the year 2030, 75,000-120,000 Californians will be affected by Parkinson’s disease. Currently, there is no cure, early detection mechanism, preventative treatment, or effective way to slow disease progression. The increasing disability caused by the progression of disease burdens the patients, their caregivers as well as society in terms of healthcare costs. The majority of PD patients suffer from sporadic disease with no clear etiology, and a in a handful of these patients specific inherited mutations have been shown to cause PD. The most frequently mutated gene is called Leucine Rich Repeat Kinase 2 (LRRK2). Our goal is to study the mutated gene product in patient based models of Parkinson’s disease. In previous CIRM funding, we have developed patient derived induced pluripotent stem cells (iPSCs) from patients harboring mutations in LRRK2. We have been successful in differentiating populations these iPSCs into the neurons that are depleted in PD. The next step is to utilize these cells as models of mutation induced PD ‘in a dish’. We will employ these pertinent disease models to answer basic biology questions that remain about the function of LRRK2. This project brings together scientists previously funded by CIRM with scientists well versed in the study of LRRK2. This multidisciplinary approach to studying the causes of PD is a natural benefit to the State of California and its citizens. By bringing a better understanding of the role of LRRK2 in the cells that are lost in the progression of PD, we will bring more concrete knowledge of PD as a whole, bringing more hope for the development of a therapeutic for disease.
Progress Report: 
  • The overarching goal of this work is to utilize models of Parkinson's disease (PD) that originate from cells of PD affected patients harboring mutations within the LRRK2 gene so that we may discern the role of mutated LRRK2 in disease. Mutations in LRRK2 are the most common cause of familial PD. The disease presentation of patients with LRRK2 mutation is typically clinically indistinguishable from sporadic PD cases, making the onset of disease due to LRRK2 dysfunction clinically relevant. We have employed stem cells derived from these patients to generate neuronal cells in which we can determine the roles of LRRK2 in the PD mutated and the unmutated state. We have focused on a cellular process called autophagy that regulates the cell response to nutrient deprivation and plays a role in the selective degradation of proteins within the cell.
  • In the first year of funding we have analyzed the expression of the protein LRRK2 in induced pluripotent stem cells, neuronal precursor cells and have begun to differentiate the neuronal precursors to dopaminergic cells of the type lost in PD (a difficult task in itself). We have applied a novel method for detection of LRRK2 in situ by marrying the protein detection of antibodies and the sensitivity of nucleic acid amplification. We will continue to develop this methodology for maximum sensitivity to LRRK2. We have established assays to assess the effects of the LRRK2 mutant on autophagy that are relevant to PD and neurological diseases in general. We have met or made great progress on most of our anticipated milestones and are eager to proceed to the next phase of the project.
  • The overarching goal of this work is to utilize stem cell based models of Parkinson's disease (PD) derived from cells of PD affected patients that harbor mutations in the LRRK2 gene so that we may elucidate the deleterious role of mutated LRRK2 in disease. Mutations in LRRK2 are the most common cause of familial PD. The disease presentation for these patients with LRRK2 mutation is typically clinically similar to those with sporadic disease, making the onset of disease due to LRRK2 dysfunction clinically relevant. We have utilized stem cells harboring a mutation in LRRK2 and also daughter cells of that line in which genomic editing techniques have been applied to correct the PD mutation or disrupt the LRRK2 gene. We have generated the same kind of cells in culture that are lost during PD and hope that next, we can determine how these mutations that eventually cause disease disrupt normal neuronal function. We have made great progress in the understanding the expression of LRRK2 in early differentiation of stem cells to neurons and his will inform our future studies on mutation caused dysfunctions.
  • The overarching goal of this work is to utilize stem cell based models of Parkinson's disease (PD) derived from cells of PD affected patients that harbor mutations in the LRRK2 gene so that we may elucidate the deleterious role of mutated LRRK2 in disease. Mutations in LRRK2 are the most common cause of familial PD. The disease presentation for these patients with LRRK2 mutation is typically clinically similar to those with sporadic disease, making the onset of disease due to LRRK2 dysfunction clinically relevant. We have utilized stem cells harboring a mutation in LRRK2 and also daughter cells of that line in which genomic editing techniques have been applied to correct the PD mutation or disrupt the LRRK2 gene. We have generated the same kind of cells in culture that are lost during PD and hope that next, we can determine how these mutations that eventually cause disease disrupt normal neuronal function. We have made great progress in the understanding the expression of LRRK2 in early differentiation of stem cells to neurons and this will inform our future studies on mutation caused dysfunctions.
  • During our project we achieved several scientific goals. Our project was to utilize Parkinson’s disease patient derived stem cells to model their disease. The particular cells we used were derived from patients with mutations in the LRRK2 gene, which is the most common cause of inherited Parkinson’s disease. At the end of our funding we can report proficiency in induced pluripotent stem cell (iPSC) differentiation to dopaminergic cells protocols in a workflow that allows higher throughput analysis. Dopaminergic cells are types of cells lost in the brain in Parkinson’s disease and if we study cells from patients with this mutation it will likely yield insight into the processes of disease onset and progression. This funding enabled collaboration with the laboratory of Dr. Schuele here at the Parkinson’s Institute to use cells generated in other CIRM funding (ETI-0246 and RT2-019665). The cell lines we used were edited to “fix” the genetic mutation in the LRRK2 gene and also to delete (or knockout) the LRRK2 gene. We used these cells and determined that loss of LRRK2 imparts an increased propensity for dopaminergic differentiation potential for knockouts over mutant and wild-type cells. Also, we determined that LRRK2 inhibitors do not negatively impact the generation of dopaminergic cells in cell culture modeling. We will continue to unravel the roles of LRRK2 in Parkinson’s disease using these pertinent, patient centric models.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-05022
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 755 861
Disease Focus: 
Huntington's Disease
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Over twenty human genetic diseases are caused by expansion of simple DNA sequences composed of repeats of three nucleotides (such as CAG, CTG, CGG and GAA) within essential genes. These repeats can occur within the region of a gene that encodes the protein, generally resulting in proteins with large stretches of repeats of just one amino acid, such as runs of glutamine. These proteins are toxic, cause the death of specific types of brain cells and result in diseases such as Huntington’s disease (HD) and many of the spinocerebellar ataxias (a type of movement disorder). Other repeats can be in regions of genes that do not code for the protein itself, but are copied into messenger RNA, which is a copy of the gene that serves to generate the protein. These RNAs with expanded repeats are also toxic to cells, and sometimes these RNAs sequester essential cellular proteins. One example of this type of disease is Myotonic Dystrophy type 1, a form of muscular dystrophy. Lastly, there are two examples of repeat disorders where the repeats silence the genes harboring these mutations: these are Friedreich’s ataxia (FRDA) and Fragile X syndrome (FXS). One limitation in the development of drugs to treat these diseases is the lack of appropriate cell models that represent the types of cells that are affected in these human diseases. With the advent of the technology to produce induced pluripotent stem cells from patient skin cells, and our ability to turn iPSCs into any cell type, such as neurons (brain cells) that are affected in these triplet repeat diseases, such cellular models are now becoming available. Our laboratories have generated iPSCs from fibroblasts obtained from patients with HD, FXS and FRDA. By comparing cells before and after reprogramming, we found that triplet repeats were expanded in the FRDA iPSCs, but not in HD iPSCs. This application is aimed at the understanding the molecular basis underlying triplet repeat expansion/instability that we have observed during the establishment and propagation of iPSCs from disease-specific fibroblasts. While artificial systems with reporter gene constructs have reproduced triplet repeat expansion in bacteria, yeast and mammalian cells, no cellular models have previously been reported that recapitulate repeat expansions at the endogenous cellular genes involved in these diseases. Therefore, our observations that repeat expansion is found in FRDA iPSCs provides the first opportunity to dissect the mechanisms involved in expansion at the molecular level for the authentic cellular genes in their natural chromatin environment. Repeat expansion is the central basis for these diseases, no matter what the outcome of the expansion (toxic protein or RNA or gene silencing), and a fuller understanding of how repeats expand may lead to new drugs to treat these diseases.
