Neurological Disorders

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

Cellular Reprogramming: Dissecting the Molecular Mechanism and Enhancing Efficiency

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01628
ICOC Funds Committed: 
$1 458 000
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Pluripotent stem cells have a remarkable potential to develop into virtually any cell type of the body, making them a powerful tool for the study or direct treatment of human disease. Recent demonstration that induced pluripotent stem (iPS) cells may be derived from differentiated adult cells offers unprecedented opportunities for basic biology research, regenerative medicine, disease modeling, drug discovery and toxicology. For example, using patient-derived iPS cells, one can model diseases in vitro and screen for drugs in ways never before possible, enabling the identification of promising new therapeutic candidates earlier in the drug discovery process. In addition, iPS cell derivatives represent an ideal source for autologous cell replacement therapies, as they would not be rejected upon transplantation back into the patient. While it is clear that iPS cells hold great promise for finding therapies for diseases, there are significant hurdles that need to be overcome before full clinical potential is realized. The mechanism of iPS cell derivation is largely elusive, and the process used to generate them is very inefficient and needs to be improved in significant ways. Currently, iPS cells are generated by forced expression of four molecular factors using genome-integrating viruses. This may lead to mutations and altered differentiation potential of iPS cells, as well as tumorogenesis if transplanted back into the patient. The inefficient and stochastic nature of the reprogramming process indicates that additional, as yet unidentified mechanisms may exist and contribute to iPS cell generation. Finally, increasing the efficiency of current iPS cell derivation protocols will increase the ability to generate large panels of patient-specific iPS cell lines. We propose to use a human cell-based assay to identify small molecules that can enhance the efficiency of iPS cell generation. Our strategy will allow us to identify small molecules that target events essential for derivation of iPS cells, as well as those that replace one or more of the four virally-delivered factors. We will use the identified small molecules to discover regulatory pathways and molecular targets involved in induction of pluripotency, gaining valuable insight into the mechanism of cellular reprogramming. Application of these small molecules themselves, as well as novel approaches derived from mechanism of action studies, will help overcome issues associated with viral integration and has the potential to transform personalized cell-replacement therapies as well as accelerate drug discovery based on iPS cell-derived disease models.
Statement of Benefit to California: 
California’s health care system faces significant challenges as millions of children and adults suffer from a host of incurable illnesses. It is expected that health care costs will continue to rise as California’s citizenry ages and requires treatments for age-related, chronic metabolic, cardiovascular, and neurodegenerative disorders. Both the measureable economic impact on California’s health care system and the incalculable emotional suffering of affected individuals, their families and communities, make it an imperative to develop novel therapeutic treatments to address these mounting medical and economic societal challenges. Recognizing the potential utility of novel stem cell technologies to address California’s unmet medical needs, California voters approved Proposition 71 which created the California Institute for Regenerative Medicine (CIRM), an agency that administers funds to support stem cell research that has the greatest potential for development of novel regenerative medical treatments and cures. The CIRM Basic Biology Awards II program is intended to fund studies that will lay the foundation for future stem cell-based translational and clinical advances. In keeping with this mission, our proposed research program aims to discover new methods for producing human induced pluripotent stem cells (iPS cells) on an industrial scale and in an efficient manner; and to develop a better understanding of the mechanisms underlying cellular reprogramming. As such, our research program will help accelerate the realization of the full potential of iPS cells in cell-based regenerative medicine therapies and drug discovery. Our proposed research program will benefit the State of California and its citizens in several ways. Firstly, our research program will lay the foundation for future stem cell-based clinical and translational advances to treat and manage one of California’s most pressing unmet medical needs. Secondly, execution of our research program will create new jobs in the academic, biotechnology and pharmaceutical sectors throughout California. Funding from CIRM will be expanded with additional funding from the applicant to augment achievement of the aims of this project. CIRM funding will leverage other sources of investment in this project to help ensure California’s continued future as a world leader in biomedical innovation and translational medicine for the benefit of human health. Lastly, our proposed research program will stimulate California’s economy by creating new enabling tools and technologies that can be broadly adopted across the life science industry, thus promoting development across the academic institutions and biopharmaceutical companies that create biomedical discoveries and advances. These activities will continue to strengthen California’s leadership position at the forefront of the stem cell and regenerative medical revolution of the 21st century.
Progress Report: 
  • iPS cell lines display high variability with respect to their growth properties, differentiation ability and disease phenotype manifestation; this is a major challenge for both in vitro disease modeling and drug screening, as well as cell replacement therapy. The cause for this variability is currently unknown and heterogeneity of the starting fibroblast population, composition of reprogramming factors, and viral integration into the genome have all been proposed to contribute to variability among different iPSC clones derived from the same starting population. In particular, the use of DNA viral vectors to deliver the reprogramming factors was suggested to be the key obstacle for eventual use of iPSCs in cell replacement therapies. Recently, several methods have been developed to derive integration-free iPS cells, but they mostly suffer from unacceptably low reprogramming efficiency or labor-intensive delivery of reprogramming factors.
  • The goal of this work is to produce integration-free iPS cells with high efficiency. To date, we developed a high-throughput platform to screen small molecules that enhance reprogramming in the presence of 3 transcription factors required for reprogramming of fibroblast cells - KLF4, SOX2, and OCT4. These efforts included assay development, characterization of our engineered stable cell lines expressing reprogramming factors under control of an inducible promoter, and optimization for high-throughput screening. We screened over 100,000 compounds from our small molecule library, comprising compounds with broad chemical diversity and covering multiple cellular target classes. We identified 130 hit candidates in our primary screen, however, in follow-up assays none of these compounds showed an effect comparable with that of a positive control compound. Moving forward, we plan on detailed characterization of other integration-free reprogramming methods to determine the differences between integrating vs. non-integrating methods for reprogramming fibroblasts. We will assess the quality and variability of iPS cell lines, as well as their potential and variability of differentiation and disease phenotype manifestation in integration-free iPSCs, and compare them to DNA virus-derived iPSCs. Our results should contribute to the understanding of the source of variability between iPS cells and bring us closer to reaching the ultimate goal: production of integration-free human iPS cells with high efficiency.
  • The use of stem cell technology to study neurodegenerative diseases has been a burgeoning area of research in recent years. Recent work by us and others have demonstrated that stem cell derived cortical neurons (CN) from Alzheimer's disease patients demonstrate key disease differentials when compared to CN’s derived from healthy controls. However, most of this work has been limited to the use of simple cultures of iPSC-derived cortical or motor neurons, or in some cases, co-culture of iPSC-derived neurons with primary human astrocytes. We have demonstrated that co-culturing of iPSC-derived neurons with primary fetal astrocytes facilitates the maturation of neurons (as assessed by electrophysiology) beyond what is observed in neuronal cultures alone. We have developed an astrocyte differentiation protocol from iPSC that allows for the generation of cells with gene expression, glutamate uptake capacity and kinetics, and neurotropic factor secretion consistent with primary fetal astrocytes. Importantly, these iPSC astrocytes can be directed to an anterior versus posterior (brain versus spinal cord) identity. The goal of this study is to compare the ability of iPSC-derived astrocytes to primary fetal astrocytes in their ability to facilitate the maturation of iPSC-derived neurons, in order to identify the appropriate co-culture conditions where iPSC-derived neurons reach functional maturity as assessed by neuronal markers and electrophysiological activity. Achievement of this goal would allow for further assessment of the contribution of neurons and astrocytes to specific disease processes in neurodegeneration and neuroinflammation through the ability to combine in co-culture either disease versus healthy iPSC-derived astrocytes and/or neurons to further elucidate the underlying mechanisms contributing to these disease phenotypes. These studies should facilitate the development of co-culture systems which drive neuronal development to a more mature phenotype, and improve the availability of stem cell-based model systems to better replicate in vivo physiological and pathological processes.
  • The use of stem cell technology to study neurodegenerative diseases has been a burgeoning area of research in recent years. Recent work by us and others have demonstrated that stem cell derived cortical neurons (CN) from Alzheimer's disease patients demonstrate key disease differentials when compared to CN's derived from healthy controls. However, most of this work has been limited to the use of simple cultures of iPSC-derived cortical or motor neurons, or in some cases, co-culture of iPSC-derived neurons with primary human astrocytes. We have demonstrated that co-culturing of iPSC-derived neurons with primary fetal astrocytes facilitates the maturation of neurons (as assessed by electrophysiology) beyond what is observed in neuronal cultures alone. We have developed an astrocyte differentiation protocol from iPSC that allows for the generation of cells with gene expression, glutumate uptake capacity and kinetics, and neurotropic factor secretion consistent with primary fetal astrocytes. The goal of this study is to compare the ability of iPSC-derived astrocytes to primary fetal astrocytes in their ability to facilitate the maturation of iPSC-derived neurons. Achievement of this goal would allow for further assessment of the contribution of neurons and astrocytes to specific diseases processes in neurodegeneration and neuroinflammation through the ability to combine co-culture either in disease vs. healthy iPSC-derived astrocytes and/or neurons to further elucidate the underlying mechanisms contributing to these disease phenotypes. These studies should facilitate the development of co-culture systems which drive neuronal development to a more mature phenotype, and improve the availability of stem-cell based model systems.