Statement of Benefit to California: 
A major obstacle in the development of new drugs for human diseases is our lack of cell models that represent the tissues or organs that are affected in these diseases. Examples of such diseases are the triplet-repeat neurodegenerative diseases, such as Huntington’s disease, the spinocerebellar ataxias, forms of muscular dystrophy, Fragile X syndrome and Friederich’s ataxia. These diseases, although relatively rare compared to cancer or heart disease, affect thousands of individuals in California. Recent advances in stem cell biology now make it possible to generate cells that reflect the cell types at risk in these diseases (such as brain, heart and muscle cells), starting from patient skin cells. Skin cells can be turned into stem cell-like cells (induced pluripotent stem cells or iPSCs), which can then give rise to just about any cell type in the human body. During the course of our studies, we found that iPSCs derived from Friedreich’s ataxia patient skin cells mimic the behavior of the genetic mutation in this disease. A simple repeat of the DNA sequence GAA is found in the gene encoding an essential protein called frataxin, and this repeat increases in length between generations in human families carrying this mutation. Over a certain threshold, the repeats silence this gene. It is also known that the repeats expand in brain cells in individuals with this disease. With the advent of patient derived iPSCs and neurons, we now have human model systems in which to study the mechanisms responsible for repeat expansion. We have already identified one set of proteins involved in repeat expansion and we now wish to delve more deeply into how the repeats expand. In this way, we may be able to identify new targets for drug development. We will extend our studies to Huntington’s disease and Fragile X syndrome. We have identified two possible therapeutic approaches for Friedreich’s ataxia, and identified molecules that either reactivate the silent gene or block repeat expansion. Our studies in related diseases may provide possible therapeutic strategies for these other disorders as well, which will be of benefit to patients suffering from these diseases, both in California and world-wide.
Progress Report: 
  • Over twenty human genetic diseases are caused by expansion of simple trinucleotide repeat sequences within essential genes, resulting in toxic proteins (as in the polyglutamine expansion diseases, such as Huntington’s disease (HD)), toxic RNAs (as in Myotonic Dystrophy type 1), or gene repression (as in Friedreich’s ataxia (FRDA) and Fragile X syndrome (FXS)). Our laboratories have generated induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with Huntington’s disease (HD), Fragile X syndrome (FXS), Myotonic dystrophy type 1 (DM1) and Friedreich’s ataxia (FRDA). By comparing cells before and after reprogramming, we found that triplet repeats were expanded in the FRDA and DM1 iPSCs, but not in HD iPSCs. During growth of the iPSCs in culture, the repeats continue to expand, suggesting that expansion might be linked to DNA replication in these cells. The expansion we observe in iPSCs does not occur in the fibroblast (skin cells) from which the iPSCs were derived. Similarly, on differentiation of the FRDA iPSCs into neurons (brain cells), repeat expansion stops. This observation suggests that some cellular factors necessary for expansion may be selectively expressed in iPSCs, but not in fibroblasts or neurons.
  • Over the past year, our studies have been aimed at the understanding the molecular basis underlying triplet repeat expansion/instability that we have observed during the establishment and propagation of iPSCs from disease-specific fibroblasts. Previous studies have implicated the mismatch repair (MMR) enzymes in repeat expansion in mouse models for HD and DM1. We find that silencing of the MSH2 gene, encoding one of the subunits of the MMR enzymes, impedes repeat expansion in human FRDA iPSCs. We find that components of the human mismatch repair (MMR) system are associated with the disease alleles in the FRDA and DM1 iPSCs, and that silencing of these genes at the level of their messenger RNAs is sufficient to suppress repeat expansion. Moreover, we have monitored the levels of the MMR enzymes in fibroblasts, iPSCs and neurons, and as expected these enzymes are present at higher amounts in the iPSCs, suggesting that it is the availability of these enzymes in iPSCs that may be responsible for repeat expansion.
  • We wish to determine whether it is the DNA structure of triplet-repeats or protein recognition of the repeats that recruits the MMR enzymes to triplet repeats in iPSCs. To this end, we used a series of small molecule probes that can be designed to target particular DNA sequences in the human genome, and we find that a molecule that targets the GAA-TTC repeats in the FRDA frataxin gene displaces MMR enzymes and prevents repeat expansion. We are currently exploring the mechanism whereby this molecule displaces the MMR enzymes. A deeper understanding of the molecular events that lead to repeat expansion at the endogenous cellular genes responsible for these diseases will likely lead to discoveries of new therapeutic strategies for these currently untreatable disorders.
  • Over the past year, our research efforts have focused on the generality of the results we found in human induced pluripotent stem cells derived from patients with the neurodegenerative disease Friedreich's ataxia (FRDA). FRDA is one of the trinucleotide repeat (TNR) diseases, and our major previous finding was that the GAA•TCC trinucleotide repeats that cause FRDA expand during isolation and propagation of FRDA hiPSCs. This expansion was shown to be dependent on enzymes that are involved in the repair of mismatches in the human genome. To extend these studies, we have now focused on hiPSCs from the related TNR diseases myotonic dystrophy, Huntington's disease and Fragile X syndrome. Myotonic dystrophy type 1 (DM1) is an inherited dominant muscular dystrophy caused by expanded CTG•CAG triplet repeats in the 3’ UTR of the DMPK1 gene, which produces a toxic gain-of-function CUG RNA. It has been shown that the severity of disease symptoms, age of onset and progression are related to the length of the triplet repeats. However, the mechanism(s) of CTG•CAG triplet-repeat instability is not fully understood. Human induced pluripotent stem cells (iPSCs) were generated from DM1 and Huntington’s disease (HD) patient fibroblasts. We isolated 41 iPSC clones from DM1 fibroblasts, all showing different CTG•CAG repeat lengths, thus demonstrating somatic instability within the initial fibroblast population. During propagation of the iPSCs, the repeats expanded in a manner analogous to the intergenerational expansion observed in DM1 patient families. The correlation between repeat length and expansion rate identified the interval between 57 and 126 repeats as being an important length threshold where expansion rates dramatically increased. Moreover, longer repeats showed faster triplet-repeat expansion. The relatively short repeats in the gene responsible for Huntington's disease are below this threshold and hence do not expand in the iPSCs. The overall tendency of triplet repeats to expand ceased on differentiation into differentiated embryoid body or neurospheres. The mismatch repair components MSH2, MSH3 and MSH6 were highly expressed in iPSCs compared to fibroblasts, and only occupied the DMPK1 gene harboring longer CTG•CAG triplet repeats. In addition, shRNA silencing of MSH2 impeded CTG•CAG triplet-repeat expansion. We have also generated hiPSC lines from seven male subjects clinically diagnosed with fragile X syndrome. These hiPSCs have been thoroughly characterized with respect to pluripotency, DNA methylation status at the FMR1 gene, CGG repeat length, FMR1 expression and neuronal differentiation. The information gained from these studies provides new insight into a general mechanism of triplet repeat expansion in iPSCs.
  • Over the past year, our research efforts have focused on the generality of the results we found in human induced pluripotent stem cells derived from patients with the neurodegenerative disease Friedreich's ataxia (FRDA). FRDA is one of the trinucleotide repeat (TNR) diseases, and our major previous finding was that the GAA•TCC trinucleotide repeats that cause FRDA expand during isolation and propagation of FRDA hiPSCs. This expansion was shown to be dependent on enzymes that are involved in the repair of mismatches in the human genome. To extend these studies, we have focused on hiPSCs from the related TNR diseases myotonic dystrophy type 1 (DM1), Huntington's disease (HD), Fragile X syndrome (FXS), and Fuchs endothelial corneal dystrophy (FECD). DM1 is an inherited dominant muscular dystrophy caused by expanded CTG•CAG triplet repeats in the DMPK gene, which produces a toxic gain-of-function CUG RNA. It has been shown that the severity of disease symptoms, age of onset and progression are related to the length of the triplet repeats. However, the mechanism(s) of CTG•CAG triplet-repeat instability is not fully understood. hiPSCs were generated from DM1 and HD patient fibroblasts. Similar to our results in FRDA, DM1 hiPSCs show repeat instability, and repeat expansion is again dependent on the DNA mismatch repair system. We defined a threshold of repeat lengths where repeat expansion occurs. The relatively short repeats in the gene responsible for Huntington's disease are below this threshold and hence do not expand in the iPSCs. We have also generated hiPSC lines from seven male subjects clinically diagnosed with fragile X syndrome. These hiPSCs have been thoroughly characterized with respect to pluripotency, DNA methylation status at the FMR1 gene, CGG repeat length, FMR1 expression and neuronal differentiation. In recent studies, we have turned our attention to the common eye disease FECD, where ~75% or so of Caucassian patients have a CTG•CAG triplet-repeat in an intron of the gene encoding the essential transcription factor TCF4. We find repeat instability in fibroblasts from FECD patient fibroblasts, and repeat expansion in the corresponding hiPSCs. Importantly, similar to DM1 with the same repeat sequence as in FECD, the pathological mechanism in both diseases appear to be similar, namely RNA toxicity caused by sequestering essential messenger RNA processing factors. We have also identified a potential small molecule therapeutic that binds CTG•CAG triplet-repeats and are currently testing this molecule in the relevant patient iPSC-derived cell types. The information gained from these studies provides new insight into a general mechanism of triplet repeat expansion in iPSCs and has revealed a new therapeutic approach for these diseases.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-05229
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 391 400
Disease Focus: 
Autism
Neurological Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Autism spectrum disorders (ASD) are a group of neurodevelopmental diseases that occur in as many as 1 in 150 children in the United States. Three hallmarks of autism are dysfunctional communication, impaired social interaction, and restricted and repetitive interests and activities. Even though no single genetic defect has been ascribed to having a causative role in the majority of ASD cases, twin concordance studies and rare familial forms of the disease strongly support a genetic malfunction and a combinatorial effect of genetic risk factors may contribute to the variability in the symptoms. One major obstacle to ASD research is the difficulty in obtaining human neural tissue to model the disease in vitro. Mouse models of ASD are limited since only rare genetic mutations have been identified so far, and single mutations in those genes cannot fully reproduce the range of critical behaviors characteristic of ASD. Direct reprogramming of patient tissues to induced pluripotent stem (iPS) cells and derivation of forebrain neurons from them will provide much needed insight into the molecular mechanism of neuronal dysfunction in diverse individuals on the autism spectrum. The use of patient-derived stem cells to characterize cellular defects brings together two investigative approaches. One is the identification of common cellular and molecular mechanisms that are central to deficiencies across diverse populations of patients. The other is quantitative comparison of pathological features that address differences amongst diverse patients. Our major goal is to characterize the synaptic dysfunction using concrete, quantifiable parameters in human neurons that have specific mutations in key synaptic proteins. This approach will give us a handle into the molecular synaptic complexes that may also be altered in sporadic ASD cases and could help us develop drug strategies that can normalize synaptic function. Although several groups are interested in generating iPS cells from autistic patients, these efforts generally do not have genomic information on the patients, and the large diversity of mutations associated with autism could lead to large variation in synaptic phenotypes. By focusing on generating iPS cells from patients carrying mutations in a small number of critical synaptic proteins and characterizing the molecular components of this complex, we are likely to be in a strong position to identify novel molecular defects associated with autistic synapses. Relative biochemical comparisons of wildtype and mutant protein complexes could help us find ways to restore synaptic function in ASD.
Statement of Benefit to California: 
Many children in California are affected by autism spectrum disorders, which include monogenic syndromes such as Fragile X syndrome and Rett syndrome. However, the majority of cases are idiopathic and an interplay of multiple genetic risk factors is suspected. Since no current drug therapies exist for autism and an accurate diagnosis can only be made in early childhood by largely behavioral criteria, the cost of care and social burden for such a disorder is high, not to mention the devastation to the quality of life for the families of affected children. We would like to identify a core set of proteins found in synapses that are disrupted or dysregulated in autism by a biochemical approach. If we succeed in this effort, we may be able to identify novel biomarkers and molecular targets for specific patient profiles, and by cross-correlating the genetic background to specific behavioral traits in specific individuals, we may come up with molecular targets that are able to address particular symptoms, which should greatly aid in therapeutic regimens that complement existing behavioral therapies. Generating iPS neurons with known copy number variations associated with autism would be a major resource for other laboratories in California and in the field in general. The economic benefit to California is manifold, as many pharmaceutical and biotech companies in California will want to exploit these novel cell lines and the therapeutic targets identified through them in order to design better drugs for autism.