Role of the microenvironment in human iPS and NSC fate and tumorigenesis

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01496
ICOC Funds Committed: 
$1 284 921
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Collaborative Funder: 
Japan
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Multipotent Neural Stem Cells (NSC) can be derived from adult central nervous system (CNS) tissue, embryonic stem cells (ESC), or iPSC and provide a partially committed cell population that has not exhibited evidence of tumorigenesis after long term CNS transplantation. Transplantation of NSC from these different sources has been shown by multiple investigators in different CNS injury and disease paradigms to promote recovery or ameliorate disease. Additionally, both {REDACTED} groups have shown that human NSCs transplanted in the subacute period after spinal cord injury promote functional recovery. While the role of the host immune response has been considered in the context of immune-rejection, predominantly regarding the T-cell response, the consequence of an ongoing inflammatory response within the context of the tissue microenvironment for cell fate, migration, and integration/efficacy has been largely overlooked. Critically, the tumorigeneis, fate, migration, and integration/repair potential of a stem cell is driven by: 1) the intrinsic properties of cell programming, e.g., the type and source of cell / means used to derive the cell, and maintenance/differentiation of the cell in vitro; and 2) the extrinsic factors the cell encounters. Variations in the intrinsic properties of the cell may affect the potential of that cell for uncontrolled proliferation or the response of the cell to extrinsic factors that it later encounters, defining its fate, migration, and integration/repair potential. The {REDACTED} group has demonstrated that iPS-derived neurospheres (iPS-NS) exhibit a surprisingly large degree of variation in tumorigenesis potential after CNS transplantation, which is correlated with tissue source as well as differentiation and NS forming capacity. Moreover, the intrinsic properties of hNSC populations derived from different cell sources have not been broadly characterized; in fact, {REDACTED} has published the first data in the field demonstrating the differences in fate and integration/repair potential between primary and secondary neurospheres generated via in vitro differentiation of mouse or human ESC and iPSC. In parallel, {REDACTED} has shown profound differences in the response of NSC derived from human tissue versus hESC to extrinsic signals. Together, these data suggest that both characterization of the intrinsic properties of NSCs derived from different sources is essential for our understanding of the basic biology of these cells. Investigation of molecules and signaling pathways directing hNSC fate choices in the injured CNS microenvironment will yield new insight into the mechanisms of fate and migration decisions in these cell populations.
Statement of Benefit to California: 
Multipotent Neural Stem Cells (NSC) can be derived from adult central nervous system (CNS) tissue, embryonic stem cells (ESC), or induced pluripotent cells (iPSC) and provide a partially committed cell population that has not exhibited evidence of tumorigenesis after long term CNS transplantation. Transplantation of NSC from these different sources has been shown by multiple investigators in different CNS injury and disease paradigms to promote recovery or ameliorate disease. Accordingly, stem cell based therapeutics such as these have the potential to treat a variety of traumatic, congenital, and acquired human conditions. However, while much progress has been made, translational research with human stem cell populations will remain limited by the progress of the fundamental understanding of the basic biology of these cells. The {REDACTED} group has pioneered understanding the critical role of timing in considering cell transplantation therapies. More recently, this group has focused on the neural induction of mouse- and human-derived iPSC and tested the potential of these cell populations for spinal cord injury treatment in animal models. {REDACTED} has established the NOD-scid mouse as a model for experimental neurotransplantation for xenograft studies, characterizing the relationship between transplant timing, engraftment outcome, cell fate, host remyelination, and functional recovery. Recently, this group has focused on how the innate inflammatory response influences cell fate and migration. In this collaborative proposal, researchers from California and Japan propose to combine their expertise to characterize and investigate some of the most fundamental aspects of the intrinsic properties of, and extrinsic factors influencing, human induced pluripotent (hiPSC) and human embryonic (hESC) stem cells, pooling knowledge and expertise in stem cell and animal model paradigms. The experiments proposed investigate the basic cellular and molecular mechanisms underlying the role of the host environment in stem cell fate regulation, and the relationship between reprogramming and tumorigenic/fate potential of hiPS-NSC in vitro and after transplantation, and key to this collaborative effort, the interface of these two aspects of basic stem cell biology. Critically, this international collaboration combines the expertise of two of the most advanced laboratories in translational stem cell biology to address several key unresolved questions in the control of cell fate, and will promote sharing of resources, data, and techniques between these labs to advance the field. Ultimately, the collaborative work proposed may permit the development of strategies to refine cellular reprogramming techniques, alter in vitro differentiation strategies, or manipulate the microenvironment to maximize the window for potential stem cell-based neurotherapeutics.
Progress Report: 
  • Multipotent Neural Stem Cells (NSC) can be derived from adult and fetal central nervous system (CNS) tissue, embryonic stem cells (ESC), or iPSC and provide a partially committed cell population that has not exhibited evidence of tumorigenesis after long term CNS transplantation. Transplantation of NSC from these different sources has been shown by multiple investigators in different CNS injury and disease paradigms to promote recovery or ameliorate disease. Additionally, both Dr. Okano and Dr. Anderson’s groups have shown that human NSCs transplanted in the subacute period after spinal cord injury promote functional recovery. While the role of the host immune response has been considered in the context of immune-rejection, predominantly regarding the T-cell response, the consequence of an ongoing inflammatory response within the context of the tissue microenvironment for cell fate, migration, and integration/efficacy has been largely overlooked. Critically, the tumorigeneis, fate, migration, and integration/repair potential of a stem cell is driven by: 1) the intrinsic properties of cell programming, e.g., the type and source of cell / means used to derive the cell, and maintenance/differentiation of the cell in vitro; and 2) the extrinsic factors the cell encounters. Variations in the intrinsic properties of the cell may affect the potential of that cell for uncontrolled proliferation or the response of the cell to extrinsic factors that it later encounters, defining its fate, migration, and integration/repair potential. The Nakamura/Okano group has demonstrated that iPS-derived neurospheres (iPS-NS) exhibit a surprisingly large degree of variation in tumorigenesis potential after CNS transplantation, which is correlated with tissue source as well as differentiation and NS forming capacity. Moreover, the intrinsic properties of hNSC populations derived from different cell sources have not been broadly characterized; in fact, Dr. Okano’s group has published the first data in the field demonstrating the differences in fate and integration/repair potential between primary and secondary neurospheres generated via in vitro differentiation of mouse or human ESC and iPSC. In parallel, Dr. Anderson’s group has shown profound differences in the response of NSC derived from human fetal tissue versus hESC to extrinsic signals. Together, these data suggest that both characterization of the intrinsic properties of NSCs derived from different sources is essential for our understanding of the basic biology of these cells. Investigation of molecules and signaling pathways directing hNSC fate choices in the injured CNS microenvironment will yield new insight into the mechanisms of fate and migration decisions in these cell populations.
  • Progress has been excellent in the first year, as has communication between the groups.
  • The Nakamura/Okada/Okano laboratory has regularly shared and updated us on these important findings and the progress of Aim 1 at Keio University via emails, live phone conferences and face-to-face meetings. The latest meeting occurred at the International Stem Cell Meeting in Toronto (ISSCR, June 2011), where safety and efficacy data of the initial screenings of numerous hiPS cell lines are shared and discussed which will have a significant impact on which cell lines we will work with under Aims 2 and 3.
  • Additionally, the Anderson laboratory took the additional step of focusing on xeno-free cells for this grant, with the goal of advancing future knowledge of utility for clinical translation based on CIRM funding. Xeno-free cells are cells that are cultured under conditions in which they are not exposed to animal proteins. Towards this goal, we have successfully transitioned multiple ESC and iPSC lines to xeno-free conditions for both maintenance, and successfully differentiated these lines to a neural stem cell lineage under parallel conditions. Moreover, by taking this step we have significantly enhanced the comparability of different cell lines for intrinsic properties and extrinsic influences, enhancing the potential impact of this work in increasing our basic understanding of stem cell biology, and how to harness it. Finally, we have conducted the first of our experiments testing the role of cell intrinsic properties in defining responses to the in vitro and in vivo microenvironment. Our data suggest that there are clear differences in intrinsic properties between cell lines, consistent with our initial hypothesis.
  • Although the role of the host immune response has been considered in the context of immune-rejection, predominantly regarding the T-cell response, the consequence of an ongoing inflammatory response within the context of the tissue microenvironment for cell fate, migration, and integration/efficacy has been largely overlooked. While classical immunosuppressants alter the T-cell response, these drugs have minimal impact on other immune cells such as neutrophils (polymorphonuclear (PMN) leukocytes) and macrophages (MACs)/microglia, which makes up a significant part of the host environment after traumatic injuries to the CNS, such as spinal cord injury (SCI). Accordingly, there is little known about the basic biology of either the host microenvironment or inflammatory microenvironment in influencing and interacting with either endogenous or transplanted stem cell populations. Understanding the molecules and signaling pathways directing hNSC fate choices in the injured CNS microenvironment is critical. hNSC derived from hiPS-NSC and hESC will be tested. We have therefore established and characterized hiPS-NSC and hES-NSC derived from multiple origins and tested the specific role of innate inflammatory cells (i.e. PMNs and macrophages) and molecules in cell fate, migration and proliferation of these hiPS-NSC and hES-NSC lines in vitro. Thus far, these data have revealed clear cell line specific intrinsic differences in response to inflammatory factors, which we will further investigated in the coming funding period both in vitro and in vivo.
  • The fate, migration, and repair potential of a stem cell is driven by a combination of intrinsic properties, such as the type, source, and maintenance/differentiation of the cell in vitro, as well as extrinsic factors the cell encounters in the in vivo environment, such as proteins related to inflammation or the growth matrix. Variations in the intrinsic properties of the cell may affect the potential of that cell for uncontrolled proliferation or the response of the cell to extrinsic factors that it encounters in its environment. We have previously shown that neural stem cells derived from human fetal tissue are highly sensitive to extrinsic inflammatory signals in vitro and in vivo. In the current studies, we sought to determine whether neural stem cell populations derived from different sources respond to the same sorts of inflammatory signals, in other words, whether these extrinsic factors affect stem cells as a general principal. Accordingly, we sought to characterize the intrinsic properties of neural stem cells derived from different sources and exposed to extrinsic inflammatory signals, including human embryonic and induced pluripotent cell, as an essential component of understanding of basic stem cell biology. We found that, in fact, all neural stem cells derived from embryonic and induced pluripotent populations responded to inflammatory signals. However, we also found that cell line intrinsic properties exert a strong degree of control, in some cases resulting in opposing consequences for cell proliferation and fate. Critically, we found that in vitro characteristics of response to extrinsic inflammatory signals were predictive for the way different cell populations behaved in vivo after transplantation. These data may offer a new opportunity to screen stem cell populations in vitro for comparability and predicted in vivo translational properties, and reveal a new and critical set of interactions between intrinsic cell programming and response to the environment.

Sustained siRNA production from human MSC to treat Huntingtons Disease and other neurodegenerative disorders

Funding Type: 
Early Translational I
Grant Number: 
TR1-01257
ICOC Funds Committed: 
$2 753 559
Disease Focus: 
Huntington's Disease
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Closed
Public Abstract: 
One in every ten thousand people in the USA have 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. HD is a challenging disease to treat. Not only do the affected, dying neurons need to be salvaged or replaced, but also the levels of the toxic mutant protein must be diminished to prevent further neural damage and to halt progression of the movement disorders and physical and mental decline that is associated with HD. Our application is focused on developing a safe and effective therapeutic strategy to reduce levels of the harmful mutant protein in damaged or at-risk neurons. We are using an RNA interference strategy – “small interfering RNA (siRNA)” to prevent the mutant protein from being produced in the cell. This strategy has been shown to be highly effective in animal models of HD. However, the inability to deliver the therapeutic molecules into the human brain in a robust and durable manner has thwarted scale-up of this potentially curative therapy into human trials. We are using mesenchymal stem cells, the “paramedics of the body”, to deliver the therapeutic siRNA directly into damaged cells. We have discovered that these stem cells 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 a new tool to also reduce mutant protein levels. Our novel system will allow the therapy to be carefully tested in preparation for future human cellular therapy trials for HD. The significance of our studies is very high because there are currently no treatments to diminish the amount of toxic mutant htt protein in the neurons of patients affected by Huntington’s Disease. There are no cures or successful clinical trials for HD. Our therapeutic strategy is initially examining models to treat HD, since the need is so acute. But this biological delivery system could also be used, in the future, for other neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia (SCA1), Alzheimer's Disease, and some forms of Parkinson's Disease, where reduction of the levels of a mutant or disease-activating protein could be curative. Development of this novel stem cell therapeutic and effective siRNA delivery system is extremely important for the community of HD and neurodegenerative disease researchers, patients, and families.
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 Huntington’s Disease is estimated to be in the billions, the emotional burden on the friends and families of HD patients is immeasurable. Health care costs are extremely high for HD patients due to the decline in both body and mind. The lost ability of HD patients to remain in the CA workforce and to support their families 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. Since there are currently no cures or successful clinical trials for HD, many are reluctant to be tested. The proposed project is designed in an effort to reach out to these individuals who, given that HD is given an orphan disease designation, may feel that they are completely forgotten and thus have little or no hope for their future or that of their families. To combat this devastating disease, we are using an RNA interference strategy, “small interfering RNA (siRNA),” to prevent the mutant htt protein from being produced in the cell. This strategy has been shown to be highly effective in animal models of HD. However the siRNA needs to be delivered to the brain or central nervous system in a continual manner, to destroy the toxic gene products as they are produced. There are currently no methods to infuse or produce siRNA in the brain, in a safe and sustained manner. Therefore the practical clinical use of this dramatically effective potential therapeutic application is currently thwarted. Here we propose a solution, using adult mesenchymal stem cells (MSC) modified to infuse siRNA directly into diseased or at-risk neurons in the striata of HD patients, to decrease the levels of the toxic mutant htt protein. MSC are known as the “paramedics of the body" and have been demonstrated through clinical trials to be safe and to have curative effects on damaged tissue. Even without the modification to reduce the mutant protein levels, the infused MSC will help repair the damaged brain tissue by promoting endogenous neuronal growth through secreted growth factors, secreting anti-apoptotic factors, and regulating inflammation. Our therapeutic strategy will initially examine models to treat HD, since the need is so acute. But our biological delivery system could also be applied to other neurodegenerative disorders such as ALS, some forms of Parkinson’s Disease, and Alzheimer’s Disease, by using siRNA to interfere with key pathways in development of the pathology. This would be the first cellular therapy for HD patients and would have a major impact on those affected in California. In addition, the methods that we are developing will have far-reaching effects for other neurodegenerative disorders.
Progress Report: 
  • During the first year of funding we have made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders.
  • The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. There is mounting evidence in trinucleotide repeat disorders that the RNA, as well as the protein, is toxic and thus we will need to significantly reduce levels of both in order to have a durable impact on this devastating disease.
  • This year we have shown that human MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neurons in vitro to achieve significant reduction in levels of the htt protein. This is a significant achievement and a primary goal of our proposed studies, and demonstrates that the hypothesis for our proposed studies is valid. The transfer occurs through direct cell-to-cell transfer of siRNA, and we have filed an international patent for this process, working closely with our Innovation Access Program at UC Davis. A manuscript documenting the results of these studies is in preparation.
  • We continue to explore the precise methods by which the cell-to-cell transfer of small RNA molecules occurs, working in close collaboration with the national Center for Biophotonics Science and Technology at UC Davis. This Center is located across the street from our CIRM-funded Institute for Regenerative Cures (IRC) where our laboratory is located, and has equipment that allows visualization of protein-protein interactions in high clarity and detail. The proximity of our HD team researchers in the IRC to the Center for Biophotonics has been an important asset to our project and a collaborative manuscript is in preparation.
  • During year two of the proposed studies we will continue to document levels of reduction of the toxic htt protein in different types of neurons, including medium spiny neurons (MSN) derived from HD patient induced pluripotent stem cells (iPSC). We have made significant advances in developing the tools for these studies, including HD iPSC line generation and MSN maturation from human pluripotent cells in culture. A manuscript on improved techniques for generating MSN from pluripotent cells is in preparation. We have also worked closely with our colleagues at the UC Davis MIND Institute to achieve improved maturation and electrical activity in neurons derived from human pluripotent stem cells in vitro, and we are examining the impact of human MSC on enhancing survival of damaged human neurons.
  • In the second year of funding we will test efficacy of the siRNA-mediated knockdown of the mutant human htt RNA and protein in the brains of our newly developed strain of immune deficient Huntington's disease mice. This strain was developed by our teams at UC Davis to allow testing of human cells in the mice, since the current strains of HD mice will reject human stem cells. A manuscript describing generation of this novel HD mouse strain is in preparation, in collaboration with our nationally prominent Center for Mouse Biology.
  • Behavioral studies will be conducted in this strain with and without the MSC/siRNA-mediated knockdown of the mutant protein, through years 2-3, in collaboration with our well established mouse neurobehavioral core at the UC Davis Center for Neurosciences. We have documented the safety of intrastriatal injection of human MSC in immune deficient mice and will next test the efficacy of human MSC engineered to continually produce the siRNA to knock down the mutant htt protein in vivo.