Progress Report: 
  • The main goal of this project is to establish a cell culture model human neuronal model of autism spectrum disorders (ASD) by generating induced pluripotent stem (iPS) cell lines from patients harboring mutations in genes associated with autism, differentiating them into forebrain neurons, and characterizing their synaptic defects at the cellular and molecular level. We have successfully obtained iPS cells from two autism spectrum disorders, tuberous sclerosis complex (TSC) and Rett syndrome (RTT). We obtained fibroblasts from patients with mutations in the TSC1 and TSC2 genes through the Coriell biorepository. We then reprogrammed them into several TSC patient-specific iPS cell lines. Furthermore, we have obtained male MECP2 mutant iPS cell lines from the lab of Dr. Alysson Muotri to study in parallel with the TSC lines.
  • We differentiated ASD iPS cell lines into neural progenitor cell (NPCs) and have been examining differences in protein levels and signaling pathways in these cells. Pathway analyses from MECP2 mutant NPCs suggest there may be a marked deficit in several major intracellular signaling pathways, and we are validating those deficits by biochemical analyses and genetic manipulations. Both TSC and RTT forebrain neurons show significant differences in synaptic regulation compared to their respective controls. Alterations in synaptic regulation are being assessed by gene expression analysis, staining for synaptic markers, and electrophysiology. We have made major progress toward realizing our goal of establishing novel iPS cell models for ASD. Furthermore, we obtained very interesting data that should help us elucidate the cell signaling deficits that lead to neuronal dysfunction.
  • We set out to establish an in vitro human neuronal model of autism spectrum disorders (ASD) by generating induced pluripotent stem (iPS) cell lines from patients harboring specific genetic mutations in syndromic forms of autism, such as Rett Syndrome (RTT) and Tuberous sclerosis (TS). We then differentiated them into neural progenitor cells (NPCs) and forebrain neurons, in order to compare their differentiation potential and to characterize mutation-associated deficits at the cellular and molecular level. Previously published data on cellular and animal models indicate that synaptic deficits are a major feature of the pathophysiology of RTT and TS.
  • We employed patient-derived induced pluripotent stem cells (iPSCs) from male RTT patients and gender-matched parental controls to probe for functional and molecular deficits in RTT. A similar approach was taken for TS.
  • As MECP2 is expressed in both the developing and mature central nervous system, we investigated deficits that may arise during early developmental stages (i.e. at the neural progenitor cell or NPC stage), which could then significantly affect neurodevelopmental processes such as neurogenesis and gliogenesis. By quantitative proteomics, we showed that the RTT cells have changes on the molecular level, at both the NPC and neuron stage, compared to their WT control, and that these changes may reflect some of the deficits in the developmental process. We report delays in maturation, such as misregulation of LIN28 at the NPC stage and subsequent deficits in glial differentiation.
  • Taken together, these results provide a framework for identifying novel early pathways that are perturbed in RTT, as well as potential therapeutics to minimize functional deficits. More generally, it will be of interest to see if these pathways and possible therapeutics may carry over to other related forms of neurodevelopmental disorders, in particular, idiopathic autism.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-02143
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 355 063
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
Public Abstract: 
A major goal of stem cell research is to generate various functional human cell types that can be used to better understand how these cells work and to use them directly in therapies. There are currently no effective treatments, let alone a cure, for many neurological conditions. Two particular devastating neurological conditions, spinal cord injury and amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease) share a common element. That is, in both conditions, the corticospinal motor neurons that control skilled voluntary movement are severely damaged, leading to significant loss of motor control. There has been extensive research on spinal cord injury and ALS in recent years. In the field of spinal cord injury, much effort has been devoted to repairing the damaged nerve paths, but this has turned out to be extremely challenging. The work on ALS, on the other hand, has mostly focused on the spinal motor neurons (often referred to as the lower motor neurons in the context of ALS). Our proposed study focuses on the corticospinal motor neurons (or the upper motor neurons) and, more broadly, the subcerebral projection neurons. Taking clues from studies in mice, we aim to understand how the subcerebral projection neurons including the corticospinal motor neurons can be made from human embryonic stem cells. We will focus on the later steps in differentiation that are not well understood, which gave rise to different types of neurons in the cerebral cortex. To aid in this process, we have engineered a fluorescent reporter in human embryonic stem cells, which, when the stem cells are turned into corticospinal motor neurons and related subcerebral projection neurons, will light up – literally. We will probe the molecular control of this process and determine if corticospinal motor neurons made in a culture dish, when introduced back into an organism, can send projections to the spinal cord, as they would normally do during development. Most of our knowledge about the development of corticospinal motor neurons comes from studies with mouse models. As there are likely to be important differences between humans and mice, we will pay special attention to the similarities and differences between mouse and human corticospinal motor neurons. Knowledge gained from this study will pave the way to make better disease-models-in-a-dish for neurological conditions such as ALS and to develop therapies for ALS, spinal cord injury, traumatic brain injury, stroke and other neurological conditions when corticospinal motor neurons are damaged.
Statement of Benefit to California: 
Neurological conditions affect millions of Californians each year. Spinal cord injury is one particularly debilitating neurological condition. The disability, loss of earning power, and loss of personal freedom associated with spinal cord injury is devastating for the injured individual, and creates a financial burden of an estimated $400 million annually for the state of California. Research is the only solution as currently there is no cure for spinal cord injury. A major functional deficit for patients of spinal cord injury is the loss of motor control. Corticospinal motor neurons mediate skilled, voluntary movement in humans and damage to these neurons leads to severe disability. Our proposed study focuses on the understanding of how corticospinal motor neurons and, more broadly, subcerebral projection neurons can be made from human embryonic stem cells under culture conditions, and how they can be introduced back to central nervous system. Understanding this process will allow scientists to design ways to use these cells for transplantation therapies not only for spinal cord injury, but also for other neurological conditions such as amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease). Effective treatments promoting functional repair will significantly increase personal independence for people with spinal cord injury and decrease the financial burden for the State of California. More importantly, treatments that enhance functional recovery will improve the quality of life for those who are directly or indirectly affected by spinal cord injury, ALS and other neurological conditions.