  • As added leverage for this grant program, and supported entirely by philanthropic donations from the community committed to curing HD, we have performed IND-enabling studies in support of an initial planned clinical trial that will use normal donor MSC (non-engineered) to validate their significant neurotrophic effects in the brain. These trophic effects have been documented in animal models. The planned study will be a phase 1 safety trial. We have completed the clinical protocol design and have received feedback from the Food and Drug Administration. We will be conducting additional studies in response to their queries, over the next 6-10 months, through a pilot grant obtained from our Clinical Translational Science Center (CTSC), which is located in the same building as our Institute. Upon completion of these additional studies we will submit the updated IND application to the FDA. MSCs for this project have been expanded and banked using standard operating procedures in place in the Good Manufacturing Practice Facility in the CIRM/UC Davis Institute for Regenerative Cures.
  • From the funded studies 4 manuscripts are now in preparation, a chapter is in press and a review paper on MSC to treat neurodegenerative diseases is in press.
  • During the second year of funding we have made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders.
  • The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. During the second year we have more fully characterized our development candidate; MSC/anti-htt. We have documented that normal human donor MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neurons in vitro to achieve significant reduction in levels of the htt protein. We reported this work at the Annual meeting of the American Academy of Neurology (G Mitchell, S Olson, K Pollock, A Kambal, W Cary, K Pepper, S Kalomoiris, and J Nolta. Mesenchymal Stem Cells as a Delivery Vehicle for Intercellular Delivery of RNAi to Treat Huntington's disease. AAN IN10-1.010, 2011) and have recently completed and submitted a manuscript describing these results (S Olson, A Kambal, K Pollock, G Mitchell, H Stewart, S Kalomoiris, W Cary, C Nacey, K Pepper, J Nolta. Mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin. Submitted, In Review, July 2011).
  • We have explored the molecular methods by which the cell-to-cell transfer of small RNA molecules occurs, working in close collaboration with the national Center for Biophotonics Science and Technology at UC Davis. This Center is located across the street from our CIRM-funded Institute for Regenerative Cures (IRC) where our laboratory is located, and has equipment that allows visualization of protein-siRNA interactions in high clarity and detail. The proximity of our HD team researchers in the IRC to the Center for Biophotonics has been an important asset to our project. This work was also presented at AAN 2011, and a collaborative manuscript is in preparation for submission (S Olson, G McNerny, K Pollock, F Chuang, T Huser and J Nolta, Visualization of siRNA Complexed to RISC Machinery: Demonstrating Intercellular siRNA Transfer by Imaging Activity. MS in preparation, Presented at AAN 2011: IN4-1.014).
  • In the second year of funding we developed the models for in vivo efficacy testing of the siRNA-mediated knockdown of the mutant human htt RNA and protein in the brains of established and new strains of Huntington's disease mice. Behavioral studies were conducted in two strains, the R6/2 immune competent mice and our new immune deficient strain, the NSG/HD, in comparison to normal littermate controls that are not affected by HD. We established the batteries of behavioral tests that are now needed to test efficacy of our development candidate in the brain, in year three. Established tests include rotarod, treadscan, pawgrip, spontaneous activity, nesting, locomotor activity, and the characteristic HD mouse hindlimb clasping phenotype. In addition we monitor the status of weight and tremor, grooming, eyes, hair, body position, and tail position, which all change over time in HD mice. These tests are conducted at 48 hour intervals by two highly trained technicians who are blinded to the treatment that the mouse had received. These behavioral and phenotypic tests have been established at the level of Good laboratory practices in our new Institute for Regenerative Cures shower-in barrier facility vivarium. We have documented the biosafety of intrastriatal injection of human MSC in immune deficient mice and are now examining the in vivo efficacy of the development candidate: human MSC engineered to continually produce the siRNA to knock down the mutant htt protein in vivo, which will be completed in year three.
  • As added leverage for this funded grant program, and supported entirely by philanthropic donations from the community committed to curing HD, we have performed IND-enabling studies in support of an initial planned clinical trial that will use normal donor MSC (non-engineered) to validate their significant neurotrophic effects in the brain. These trophic effects have been documented in animal models. The planned study will be a phase 1 safety trial. We have completed the clinical protocol design and have received feedback from the Food and Drug Administration. We will be conducting additional studies in response to their queries, over the next 6-10 months, through a pilot grant obtained from our Clinical Translational Science Center (CTSC), which is located in the same building as our Institute. Upon completion of these additional studies we will submit the updated IND application to the FDA. MSCs for this project have been expanded and banked using standard operating procedures in place in the Good Manufacturing Practice Facility in the CIRM/UC Davis Institute for Regenerative Cures.
  • During the three years of funding we made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders.
  • The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. There is mounting evidence in trinucleotide repeat disorders that the RNA, as well as the protein, is toxic and thus we will need to significantly reduce levels of both in order to have a durable impact on this devastating disease.
  • We initially demonstrated that human MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neuronal cells in vitro to achieve significant reduction in levels of the htt protein. This is a significant achievement and a primary goal of our proposed studies, and demonstrates that the hypothesis for our proposed studies is valid. The transfer occurs either through direct cell-to-cell transfer of siRNA or through exosome transfer, and we filed an international patent for this process, working closely with our Innovation Access Program at UC Davis. This patent has IP sharing with CIRM.
  • An NIH transformative grant was awarded to Dr. Nolta to further explore these exciting findings. This provides funding for five years to further define and optimize the siRNA transfer mechanism.
  • A manuscript documenting the results of these studies was published:
  • S Olson, A Kambal, K Pollock, G Mitchell, H Stewart, S Kalomoiris, W Cary, C Nacey, K Pepper, J Nolta. Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin. Molecular and Cellular Neuroscience; 49(3):271-81, 2012.
  • Also a review was published with our collaborator Dr. Gary Dunbar:
  • S Olson, K Pollock, A Kambal, W Cary, G Mitchell, J Tempkin, H Stewart, J McGee, G Bauer, T Tempkin, V Wheelock, G Annett, G Dunbar and J Nolta, Genetically Engineered Mesenchymal Stem Cells as a Proposed Therapeutic for Huntington’s disease. Molecular Neurobiology; 45(1):87-98, 2012.
  • We examined the potential efficacy of injecting relatively small numbers of MSCs engineered to produce ant-htt siRNA into the striata of the HD mouse strain R6/2, in three series of experiments. Results of these experiments did not reach significance for the test agent as compared to controls. The slope of the decline in rotarod performance was less with the test agent, and development of clasping behavior was slightly delayed after injection of MSC/aHtt, but this caught up to the controls and was not significant after day 60.
  • Our conclusions are that the R6/2 strain is too rapidly progressing to see efficacy with the test agent, and also that improved methods of siRNA transfer from cell to cell are needed. We are currently working on this problem through the NIH transformative award, and will use the YAC 128 strain, which has a more slowly progressing phenotype, for all future studies. These mice are now bred and in use in our vivarium, for the MSC/BDNF studies funded through our disease team grant.
  • Through this translational grant funding we have also developed in vitro potency assays, using human embryonic stem cell-derived neurons and medium spiny neurons, as we have described in prior reports. The differentiation techniques (funded through other grants to our group) have now been published:1-3
  • 1. Liu J, Githinji J, McLaughlin B, Wilczek K, Nolta J. Role of miRNAs in Neuronal Differentiation from Human Embryonic Stem Cell-Derived Neural Stem Cells. Stem Cell Rev;8(4):1129-37, 2012.
  • 2. Jun-feng Feng, Jing Liu, Xiu-zhen Zhang, Lei Zhang, Ji-yao Jiang, Nolta J, Min Zhao. Guided Migration of Neural Stem Cells Derived from Human Embryonic Stem Cells by an Electric Field. Stem Cells. Feb; 30(2):349-55, 2012.
  • 3. Liu J, Koscielska KA, Cao Z, Hulsizer S, Grace N, Mitchell G, Nacey C, Githinji J, McGee J, Garcia-Arocena D, Hagerman RJ, Nolta J, Pessah I, Hagerman PJ. Signaling defects in iPSC-derived fragile X premutation neurons. Hum Mol Genet. 21(17):3795-805. 2012.