Progress Report: 
  • A major goal of stem cell research is to generate various functional human cell types to promote repair or replacement in injury or disease. Our lab studies the repair of central nervous system after injury such as a spinal cord injury. We have been utilizing a fluorescent reporter line we developed with CIRM funding to enrich and characterize human corticospinal motor neurons, a neuronal population that is damaged or lost in spinal cord injury and amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease). These neurons control skilled voluntary movement in humans, the loss or damage of which leads to paralysis and disability. We have made significant progress in this funding period. We validated that our fluorescent reporter works as intended. We found that reporter gene expression represents cells of different developmental stages at different times of differentiation. We have done the first batches of transplantation studies to show that it is possible to use the reporter gene to track the cells and cellular processes in the host central nervous system. In addition, we have developed a separate reporter gene to universally mark all embryonic stem-derived cells, a tool that may be useful to other stem cell researchers. We are now ready to move to the next phase of the project: to characterize corticospinal motor neurons in more detail in vitro and in vivo. Knowledge gained from this study will pave the way to make better disease-models-in-a-dish for neurological conditions such as ALS and to develop therapies for ALS, spinal cord injury, traumatic brain injury, stroke and other neurological conditions when corticospinal motor neurons are damaged of lost.
  • A major goal of stem cell research is to generate various functional human cell types to promote repair or replacement in injury or disease. Our lab studies the repair of central nervous system after injury such as a spinal cord injury. We have been utilizing a fluorescent reporter line we developed with CIRM funding to derive and characterize human corticospinal motor neurons, a neuronal population that is damaged or lost in spinal cord injury and amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease). These neurons are of paramount importance to skilled voluntary movement in humans, the loss or damage of which leads to paralysis and disability. The goal for making a reporter line is that whenever the cells light up (literally), we will know what they have become the type of cells that we would wish to get. Following last year’s initial progress, we have made significant progress in this funding period. We found that our fluorescent reporter is useful in following the desired cell types throughout cell growth in culture dishes or after we introduce these cells into animal models by transplantation. We have performed experiments to validate the identity and usefulness of these cells. In culture, these cells exhibit the desired signature gene expression pattern, electrophysiological properties and morphologies as well. We will continue to improve our culture condition to maximize efficiency and purity. Meanwhile, we have transplanted these cells into the mouse brain to study them in the complex central nervous system because many of the properties cannot be studied in cell culture such as the connection of nerve cells to other brain area or spinal cord. We were excited to find that these cells, once transplanted, can survive, integrate into the mouse central nervous system, and send out long neuronal processes characteristic of endogenous nerve cells. Some of the projections appear to take the path of the projections of the corticospinal motor neurons, indicating that our approach will likely succeed. Thanks to CIRM’s support, we will continue to investigate the various parameters to improve our transplantation studies. Knowledge gained from this study will pave the way to make better disease-models-in-a-dish for neurological conditions such as ALS and to develop therapies for ALS, spinal cord injury, traumatic brain injury, stroke and other neurological conditions when corticospinal motor neurons are damaged of lost.
  • A major goal of stem cell research is to generate various functional human cell types from stem cells both for developing cell transplantation therapies and for better understanding human biology. Our lab studies the repair of central nervous system after injury and in particular spinal cord injury. To complement our studies of the molecular control of axon regeneration using animal models of spinal cord injury, we have been developing ways to derive human corticospinal motor neurons from human embryonic stem cells through this CIRM funded project. These neurons are of paramount importance to skilled voluntary movement in humans, loss or damage of which leads to paralysis and disability in patients of spinal cord injury and amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease). We took advantage of a reporter line we developed with a prior CIRM SEED grant to generate human corticospinal motor neurons. This reporter line carries a fluorescent reporter gene under the control of an endogenous gene encoding a molecular marker and determinant of corticospinal motor neurons, Fezf2. The idea was that whenever the cells carrying the reporter gene lights up – literally, we would know the cells are expressing Fezf2. Using this approach, we have learned quite a bit about human cells that express Fezf2. First, there are a large population of human neural stem cells that express Fezf2 early in neural differentiation, which is likely mirrored in human development. Fezf2 positive neural stem cells can become Fezf2 positive neurons, but they can also become Fezf2 negative neurons. On the contrary, Fezf2 negative stem/progenitor cells do not become Fezf2 positive neurons. During neural differentiation in culture starting from human embryonic stem cells, Fezf2 expression is dynamic. Earlier Fezf2-expressing neural stem cells have different properties from the late Fezf2-expressing neural stem cells in that they have different capabilities to turning into differential neuronal types. Particular in this last year of funding, we conducted in-depth characterization of the molecular signature of Fezf2 positive and negative neural stem/progenitor cells, as well as neurons that had been derived from these progenitors, at different times in differentiation. Hierarchical cluster analysis not only provided new insights on the different cell populations in the differentiation culture over time but also on the different molecular markers based on studies in mice. We have also extended our in vivo transplantation studies to determine how well these neural progenitor cells may survive and integrate into the mouse nervous system. The data indicate that neural progenitors that express the fluorescence reporter can survive, integrate into the host nervous system and send out extensive axonal trajectories. Some axons grew along the appropriate paths expected for corticospinal and related subcerebral projection neurons while others appear to wonder off the course. These data indicate that one challenge in future research will be to elucidate mechanisms of control the re-connection of transplanted stem cell derivatives with appropriate host targets when cell transplantation therapies are used to replace lost or damaged neurons in disease or injury.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-05219
Investigator: 
ICOC Funds Committed: 
$1 372 660
Disease Focus: 
Infectious Disease
Neurological Disorders
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Human cytomegalovirus (HCMV) is the major cause of birth defects, almost all of which are neuronal in origin. Approximately 1% of newborns are infected, and of the 13% that are symptomatic at birth, 50% will have severe permanent hearing deficits, vision loss, motor impairment, and mental retardation. At least 14% of asymptomatic infants also will later show disabilities. Much of this effect is likely caused by HCMV affecting neural development in the fetus. Embryonic stem cells are an excellent source of human progenitors, which are cells that can turn into mature neurons i.e. neural differentiation. We know from published cell culture studies that HCMV affects neural progenitor cells during neural differentiation, but it is unclear as to what are the underlying molecular mechanisms for its effect. A major goal of our research is to understand at a high-resolution how HCMV controls the way neural progenitors become proper neurons. Elucidation of the genes that are affected will serve as a basis for therapeutic strategies to ameliorate the effects of HCMV infection in newborns. The significance of our studies also extends to the serious problem of HCMV infection in immunocompromised individuals, with recipients of allogeneic transplants having a high risk of severe disease and allograft rejection. This potential problem in stem cell therapy has received little attention thus far. The proposed use of stem cell transplantation in treating neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.
Statement of Benefit to California: 
Human cytomegalovirus (HCMV) is the major viral cause of birth defects. In 2009, there were 526,774 births in California, resulting in congenital HCMV infection in approximately 5,200 newborns, with at least 800 infants expected to have long-lasting disabilities. Congenital cytomegalovirus infection is the most common nongenetic congenital cause of deafness. In contrast, before the development of the rubella vaccine, less than 70 infants per year in the entire US were reported to have congenital rubella syndrome, also associated with deafness. The burden to families and the economic costs to society of congenital cytomegalovirus infection are immense, and there is no vaccine available. Our proposed research serves to form the basis of future therapies to ameliorate, or even reduce this medical burden. The significance of our studies also extends to the serious problem of HCMV infection in immunocompromised individuals who receive transplants of organs and stem cells from other individuals. Infection in these transplant recipients often results in severe disease and rejection of the transplant. The California Institute for Regenerative Medicine has made a major commitment to provide funding to move stem cell-based therapies to clinical trials. The goal of using stem cell transplantation to treat neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells. Our research will provide the knowledge base to understand the genes that are changed during HCMV infection of human neural progenitors and neurons. It will also provide a foundation for studies of how other viruses will affect human neurons, and likely, other cell-types. Intellectual property from this work will feed into opportunities for antiviral strategies and increased jobs in biotech for Californians.
Progress Report: 
  • Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments in the newborn. It is likely that the timing of infection as well as the range of susceptible cells at the time of infection will affect the severity of the disease. A major goal of our research is to understand at a high-resolution the effects of HCMV infection on the neural lineage specification and maturation of stem and progenitor cells. Elucidation of the genes and cellular processes that are affected will serve as a basis for therapeutic strategies to ameliorate the effects of HCMV infection in newborns. The significance of our studies also extends to the serious problem of HCMV infection in immunocompromised individuals, with recipients of allogeneic transplants having a high risk of severe disease and allograft rejection. This potential problem in stem cell therapy has received little attention thus far. The proposed use of stem cell transplantation in treating neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.
  • This past year, we have made significant progress in accomplishing the goals of this project. We used human embryonic stem cells-derived primitive pre-rosette neural stem cells (pNSCs) maintained in chemically defined conditions to study host-HCMV interactions in early neural development. Infection of pNSCs with HCMV was largely inefficient and non-progressive. At low multiplicity of infection (MOI), we observed severe defects with regards to the proportion of cells expressing the major immediate-early proteins (IE) despite an optimal viral entry, thus indicating the existence of a blockade to specific pre-IE events. IE expression, even at high MOI, was found to be restricted to a subset of cells negative for the expression of the forebrain marker FORSE-1. Treatment of pNSCs with the caudalizing agent retinoic acid rescued IE expression, suggesting that the hindbrain microenvironment might be more permissive for the infection. Transactivation of the viral early genes was found to be severely debilitated and expression of the late genes was barely detectable even at high MOI. Differentiation of pNSCs into primitive neural progenitor cells (pNPCs) restored IE expression but not the transactivation of early and late genes. Increasing the number of viral particles bypassed this barrier to early gene expression and thus permitted expression of the late genes in pNPCs. Consequently, viral spread was only observed at high MOI but was largely restricted to one cycle of replication as secondarily infected cells failed to efficiently express early genes. Of note, virions produced in pNPCs and pNSCs were exclusively cell-associated. Finally, we found that viral genomes could persist in pNSCs culture up to a month after infection despite the absence of detectable IE expression by immunofluorescence. Clonogenic expansion of infected pNSCs revealed that the presence of viral DNA and IE proteins were insufficient to block host cell division therefore allowing the survival of viral genomes via cellular division rather than viral replication. These results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus. To study in greater depth the molecular basis of the interaction of HCMV with cell of the neural lineage, we also have initiated high-throughput genomics approaches to analyze HCMV microRNAs, alterations in cellular microRNA and gene expression profiles, and global defects in host alternative splicing in infected and uninfected pNSC-derived NPCs. Interestingly, although there are many changes in host cell gene expression in the infected cells, there was only a small overlap with the set of changes we had found in infected human fibroblasts. This highlights the importance of performing these studies in the relevant targets of the virus in the developing fetus.
  • We expect that the results of these studies will provide an unprecedented resolution of the effects on neurogenesis when HCMV infects a newborn, serve as a foundation for future therapeutic efforts in preventing the birth defects due to HCMV, and provide insight into the serious potential problem of disseminated HCMV in immunosuppressed individuals receiving transplanted allogeneic stem cells.
  • Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments in the newborn. A major goal of our research is to understand at a high-resolution the effects of HCMV infection on the neural lineage specification and maturation of stem and progenitor cells. Elucidation of the genes and cellular processes that are affected will serve as a basis for therapeutic strategies to ameliorate the effects of HCMV infection in newborns. The significance of our studies also extends to the serious problem of HCMV infection in immunocompromised individuals, with recipients of allogeneic transplants having a high risk of severe disease and allograft rejection. This potential problem in stem cell therapy has received little attention thus far. The proposed use of stem cell transplantation in treating neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.
  • This past year, we have made significant progress in accomplishing the goals of this project. We used human embryonic stem cell (hESC)-derived primitive pre-rosette neural stem cells (pNSCs) to study host-HCMV interactions in early neural development and found with several different lines of hESC-derived pNSCs that HCMV infection is inefficient and non-progressive. Differentiation of pNSCs into primitive neural progenitor cells (pNPCs) restored some viral early gene expression but not the transactivation of late genes. Impaired viral gene expression in pNSCs was not a result of inefficient viral entry or nuclear import of viral DNA but correlated with deficient nuclear import of the virion-associated protein UL82, which is believed to play a role in removing barriers to viral RNA synthesis. Additionally, we found that viral genomes could persist in pNSCs culture up to a month after infection despite the absence of detectable viral lytic gene expression, although we could also detect expression of viral latency-associated genes, suggesting that the virus becomes latent in pNSCs.
  • To study in greater depth the molecular basis of the interaction of HCMV with cells of the neural lineage, we have continued high-throughput genomics approaches to analyze HCMV microRNAs, alterations in cellular microRNA and gene expression profiles, and global defects in host alternative splicing in infected and uninfected pNSC-derived NPCs. We found that in infected NPCs, there was specific downregulation of transcripts related to neuron differentiation. These findings demonstrate the capacity of HCMV infection to alter the neural identities of key precursor cells in the developing nervous system. We also analyzed our infected NPC RNA-seq database for differences in host mRNA polyadenylation patterns and found that over a hundred transcripts were significantly altered in terms of their 3' end cleavage site preference, with the majority of these events resulting in shortened 3' UTRs.