Enhancing healing via Wnt-protein mediated activation of endogenous stem cells

Funding Type: 
Early Translational I
Grant Number: 
TR1-01249
ICOC Funds Committed: 
$6 263 086
Disease Focus: 
Bone or Cartilage Disease
Stroke
Neurological Disorders
Heart Disease
Neurological Disorders
Skin Disease
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
All adult tissues contain stem cells. Some tissues, like bone marrow and skin, harbor more adult stem cells; other tissues, like muscle, have fewer. When a tissue or organ is injured these stem cells possess a remarkable ability to divide and multiply. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to self-renew and to proliferate, which in turn hinders tissue regeneration. Our strategy is to commandeer the molecular machinery responsible for adult stem cell self-renewal and proliferation and by doing so, stimulate the endogenous program of tissue regeneration. This approach takes advantage of the solution that Nature itself developed for repairing damaged or diseased tissues, and controls adult stem cell proliferation in a localized, highly controlled fashion. This strategy circumvents the immunological, medical, and ethical hurdles that exist when exogenous stem cells are introduced into a human. When utilizing this strategy the goal of reaching clinical trials in human patients within 5 years becomes realistic. Specifically, we will target the growing problem of neurologic, musculoskeletal, cardiovascular, and wound healing diseases by local delivery of a protein that promotes the body’s inherent ability to repair and regenerate tissues. We have evidence that this class of proteins, when delivered locally to an injury site, is able to stimulate adult tissue stem cells to grow and repair/replace the deficient tissue following injury. We have developed technologies to package the protein in a specialized manner that preserves its biological activity but simultaneously restricts its diffusion to unintended regions of the body. For example, when we treat a skeletal injury with this packaged protein we augment the natural ability to heal bone by 350%; and when this protein is delivered to the heart immediately after an infarction cardiac output is improved and complications related to scarring are reduced. This remarkable capacity to augment tissue healing is not limited to bones and the heart: the same powerful effect can be elicited in the brain, and skin injuries. The disease targets of stroke, bone fractures, heart attacks, and skin wounds and ulcers represent an enormous health care burden now, but this burden is expected to skyrocket because our population is quickly aging. Thus, our proposal addresses a present and ongoing challenge to healthcare for the majority of Californians, with a novel therapeutic strategy that mimics the body’s inherent repair mechanisms.
Statement of Benefit to California: 
Californians represent 1 in 7 Americans, and make up the single largest healthcare market in the United States. The diseases and injuries that affect Californians affect the rest of the US, and the world. For example, stroke is the third leading cause of death, with more than 700,000 people affected every year. It is a leading cause of serious long-term disability, with an estimated 5.4 million stroke survivors currently alive today. Symptoms of musculoskeletal disease are the number two most cited reasons for visit to a physician. Musculoskeletal disease is the leading cause of work-related and physical disability in the United States, with arthritis being the leading chronic condition reported by the elderly. In adults over the age of 70, 40% suffer from osteoarthritis of the knee and of these nearly 80% have limitation of movement. By 2030, nearly 67 million US adults will be diagnosed with arthritis. Cardiovascular disease is the leading cause of death, and is a major cause of disability worldwide. The annual socioeconomic burden posed by cardiovascular disease is estimated to exceed $400 billion annually and remains a major cause of health disparities and rising health care costs. Skin wounds from burns, trauma, or surgery, and chronic wounds associated with diabetes or pressure ulcer, exact a staggering toll on our healthcare system: Burns alone affect 1.25M Americans each year, and the economic global burden of these injuries approaches $50B/yr. In California alone, the annual healthcare expenditures for stroke, skeletal repair, heart attacks, and skin wound healing are staggering and exceed 700,000 cases, 3.5M hospital days, and $34B. We have developed a novel, protein-based therapeutic platform to accelerate and enhance tissue regeneration through activation of adult stem cells. This technology takes advantage of a powerful stem cell factor that is essential for the development and repair of most of the body’s tissues. We have generated the first stable, biologically active recombinant Wnt pathway agonist, and showed that this protein has the ability to activate adult stem cells after tissue injury. Thus, our developmental candidate leverages the body’s natural response to injury. We have generated exciting preclinical results in a variety of animals models including stroke, skeletal repair, heart attack, and skin wounding. If successful, this early translational award would have enormous benefits for the citizens of California and beyond.
Progress Report: 
  • In the first year of CIRM funding our objectives were to optimize the activity of the Wnt protein for use in the body and then to test, in a variety of injury models, the effects of this lipid-packaged form of Wnt. We have made considerable progress on both of these fronts. For example, in Roel Nusse and Jill Helms’ groups, we have been able to generate large amounts of the mouse form of Wnt3a protein and package it into liposomal vesicles, which can then be used by all investigators in their studies of injury and repair. Also, Roel Nusse succeeded in generating human Wnt3a protein. This is a major accomplishment since our ultimate goal is to develop this regenerative medicine tool for use in humans. In Jill Helms’ lab we made steady progress in standardizing the activity of the liposomal Wnt3a formulation, and this is critically important for all subsequent studies that will compare the efficacy of this treatment across multiple injury repair scenarios.
  • Each group began testing the effects of liposomal Wnt3a treatment for their particular application. For example, in Theo Palmer’s group, the investigators tested how liposomal Wnt3a affected cells in the brain following a stroke. We previously found that Wnt3A promotes the growth of neural stem cells in a petri dish and we are now trying to determine if delivery of Wnt3A can enhance the activity of endogenous stem cells in the brain and improve the level of recovery following stroke. Research in the first year examined toxicity of a liposome formulation used to deliver Wnt3a and we found it to be well tolerated after injection into the brains of mice. We also find that liposomal Wnt3a can promote the production of new neurons following stroke. The ongoing research involves experiments to determine if these changes in stem cell activity are accompanied by improved neurological function. In Jill Helms’ group, the investigators tested how liposomal Wnt3a affected cells in a bone injury site. We made a significant discovery this year, by demonstrating that liposomal Wnt3a stimulates the proliferation of skeletal progenitor cells and accelerates their differentiation into osteoblasts (published in Science Translational Medicine 2010). We also started testing liposomal Wnt3a for safety and toxicity issues, both of which are important prerequisites for use of liposomal Wnt3a in humans. Following a heart attack (i.e., myocardial infarction) we found that endogenous Wnt signaling peaks between post-infarct day 5-7. We also found that small aggregates of cardiac cells called cardiospheres respond to Wnt in a dose-responsive manner. In skin wounds, we tested the effect of boosting Wnt signaling during skin wound healing. We found that the injection of Wnt liposomes into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed and strength of wound closure are now being measured.
  • In aggregate, our work on this project continues to move forward with a number of great successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.
  • In the second year of CIRM funding our objectives were to optimize packaging of the developmental candidate, Wnt3a protein, and then to continue to test its efficacy to enhance tissue healing. We continue to make considerable progress on the stated objectives. In Roel Nusse’s laboratory, human Wnt3a protein is now being produced using an FDA-approved cell line, and Jill Helms’ lab the protein is effectively packaged into lipid particles that delay degradation of the protein when it is introduced into the body.
  • Each group has continued to test the effects of liposomal Wnt3a treatment for their particular application. In Theo Palmer’s group we have studied how liposomal Wnt3a affects neurogenesis following stroke. We now know that liposomal Wnt3a transiently stimulates neural progenitor cell proliferation. We don’t see any functional improvement after stroke, though, which is our primary objective.
  • In Jill Helms’ group we’ve now shown that liposomal Wnt3a enhances fracture healing and osseointegration of dental and orthopedic implants and now we demonstrate that liposomal Wnt3a also can improve the bone-forming capacity of bone marrow grafts, especially when they are taken from aged animals.
  • We’ve also tested the ability of liposomal Wnt3a to improve heart function after a heart attack (i.e., myocardial infarction). Small aggregates of cardiac progenitor cells called cardiospheres proliferate to Wnt3a in a dose-responsive manner, and we see an initial improvement in cardiac function after treatment of cells with liposomal Wnt3a. the long-term improvements, however, are not significant and this remains our ultimate goal. In skin wounds, we tested the effect of boosting Wnt signaling during wound healing. We found that the injection of liposomal Wnt3a into wounds enhanced the regeneration of hair follicles, which would otherwise not regenerate and make a scar instead. The speed of wound closure is also enhanced in regions of the skin where there are hair follicles.
  • In aggregate, our work continues to move forward with a number of critical successes, and encouraging data to support our hypothesis that augmenting Wnt signaling following tissue injury will provide beneficial effects.
  • Every adult tissue harbors stem cells. Some tissues, like bone marrow and skin, have more adult stem cells and other tissues, like muscle or brain, have fewer. When a tissue is injured, these stem cells divide and multiply but only to a limited extent. In the end, the ability of a tissue to repair itself seems to depend on how many stem cells reside in a particular tissue, and the state of those stem cells. For example, stress, disease, and aging all diminish the capacity of adult stem cells to respond to injury, which in turn hinders tissue healing. One of the great unmet challenges for regenerative medicine is to devise ways to increase the numbers of these “endogenous” stem cells, and revive their ability to self-renew and proliferate.
  • The scientific basis for our work rests upon our demonstration that a naturally occurring stem cell growth factor, Wnt3a, can be packaged and delivered in such a way that it is robustly stimulates stem cells within an injured tissue to divide and self-renew. This, in turn, leads to unprecedented tissue healing in a wide array of bone injuries especially in aged animals. As California’s population ages, the cost to treat such skeletal injuries in the elderly will skyrocket. Thus, our work addresses a present and ongoing challenge to healthcare for the majority of Californians and the world, and we do it by mimicking the body’s natural response to injury and repair.
  • To our knowledge, there is no existing technology that displays such effectiveness, or that holds such potential for the stem cell-based treatment of skeletal injuries, as does a L-Wnt3a strategy. Because this approach directly activates the body’s own stem cells, it avoids many of the pitfalls associated with the introduction of foreign stem cells or virally reprogrammed autologous stem cells into the human body. In summary, our data show that L-Wnt3a constitutes a viable therapeutic approach for the treatment of skeletal injuries, especially those in individuals with diminished healing potential.
  • This progress report covers the period between Sep 01 2012through Aug 31 2013, and summarizes the work accomplished under ET funding TR1-01249. Under this award we developed a Wnt protein-based platform for activating a patient’s own stem cells for the purpose of tissue regeneration.
  • At the beginning of our grant period we generated research grade human WNT3A protein in quantities sufficient for all our discovery experiments. We then tested the ability of this WNT protein therapeutic to improve the healing response in animal models of stroke, heart attack, skin wounding, and bone fracture. These experimental models recapitulated some of the most prevalent and debilitating human diseases that collectively, affect millions of Californians.
  • At the end of year 2, we assembled an external review panel to select the promising clinical indication. The scientific advisory board unanimously selected skeletal repair as the leading indication. The WNT protein is notoriously difficult to purify; consequently in year 3 we developed new methods to streamline the purification of WNT proteins, and the packaging of the WNT protein into liposomal vesicles that stabilized the protein for in vivo use.
  • In years 3 and 4 we continued to accrue strong scientific evidence in both large and small animal models that a WNT protein therapeutic accelerates bone regeneration in critical size bony non-unions, in fractures, and in cases of implant osseointegration. In this last year of funding, we clarified and characterized the mechanism of action of the WNT protein, by showing that it activates endogenous stem cells, which in turn leads to faster healing of a range of different skeletal defects.
  • In this last year we also identified a therapeutic dose range for the WNT protein, and developed a route and method of delivery that was simultaneously effective and yet limited the body’s exposure to this potent stem cell factor. We initiated preliminary safety studies to identify potential risks, and compared the effects of WNT treatment with other commercially available bone growth factors. In sum, we succeeded in moving our early translational candidate from exploratory studies to validation, and are now ready to enter into the IND-enabling phase of therapeutic candidate development.