  • Our finding that HCMV induces major changes in the transcriptome of NPCs, particularly at the level of neural genes, suggested that the virus might affect these cells functionally. To directly evaluate this, we differentiated pNSCs into midbrain dopaminergic (mDA) neurons and infected these cells at different times of the differentiation process. Seeding pNSCs in differentiation medium for 6 weeks yields a high frequency of mature neurons with long axonal projections. Infection of pNSCs at the start of differentiation greatly reduces generation of beta III-tubulin+ neurons 4 weeks later, and prevents differentiation to mature MAP2+ neurons. Infection after 1 week of differentiation also reduces the number of beta III-tubulin+ neurons and results in massive cell death. Infection at 2 weeks after differentiation start does not reduce the number of βIII-tubulin+ or MAP2+ neurons, but the cells display major anomalies. Since neurons are highly sensitive to oxidative stresses and HCMV infection increases the production of reactive oxygen species in fibroblasts, we investigated whether the same effect occurred in neuronal cultures. When pNSCs were infected at week 4 after differentiation, high levels of ROS were detected. These results suggest that the complex effects of HCMV infection at various stages of neural cell differentiation on both cell survival and maturation may account for the broad range of birth defects.
  • We expect that the results of these studies will provide an unprecedented resolution of the effects on neurogenesis when HCMV infects a newborn, serve as a foundation for future therapeutic efforts in preventing the birth defects due to HCMV, and provide insight into the serious potential problem of disseminated HCMV in immunosuppressed individuals receiving transplanted allogeneic stem cells.
  • Congenital and childhood sensorineural hearing loss (SNHL) is a multifactorial disease that severely impacts quality of life. The single most important etiology of congenital SNHL is prenatal human cytomegalovirus (HCMV) infection, which accounts for 20-30% of all deafness in infants and children. Approximately 1 in 150 children is born with congenital HCMV, and 1 in 5 of these children will be born with or will develop permanent neural disabilities, the most common of which is SNHL. SNHL can be either bilateral or unilateral, and the severity of the hearing loss and its progression varies widely. Although the association of congenital HCMV infection and SNHL has been recognized for 50 years, how infection induces the hearing loss is unknown. Since the target of HCMV is cells of the neural lineage, a major goal of our research is to understand at high-resolution the effects of HCMV infection on neural lineage specification and maturation of stem and progenitor cells. Elucidation of the genes and cellular processes that are affected will serve as a basis for therapeutic strategies to ameliorate the effects of HCMV infection in newborns. The significance of our studies also extends to the problem of HCMV infection in immunocompromised individuals, with recipients of allogeneic transplants having a high risk of severe disease and allograft rejection. The proposed use of stem cell transplantation in treating neuronal injury and neurodegenerative diseases, as well as transplantation of other organ-specific precursors, makes it imperative to understand how disseminated HCMV infection in immunosuppressed recipients will affect the function and differentiation of the cells.
  • This past year, we made significant progress in accomplishing the goals of this project. To study in greater depth the molecular basis of the interaction of HCMV with cells of the neural lineage, we continued high-throughput genomics approaches to analyze HCMV microRNAs, alterations in cellular microRNA and gene expression profiles, and global defects in host alternative splicing in infected and uninfected pNSC-derived NPCs. We also analyzed our infected NPC RNA-seq database for differences in host mRNA polyadenylation patterns and found that over a hundred transcripts were significantly altered in terms of their 3' end cleavage site preference, with the majority of these events resulting in shortened 3' UTRs.
  • One of our most striking findings was that there was strongly enhanced expression of three miRNAs that play a critical role in auditory development. Other genes involved in cochlear development were also dysregulated by infection. These results implicate a novel molecular mechanism of damage to the developing inner ear of congenitally infected children, based on altered developmental gene regulation. These findings coupled with our success in inducing differentiation of human ES into otic progenitors that can be further differentiated to hair cell-like cells and immature auditory neurons have provided a strong foundation to pursue in depth HCMV infection of auditory progenitor cells, with the goal of determining how infection compromises the gene expression and function of human ES-derived inner ear cells and to use this information to develop new strategies for the treatment and prevention of hearing loss in congenitally infected children.
  • Regrettably, we are not able to continue these highly important studies, as this CIRM grant ended and a new proposal to CIRM was not funded. This was very disappointing because there is a gap in the portfolio of grants awarded by CIRM, which is a lack of attention to important problems relating to Child Health. One of the most important areas in stem cells that have been neglected is congenital sensorineural hearing loss. Only 3 grants relating to hearing loss have ever been awarded by CIRM. Yet the incidence of just congenital hearing loss (not including hearing loss from other causes or associated with aging) is 400 in 100,000 newborns. Moreover, the disability will last for life, causing a significant burden to the patient and family and a high economic cost to society. There is no drug or vaccine to prevent congenital hearing loss due to CMV, and thus it is essential develop new therapeutic strategies, including stem cells, gene targeting, and drug discovery. Unfortunately, there is no accepted mechanism by which HCMV infection leads to hearing loss, and a lack of public awareness regarding the serious medical problems resulting from congenital HCMV Infection has made it difficult to garner support for studies to identify an etiology.
  • We appreciate the support CIRM has provided and hope that in the future there will be sufficient public awareness of the devastating effects of congenital HCMV infection to bring pressure upon funding agencies to recognize and support this important research.
Funding Type: 
Basic Biology III
Grant Number: 
RB3-02129
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$1 382 400
Disease Focus: 
Autism
Neurological Disorders
Rett's Syndrome
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Stem cells, including human embryonic stem cells, provide extraordinary new opportunities to model human diseases and may serve as platforms for drug screening and validation. Especially with the ever-improving effective and safe methodologies to produce genetically identical human induced pluripotent stem cells (iPSCs), increasing number of patient-specific iPSCs will be generated, which will enormously facilitate the disease modeling process. Also given the advancement in human genetics in defining human genetic mutations for various disorders, it is becoming possible that one can quickly start with discovery of disease-related genetic mutations to produce patient-specific iPSCs, which can then be differentiated into the right cell type to model for the disease in vitro, followed by setting up the drug screening paradigms using such disease highly relevant cells. In the context of neurological disorders, both synaptic transmission and gene expression can be combined for phenotyping and phenotypic reversal screening and in vitro functional (synaptic transmission) reversal validation. The missing gap for starting with the genetic mutation to pave the way to drug discovery and development is in vivo validation-related preclinical studies. In order to fill this gap, in this application we are proposing to use Rett syndrome as a proof of principle, to establish human cell xenografting paradigm and perform optogenetics and in vivo recording or functional MRI (fMRI), to study the neurotransmission/connectivity characteristics of normal and diseased human neurons. Our approach will be applicable to many other human neurological disease models and will allow for a combination of pharmacokinetic, and in vivo toxicology work together with the in vivo disease phenotypic reversal studies, bridging the gap between cell culture based disease modeling and drug screening to in vivo validation of drug candidates to complete the cycle of preclinical studies, paving the way to clinical trials. A success of this proposed study will have enormous implications to complete the path of using human pluripotent stem cells to build novel paradigms for a complete drug development process.