Using patient-specific iPSC derived dopaminergic neurons to overcome a major bottleneck in Parkinson's disease research and drug discovery

Funding Type: 
Early Translational I
Grant Number: 
TR1-01246
ICOC Funds Committed: 
$3 701 766
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Collaborative Funder: 
Germany
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
The goals of this study are to develop patient-specific induced pluripotent cell lines (iPSCs) from patients with Parkinson’s disease (PD) with defined mutations and sporadic forms of the disease. Recent groundbreaking discoveries allow us now to use adult human skin cells, transduce them with specific genes, and generate cells that exhibit characteristics of embryonic stem cells, termed induced pluripotent stem cells (iPSCs). These lines will be used as an experimental pre-clinical model to study disease mechanisms unique to PD. We predict that these cells will not only serve an ‘authentic’ model for PD when further differentiated into the specific dopaminergic neurons, but that these cells are pathologically affected with PD. The specific objectives of these studies are to (1) establish a bank of iPSCs from patients with idiopathic PD and patients with defined mutations in genes associated with PD, (2) differentiate iPSCs into dopaminergic neurons and assess neurochemical and neuropathological characteristics of PD of these cells in vitro, and (3) test the hypothesis that specific pharmacologic agents can be used to block or reverse pathological phenotypes. The absence of cellular models of Parkinson’s disease represents a major bottleneck in the scientific field of PD, which, if solved in this collaborative effort, would be instantly translated into a wide range of clinical applications, including drug discovery. This research is highly translational, as the final component is aimed at testing lead compounds that could be neuroprotective, and ultimately at developing a high-throughput drug screening program to discover new disease modifying compounds. This is an essential avenue if we want to offer our patients a new therapeutic approach that can give them a near normal life after being diagnosed with this progressively disabling disease.
Statement of Benefit to California: 
Approx. 36,000-60,000 people in the State of California are affected with Parkinson’s disease (PD), a common neurodegenerative disease that causes a high degree of disability and financial burden for our health care system. It is estimated that the number of PD cases will double by the year 2030. We have a critical need for novel therapies that will prevent or even reverse neuronal cell loss of specific neurons in the brain of patients. This collaborative proposal will provide real benefits and values to the state of California and its citizens in providing new approaches for understanding disease mechanisms, diagnostic tools and drug discovery of novel treatment for PD. Reprogramming of adult skin cells to a pluripotent state is the underlying mechanism upon which this application is built upon and offers an attractive avenue of research in this case to develop an ‘authentic’ pre-clinical model of PD. The rationale for the proposed research is that differentiated pluripotent stem cells from patients with known genetic forms of PD will recapitulate in vitro one or more of the key molecular aspects of neural degeneration associated with PD and thus provide an entirely novel human cellular system for investigation PD-related disease pathways and for drug discovery. The impact of this collaborative research project, if successful, is difficult to over-estimate. The scientific field has been struggling with the inability to directly access cells that are affected by the disease process that underlies PD and therefore all research and drug discovery has relied on ”best guess” models of the disease. Thus, the absence of cellular models of Parkinson’s disease represents a huge bottleneck in the field.
Progress Report: 
  • In the first year of the CIRM Early translational research award, we established a bank of 51 cell lines derived from skin cells of patients with Parkinson’s disease that carry specific mutations in known genes that cause PD as well as sporadic PD patients. We also recruited matched healthy individuals that serve as controls.
  • In a next step, we reprogrammed (‘rejunivated’) 17 samples of skin cells to derive pluripotent stem cells (iPSC) that closely resemble human embryonic stem cells characterized by biochemical and molecular techniques. We also optimize this process by introducing factors the will be removed after successful reprogramming.
  • We have now built a foundation for the next milestones and made already progress on the differentiation into authentic dopamine producing cells, and we have developed assays to assess the Parkinson’s disease-specific pathological phenotype of the dopamine neurons.
  • The goal of this CIRM early translational grant is to develop a model for “Parkinson’s disease (PD) in a culture dish” using patient-specific induced pluripotent stem cell lines (iPS). The underlying idea is to utilize these lines as an experimental pre-clinical model to study disease mechanisms unique to PD that could lay the foundation for drug discovery.
  • Over the last year, we have expanded our patient skin cell bank to 57 cell lines and the iPS cell bank to 39 well-characterized pluripotent stem cell lines from PD patients and healthy controls individuals. We have improved current protocols of neuronal differentiation from patient-derived iPS lines into dopamine producing neurons and can show consistency and reproducibility of making midbrain dopamine expressing nerve cells.
  • In our first publication (Nguyen et al. 2011), we describe for the first time differences in iPS-derived neurons from a PD patient with a common causative mutation in the LRRK2 gene. These patient cells are more susceptible for cellular toxins leading ultimately to more cell degeneration and cell death.
  • We are also investigating a common disease mechanism implicated in PD, which is mitochondrial dysfunction. In skin cells of a patient we were able to find profound deficits of mitochondrial function compared to control lines and we are now in the process of confirming these results in neural precursors and mature dopamine neurons.
  • Overall, we have made substantial progress towards the goal of this grant which is the a new cell culture model of PD which can replicate PD-related cellular pathology.
  • The goal of this CIRM early translational grant is to develop a model for “Parkinson’s disease (PD) in a culture dish” using patient-specific induced pluripotent stem cell lines (iPS). The underlying idea is to utilize these lines as an experimental pre-clinical model to study disease mechanisms unique to PD that could lay the foundation for drug discovery.
  • Over the last year, we have expanded our patient skin cell bank to 61 cell lines and the iPS cell bank to 51 well-characterized pluripotent stem cell lines from PD patients and healthy controls individuals. We have improved current protocols of neuronal differentiation from patient-derived iPS lines into dopamine producing neurons and can show consistency and reproducibility of making midbrain dopamine expressing nerve cells. This has been now published in Mak et al. 2012. Furthermore, we also develop new protocols to also derive other neuronal subtypes and glia, which are the support cells in the brain, to build co-culture systems. These co-cultures might represent closer the physiological conditions in the brain.
  • In our first publication (Nguyen et al. 2011), we describe for the first time differences in iPS-derived neurons from a PD patient with a common causative mutation in the LRRK2 gene. These patient cells are more susceptible for cellular toxins leading ultimately to more cell degeneration and cell death. In a second publication Byers et al. 2011, we describe similar findings for a different mutation in the alpha-synuclein gene where the normal protein is overexpressed due to a triplication of the gene locus.
  • We are also investigating a common disease mechanism implicated in PD, which is mitochondrial dysfunction. In skin cells of a patient we were able to find profound deficits of mitochondrial function compared to control lines and we are now in the process of confirming these results in neural precursors and mature dopamine neurons.
  • We are expanding the assay development to other disease-related mechanisms such as deficits in outgrowth of neuronal projections and protein aggregation.
  • Overall, through this program we have developed an invaluable resource of patient-derived cell lines that will be crucial for understanding disease mechanisms and drug discovery. We also showed proof that these cell lines can indeed recapitulates important aspects of disease and are therefore valuable assets as research tools.

Neural Stem Cells as a Developmental Candidate to Treat Alzheimer Disease

Funding Type: 
Early Translational I
Grant Number: 
TR1-01245
ICOC Funds Committed: 
$3 599 997
Disease Focus: 
Aging
Alzheimer's Disease
Neurological Disorders
Collaborative Funder: 
Victoria, Australia
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Alzheimer disease (AD), the most common cause of dementia among the elderly and the third leading cause of death, presently afflicts over 5 million people in the USA, including over 500,000 in California. Age is the major risk factor, with 5% of the population over age 65 affected, with the incidence doubling every 5 years thereafter, such that 40-50% of those over age 85 are afflicted. Being told that one suffers from AD is one of the most devastating diagnoses a patient (and their family/caregivers) can ever receive, dooming the patient to a decade or more of progressive cognitive decline and eventual loss of all memory. At the terminal stages, the patients have lost all reasoning ability and are usually bed-ridden and unable to care for themselves. As the elderly represent the fastest growing segment of our society, there is an urgent need to develop therapies to delay, prevent or treat AD. If the present trend continues and no therapy is developed, over 16 million Americans will suffer from AD by 2050, placing staggering demands on our healthcare and economic systems. Thus, supporting AD research is a wise and prudent investment, particularly focusing on the power that stem cell biology offers. Currently, there is no cure or means of preventing AD. Existing treatments provide minor symptomatic relief– often associated with severe side effects. Multiple strategies are likely needed to prevent or treat AD, including the utilization of cell based approaches. In fact, our preliminary studies indicate that focusing on the promise of human stem cell biology could provide a meaningful therapy for a disease for which more traditional pharmaceutical approaches have failed. We aim to test the hypothesis that neural stem cells represent a novel therapeutic strategy for the treatment of AD. Our broad goal is to determine whether neural stem cells can be translated from the bench to the clinic as a therapy for AD. This proposal builds on extensive preliminary data that support the feasibility of neural stem cell-based therapies for the treatment of AD. Thus, this proposal focuses on a development candidate for treating Alzheimer disease. To translate our initial stem cell findings into a future clinical application for treating AD, we assembled a world class multi-disciplinary team of scientific leaders from the fields of stem cell biology, animal modeling, neurodegeneration, immunology, genomics, and AD clinical trials to collaborate in this early translational study aimed at developing a novel treatment for AD. Our broad goal is to examine the efficacy of human neural stem cells to rescue the cognitive phenotype in animal models of AD. Our studies aim to identify a clear developmental candidate and generate sufficient data to warrant Investigational New Drug (IND) enabling activity. The proposed studies represent a novel and promising strategy for treating AD, a major human disorder for which there is currently no effective therapy.
Statement of Benefit to California: 
Neurological disorders have devastating consequences for the quality of life, and among these, perhaps none is as dire as Alzheimer disease. Alzheimer disease robs individuals of their memory and cognitive abilities, such that they are no longer able to function in society or even interact with their family. Alzheimer disease is the most common cause of dementia among the elderly and the most significant and costly neurological disorder. Currently, 5.2 million individuals are afflicted with this insidious disorder, including over 588,000 in the State of California. Hence, over 10% of the nation's Alzheimer patients reside in California. Moreover, California has the dubious distinction of ranking first in terms of states with the largest number of deaths due to this disorder. Age is the major risk factor for Alzheimer disease, with 5% of the population over age 65 afflicted, with the incidence doubling every 5 years such that 40-50% of the population over age 85 is afflicted. As the elderly represent the fastest growing segment of our society, there is an urgent need to develop therapies to prevent or treat Alzheimer disease. By 2030, the number of Alzheimer patients living in California will double to over 1.1 million. All ethnic groups will be affected, although the number of Latinos and Asians living with Alzheimer will triple by 2030, and it will double among African-Americans within this timeframe. To further highlight the direness, at present, one person develops Alzheimer disease every 72 seconds, and it is estimated that by 2050, one person will develop the disease every 33 seconds! Clearly, the sheer volume of new cases will create unprecedented burdens on our healthcare system and have a major impact on our economic system. As the most populous state, California will be disproportionately affected, stretching our public finances to their limits. To illustrate the economic impact of Alzheimer disease, studies show that an estimated $8.5 billion of care were provided in one year in the state of California alone (this value does not include other economic aspects of Alzheimer disease). Therefore, it is prudent and necessary to invest resources to try and develop strategies to delay, prevent, or treat Alzheimer disease now. California has taken the national lead in conducting stem cell research. Despite this, there has not been a significant effort to utilize the power of stem cell biology for Alzheimer disease. This proposal seeks to reverse this trend, as we have assembled a world class group of investigators throughout the State of California and in [REDACTED] to tackle the most significant and critical questions that arise in translating basic research on human stem cells into a clinical application for the treatment of Alzheimer disease. This proposal is based on an extensive body of preliminary data that attest to the feasibility of further exploring human stem cells as a treatment for Alzheimer disease.
Progress Report: 
  • Over the past decade, the potential for using stem cell transplantation as a therapy to treat neurological disorders and injury has been increasingly explored in animal models. Studies from our lab have shown that neural stem cell transplantation can improve cognitive deficits in mice resulting from extensive neuronal loss and protein aggregation, both hallmarks of Alzheimer’s Disease pathology. Our results support the justification for exploring the use of human derived stem cells for the treatment of Alzheimer’s patients.
  • During the past few months, we have begun studies aimed at taking human derived stem cells from the bench top to the bed side. To identify the best possible human stem cells to use in our future studies, we have conducted comparisons between a wide array of human stem cells and a mouse neural stem cell line (the same mouse stem cells used in the studies mentioned above). Using these results, we have selected a cohort of human stem cell candidates to which we will continue to study in upcoming experiments involving our AD model mice.
  • In addition to identifying the best human stem cells to conduct further studies, we have also performed experiments to determine the optimal immune suppression regimen to use in our human stem cell engraftment studies. Similar to organ transplants in humans, we will need to administer immune suppressants to mice which receive our candidate human stem cells. Our group has identified a potential suppressant, also found to work in humans, which we will use in future studies.
  • Over the past decade, the potential for using stem cell transplantation as a therapy to treat neurological disorders and injury has been increasingly explored in animal models. Studies from our lab have shown that neural stem cell transplantation can improve cognitive deficits in mice resulting from extensive neuronal loss and protein aggregation, both hallmarks of Alzheimer’s Disease pathology. Our results support the justification for exploring the use of human derived stem cells for the treatment of Alzheimer’s patients.
  • During the past few months, we have begun studies aimed at taking human derived stem cells from the bench top to the bed side. To identify the best possible human stem cells to use in our future studies, we have conducted comparisons between a wide array of human stem cells and a mouse neural stem cell line (the same mouse stem cells used in the studies mentioned above). Using these results, we have selected a cohort of human stem cell candidates to which we will continue to study in upcoming experiments involving our AD model mice.
  • In addition to identifying the best human stem cells to conduct further studies, we have also performed experiments to determine the optimal immune suppression regimen to use in our human stem cell engraftment studies. Similar to organ transplants in humans, we will need to administer immune suppressants to mice which receive our candidate human stem cells. Our group has identified a potential suppressant, also found to work in humans, which we will use in future studies.
  • During the last reporting period the lab has made substantial advancements in determining the effects of long term human neural stem cells engraftment on pathologies associated with the advancement of Alzheimer's disease. In addition, data obtained by our lab has may provide additional insight on ways to target the immune system as a means of prolonging neural stem cell survival and effectiveness.

Stem Cell Mechanisms Governing Discrete Waves of Gliogenesis in the Childhood Brain

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06093
ICOC Funds Committed: 
$1 264 248
Disease Focus: 
Neurological Disorders
Pediatrics
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
White matter is the infrastructure of the brain, providing conduits for communication between neural regions. White matter continues to mature from birth until early adulthood, particularly in regions of brain critical for higher cognitive functions. However, the precise timing of white matter maturation in the various neural circuits is not well described, and the mechanisms controlling white matter developmental/maturation are poorly understood. White matter is conceptually like wires and their insulating sheath is a substance called myelin. It is clear that neural stem and precursor cells contribute significantly to white matter maturation by forming the cells that generate myelin. In the proposed experiments, we will map the precise timing of myelination in the human brain and changes in the populations of neural precursor cells that generate myelin from birth to adulthood and define mechanisms that govern the process of white matter maturation. The resulting findings about how white matter develops may provide insights for white matter regeneration to aid in therapy for diseases such as cerebral palsy, multiple sclerosis and chemotherapy-induced cognitive dysfunction.
Statement of Benefit to California: 
Diseases of white matter account for significant neurological morbidity in both children and adults in California. Understanding the cellular and molecular mechanisms that govern white matter development the may unlock clues to the regenerative potential of endogeneous stem and precursor cells in the childhood and adult brain. Although the brain continues robust white matter development throughout childhood, adolescence and young adulthood, relatively little is known about the mechanisms that orchestrate proliferation, differentiation and functional maturation of neural stem and precursor cells to generate myelin-forming oligodendrocytes during postnatal brain development. In the present proposal, we will define how white matter precursor cell populations vary with age throughout the brain and determine if and how neuronal activity instructs neural stem and precursor cell contributions to human white matter myelin maturation. Disruption of white matter myelination is implicated in a range of neurological diseases, including cerebral palsy, multiple sclerosis, cognitive dysfunction from chemotherapy exposure, attention deficit and hyperactivity disorder (ADHD) and even psychiatric diseases such as schizophrenia. The results of these studies have the potential to elucidate clues to white matter regeneration that may benefit hundreds of thousands of Californians.
Progress Report: 
  • Formation of the insulated fiber infrastructure of the human brain (called "myelin") depends upon the function of a precursor cell type called "oligodendrocyte precursor cells (OPC)". The first Aim of this study seeks to determine how OPCs differ from each other in different regions of the brain, and over different ages. Understanding this heterogeneity is important as we explore the regenerative capacity of this class of precursor cells. We have, in the past year, isolated OPCs from various regions of the human brain from individuals at various ages and are studying the molecular characteristics of these precursor cells at the single cell level in order to define distinct OPC subpopulations. We have also begun to study the functional capabilities of OPCs isolated from different brain regions. The second Aim of this study seeks to understand how interactions between electrically active neurons and OPCs affect OPC function and myelin formation. We have found that when mouse motor cortex neurons "fire" signals in such a way as to elicit a complex motor behavior, much as would happen when one practices a motor task, OPCs within that circuit respond and myelination increases. This affects the function of that circuit in a lasting way. These results indicate that neurons and OPCs interact in important ways to modulate myelination and supports the hypothesis that OPC function may play a role in learning.

Stem cell models to analyze the role of mutated C9ORF72 in neurodegeneration

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06045
ICOC Funds Committed: 
$1 393 200
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Dementia
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Amyotrophic lateral sclerosis (ALS) is an idiopathic adult-onset degenerative disease characterized by progressive weakness from loss of upper and lower motor neurons. Onset is insidious, progression is essentially linear, and death occurs within 3-5 years in 90% of patients. In the US, 5,000 deaths occur per year and in the world, 100,000. In October, 2011, the causative gene defect in a long sought after locus on chromosome 9 for ALS, frontotemporal dementia (FTD) and overlap ALS-FTD was identified to be a expansion of a hexanucleotide repeat in the uncharacterized C9ORF72 gene. The goal of the proposed research is to generate human stem cell models from cells derived from ALS patients with the C9ORF72 expanded repeats and relevant control cells using genome-editing technology. We will also generate a stem cell model expressing the repeat independent of the C9ORF72 gene to study if the repeat alone is causing neural defects. Using advanced genome technologies, biochemical and cellular approaches, we will study the molecular pathways affected in motor neurons derived from these stem cell models. Finally, we will use innovative technologies to rescue the abnormal phenotypes that arise from the expanded repeat in human motor neurons. Completion of the proposed research is expected to transform our understanding of the regulatory and pathogenetic mechanisms underlying ALS and FTD, and establish therapeutic options for these debilitating diseases.
Statement of Benefit to California: 
Our research provides the foundation for decoding the mechanisms that underlie the single most frequent genetic mutation found to contribute to both ALS and FTD, debilitating neurological diseases that impact many Californians. In California, the expected prevalence of ALS (the number of total existing cases) is 2,200 to 3,000 cases at any one time, and the incidence is 750-1,100 new cases each year. The number of FTD cases is five times as many. Our research has and will continue to serve as a basis for understanding deviations from normal and disease patient neuronal cells, enabling us to make inroards to understanding neurological disease modeling using neurons differentiated from reprogammed patient-specific lines. Such disease modeling will have great potential for California health care patients, pharmaceutical and biotechnology industries in terms of improved human models for drug discovery and toxicology testing. Our improved knowledge base will support our efforts as well as other Californian researchers to study stem cell models of neurological disease and design new diagnostics and treatments, thereby maintaining California's position as a leader in clinical research.
Progress Report: 
  • Expanded hexanucleotide repeats in the C9ORF72 gene were identified in Oct 2011 as a cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), thus identifying the single most frequent genetic cause of each and connecting them to repeat expansion disease. We are developing stem cell disease models to enable key hypotheses of pathogenesis and new interventions to be tested. We have successfully engineered stem cell models to analyze the effects of C9ORF72 mutations, and have differentiated these stem cell models into motor neurons which enabled us to conduct transcriptomic and biochemical studies. In addition, we have utilized antisense-oligonucleotides (ASOs) from ISIS Pharmaceuticals to deplete mutant C9ORF72 in motor neurons. We expect our efforts to provide mechanistic insights and a potential therapy in human cells.