Statement of Benefit to California: 
Rett Syndrome (RTT) is a progressive neurodevelopmental disorder caused by primarily loss-of-function mutations in the X-linked MeCP2 gene. It mainly affects females with an incidence of about 1 in 10,000 births. After up to 18 months of apparently normal development, children with RTT develop severe neurological symptoms including motor defects, mental retardation, autistic traits, seizures and anxiety. RTT is one of the Autism Spectrum Disorders (ASDs) that affects many children in California. In this application, we propose to use our hESC-based Rett syndrome (RTT) model as a proof-of-principle case to define a set of core transcriptome that can be used for drug screenings. Human embryonic stem cells (hESCs) hold great potential for cell replacement therapy where cells are lost due to disease or injury. For the diseases of the central nervous system, hESC-derived neurons could be used for repair. This approach requires careful characterization of hESCs prior to utilizing their therapeutic potentials. Unfortunately, most of the characterization of hESCs are performed in vitro when disease models are generated using hESC-derived neurons. In this application, using RTT as a proof of principle study, we will bridge the gap and perform in vivo characterization of transplanted normal and RTT human neurons. Our findings will not only benefit RTT and other ASD patients, but also subsequently enable broad applications of this approach in drug discovery using human pluripotent stem cell-based disease models to benefit the citizens of California in a broader spectrum.
Progress Report: 
  • The potential of stem cells, such as human embryonic stem cells and induced pluripotent stem cells (iPSCs), has been widely recognized for cell replacement therapy, modeling human diseases and serving as a platform for drug screening and validation. In this grant, we proposed to use Rett syndrome as a proof of principle, to establish a human cell xenografting paradigm (i.e., transplanting human cells into mouse/rat embryos) and perform in vivo analyses to study the neurotransmission characteristics of normal and diseased human neurons. We initially determined that it was feasible to use the lentiviral CamKII-ChR2 construct to drive excitatory neuronal-specific expression of ChR2 in mouse hippocampal pyramidal neurons as well as human embryonic stem cell derived neurons. Importantly, we have found that both ChR2 expressing mouse hippocampal neurons and human neurons derived from embryonic stem cells can spike action potentials when stimulated in vitro, indicating that exogenously expressed ChR2 is functional. Furthermore, we successfully transplanted human embryonic stem cell derived neural stem/progenitor cells into fetal rat forebrain at embryonic day 17. Our analysis of the recipient animals at postnatal day 21 showed that approximately 40-50% of the cells survived and began to express neuronal markers, such as NeuN, indicating the neuronal differentiation, as well as the long-term survival, of transplanted human cells in the recipient animals. As originally proposed, we will proceed with the documentation of the in vivo phenotype of Rett syndrome diseased neurons. Our approach will be particularly crucial to not only validate candidate drugs or other therapeutic interventions to treat Rett syndrome using xeno-transplanted human Rett neurons, but also to study the in vivo behavior of those neurons with and without the therapeutic intervention.
  • Stem cells, such as human embryonic stem cells and induced pluripotent stem cells (iPSCs), carry great potentials for cell replacement therapy, human diseases modeling and drug screenings. We proposed to use Rett syndrome (RTT) as a proof of principle, to establish a human cell xenografting paradigm (i.e., transplanting human cells into mouse/rat brains) to study the function of normal and diseased human neurons in vivo. During the 2nd year of funding, we gained new insights into the electrophysiological characteristics of RTT neurons. Specifically, we found that the neurotransmission phenotype of neurons derived from RTT patient-specific iPSCs was highly circuitry-dependent. On the other hand, when cell-intrinsic electrophysiological properties were measured, extremely stable abnormalities in action potential profiles, resting membrane potentials, etc. were observed, indicative of the validity of the culture system. Given that currently scientists have very limited control over the features of neuronal connections formed in culture conditions, our findings make the in vivo assessment of RTT neuronal properties even more desirable, because the circuitry features are more amenable in vivo, with anatomical cues. In light of aforementioned in vitro findings, we focused our attention to both cell-intrinsic electrophysiological characteristics of RTT neurons, as well as their connectivity or neural network properties, after neurons were integrated into host circuits in vivo following xenotransplantation. Our preliminary data demonstrated that the action-potential abnormalities of RTT neurons are preserved in vivo after xenotransplantation. So far we have established a relatively optimized system for studying human iPSC-derived RTT neurons integrated into mouse brains. We are poised to uncover not only the neuronal intrinsic electrophysiological properties but also the connectivity of wild type and RTT neurons with host circuits. Moreover, we have made substantial progress with regards to a novel technology, i.e., single neuron gene expression profiling coupled with electrophysiological recordings both in vitro and in vivo. Up to now, 8 RTT iPSC-derived neurons were profiled via RNA sequencing following electrophysiological recordings, and some interesting clues have already been revealed. Currently we are collecting more neurons and we expect to make unprecedented discoveries with mechanistic insights into RTT disease pathophysiology, which will facilitate the development of novel therapies for RTT. This paradigm is also generally applicable for studying other neurological disorders.
  • Over the last decade, the importance of the stem cells for cell replacement therapy, human disease modeling and drug toxicity/therapy screenings has been greatly appreciated by both the general public and the scientific community. In our application utilizing human embryonic stem cells and induced pluripotent stem cells (iPSCs), we proposed to use Rett syndrome (RTT) as a proof of principle to establish a human cell xenografting paradigm (i.e., transplanting human cells into mouse/rat brains) to study the function of normal and diseased human neurons in vivo. While we increased our knowledge about the electrophysiological characteristics of RTT neurons during Year 2 funding, we mainly focused on the transplantation of the normal and diseased cells, as well as the molecular signatures of transplanted cells at a single cell level, during Year 3 of the funding period. Following our initial transplantation experiments, we observed clustering of the transplanted cells at the injection site, even though there were number of cells integrating into the host brains. In order to circumvent this problem and answer our original questions, we developed an alternative approach. Specifically, we adopted the “transparent brain” methodology to better visualize the integration and the projections of the transplanted cells, as well as the circuitries that they participate, in the host environment to reveal the connectivity of wild type and RTT neurons with the host circuits. With this method, we’re able to follow the transplanted RTT neurons at a higher resolution -without the limitations of the conventional approaches- for studying human iPSC-derived RTT neurons integrated into mouse brains. As part of our last Specific Aim, we’ve performed single neuron gene expression profiling coupled with electrophysiological recordings both in vitro and in vivo. Specifically, we implemented electrophysiological recordings from the transplanted RTT iPSC-derived neurons and isolated the genomic material from the same cell to perform transcriptome analyses. We collected significant amount of data from RNA sequencing experiments and have been performing relevant bioinformatic analyses. In order to complete the gene expression profiling analysis, we obtained a no-cost-extension of the project, and upon completion of the no-cost-extension period, the relevant report will be filed outlining the outcomes of the single neuron transcriptome analysis coupled with electrophysiology. Collectively, our findings provide mechanistic insights into RTT disease pathophysiology, which will facilitate the development of novel therapies for RTT. Lastly, our approach is applicable for studying other neurological disorders in addition to RTT.

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