Mechanism and Utility of Direct Neuronal Conversion with a MicroRNA-Chromatin Switch

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-05886
ICOC Funds Committed: 
$1 392 426
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Directly Reprogrammed Cell
oldStatus: 
Active
Public Abstract: 
Many human diseases and injuries that affect the brain and nervous system could potentially be treated by either introducing healthy neurons or persuading the cells that normally provide supporting functions to become functioning neurons. A number of barriers must be traversed to bring these goals to practical therapies. Recently our laboratory and others have found ways of converting different human cell types to functioning neurons. Surprisingly, two routes for the production of neurons have been discovered. Our preliminary evidence indicates that these two routes are likely to work together and therefore more effective ways of producing neurons can likely be provided by understanding these two routes, which is one aim of this application. Another barrier to effective treatment of human neurologic diseases has been the inability to develop good models of human neurologic disease due to inability to sample tissues from patients with these diseases. Hence we will understand ways of making neurons from blood cells and other cells, which can be easily obtained from patients with little or no risk. Our third goal will be to understand how different types of neurons can be produced from patient cells. We would also like to understand the barriers and check points that keep one type of cell from becoming another another type of cell. Understanding these mysterious processes could help provide new sources of human cells for replacement therapies and disease models.
Statement of Benefit to California: 
The state of California and its citizens are likely to benefit from the work described in this proposal by the development of more accurate models for the testing of drugs and new means of treatment of human neurologic diseases. Presently these diseases are among the most common afflicting Californians, as well as others and will become more common in an aging population. Common and devastating diseases such as Alzheimer’s, Schizophrenia, Parkinson's Disease, and others lack facile cell culture models that allow one to probe the basis of the disease and to test therapies safely and without risk to the patient. Our work is already providing these models, but we hope to make even better ones by understanding the fundamental processes that allow one cell type (such as a skin cell or blood cell) to be converted to human neurons, where the disease process can be investigated. In the past the inability to make neurons from patients with specific diseases has been a major roadblock to treatment. In the future the studies described here might be able to provide healthy neurons to replace ones loss through disease or injury.
Progress Report: 
  • During the past year, our laboratory has investigated the way that human skin cells can be changed to neurons. To do this, we have used a natural switch that occurs as embryonic cells decide to become neurons. We have found that this process proceeds in a highly ordered series of stages that involve first a resetting of fundamental cell biologic processes characteristic of neurons. This is followed by activation of genes encoding proteins that allow different types of neurons to interact and develop communication between one another. This finding surprised us since we expected to find changes in transcription factors, which instruct the formation of neurons. Instead, we find that the natural switching mechanism in neurons first regulates cell-to-cell communication.

Role of the NMD RNA Decay Pathway in Maintaining the Stem-Like State

Funding Type: 
Basic Biology IV
Grant Number: 
RB4-06345
ICOC Funds Committed: 
$1 360 450
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
A subset of intellectual disability cases in humans are caused by mutations in an X-linked gene essential for a quality control mechanism called nonsense-mediated RNA decay (NMD). Patients with mutations in this gene—UPF3B—commonly have not only ID, but also schizophrenia, autism, and attention-deficit/hyperactivity disorder. Thus, the study of UPF3B and NMD may provide insight into a wide spectrum of cognitive and psychological disorders. To examine how mutations in UPF3B can cause mental defects, we will generate and characterize induced-pluripotent stem cells from intellectual disability patients with mutations in the UPF3B gene. In addition to having a role in neural development, our recent evidence suggests that NMD is important for maintaining the identity of ES cells and progenitor cells. How does NMD do this? While NMD is a quality control mechanism, it is also a well characterized biochemical pathway that serves to rapidly degrade specific subsets of normal messenger ribonucleic acids (mRNAs), the transiently produced copies of our genetic material: deoxyribonucleic acid (DNA). We have obtained evidence that NMD preferentially degrades mRNAs that interfere with the stem cell program (i.e., NMD promotes the decay mRNAs encoding proteins that promote differentiation and inhibit cell proliferation). In this proposal, we will identify the target mRNAs of NMD in stem and progenitor cells and directly address the role of NMD in maintaining the stem-like state.
Statement of Benefit to California: 
iPS cells provide a means to elucidate the mechanisms underlying diseases that afflict a growing number of Californians. Our proposed project concerns making and testing iPS cells from patients with mutations in the UPF3B gene, all of whom have intellectual disabilities. In addition, many of these patients have autism, attention-deficit disorders, and schizophrenia. By using iPS cells to identify the cellular and molecular defects in these patients, we have the potential to ultimately ameliorate the symptoms of many of these patients. This is important, as over 1.6 million people in California have serious mental illness. Moreover, a large proportion of patients with UPF3B mutations have autism, a disorder that has undergone an alarming 12-fold increase in California between 1987 and 2007. The public mental health facilities in California are inadequate to meet the needs of people with mental health disorders. Furthermore, what is provided is expensive: $4.4 billion was spent on public mental health agency services in California in 2006. Mental health problems also exert a heavy burden on California’s criminal justice system. In 2006, over 11,000 children and 40,000 adults with mental health disorders were incarcerated in California’s juvenile justice system. Our research is also directed towards understanding fundamental mechanisms by which all stem cells are maintained, which has the potential to also impact non-psychiatric disorders suffered by Californians.
Progress Report: 
  • A key quality of stem cells is their ability to switch from a proliferative cell state in which they reproduce themselves to a differentiated cell state that ultimately allows them to carry out the functions of a fully mature cell. Most research on the nature of this switch has focused on the role of proteins that determine whether the genetic material—DNA—generates a copy of it itself in the form of messenger RNA, a process called transcription. In stem cells, such proteins—which are called transcription factors—activate the production of messenger RNAs encoding proteins that promote the proliferative and undifferentiated cell state. They also increase the production of messenger mRNAs that encode inhibitors of differentiation and cell proliferation. The level and profile of such transcription factors are altered in response to signals that trigger stem cells to differentiate. For example, transcription factors that promote the undifferentiated cell state are decreased in level and transcription factors that drive differentiation down a particular lineage are increased in level. While this transcription factor-centric view of stem cells explains some aspects of stem cell biology, it is, in of itself, insufficient to explain many of their behaviors, including both their ability to maintain the stem-like state and to differentiate. We hypothesize that a molecular pathway that complements transcription-base mechanisms in controlling stem cell maintenance vs. differentiation decisions is an RNA decay pathway called nonsense-mediated RNA decay (NMD). Messenger RNA decay is as important as transcription in determining the level of messenger RNA. Signals that trigger increased decay of a given messenger RNA leads to decreased levels of its encoded protein, while signals that trigger the opposite response increase the level of the encoded protein. Our project revolves around two main ideas. First, that NMD promotes the stem-like state by preferentially degrading messenger RNAs that encode differentiation-promoting proteins and proliferation inhibitor proteins. Second, that NMD must be downregulated in magnitude to allow stem cells to differentiate. During the progress period, we obtained substantial evidence for both of these hypotheses. With regard to the first hypothesis, we have used genome-wide approaches to identify hundreds of messenger RNAs that are regulated by NMD in both in vivo (in mice) and in vitro (in cell lines). To determine which of these messenger mRNAs are directly degraded by NMD, we have used a variety of approaches. This work has revealed that NMD preferentially degrades messenger RNAs encoding neural differentiation inhibitors and proliferation inhibitors in neural stem cells. In contrast, very few messenger RNAs encoding pro-stem cell proteins or pro-proliferation proteins are degraded by NMD. Together this provides support for our hypothesis that NMD promotes the stem-like state by shifting the proportion of messenger RNAs in a cell towards promoting an undifferentiated, proliferative cell state. With regard to the second hypothesis, we have found that many proteins that are directly involved in the NMD pathway are downregulated upon differentiation of stem and progenitor cells. Not only are NMD proteins reduced in level, but we find that the magnitude of NMD itself is reduced. We have used a variety of molecular techniques to determine whether this NMD downregulatory response has a role in neural differentiation and found that NMD downreglation is both necessary and sufficient for this event. Such experiments have also revealed particular messenger mRNAs degraded by NMD that are crucial for the NMD downregulatory response to promote neural differentiation. Our research has implications for intellectual disability cases in humans caused by mutations in an X-linked gene essential for NMD. Patients with mutations in this gene—UPF3B—not only have intellectual disability, but also schizophrenia, autism, and attention-deficit/hyperactivity disorder. Thus, the study of NMD may provide insight into a wide spectrum of cognitive and psychological disorders. We are currently in the process of generating induced-pluripotent stem (iPS) cells from intellectual disability patients with mutations in the UPF3B gene towards this goal.

Pages

Subscribe to RSS - Neurological Disorders

© 2013 California Institute for Regenerative Medicine