Neurological Disorders

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

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: 
Statement of Benefit to California: 
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.

Role of HLA in neural stem cell rejection using humanized mice

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01735-A
ICOC Funds Committed: 
$1 472 634
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
Public Abstract: 
One of the key issues in stem cell transplant biology is solving the problem of transplant rejection. Despite over three decades of research in human embryonic stem cells, little is known about the factors governing immune system tolerance to grafts derived from these cells. In order for the promise of embryonic stem cell transplantation for treatment of diseases to be realized, focused efforts must be made to overcome this formidable hurdle. Our proposal will directly address this critically important issue by investigating the importance of matching immune system components known as human leukocyte antigens (HLA). Because mouse and human immune systems are fundamentally different, we will establish cutting-edge mouse models that have human immune systems as suitable hosts within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human blood-derived stem cells. These mice go on to develop a human immune system, complete with HLAs, and can subsequently be used to engraft embryonic stem cell-derived brain cells that are either HLA matched or mismatched. Due to our collective expertise in the central nervous system and animal transplantation studies for Parkinson’s disease, our specific focus will be on transplanting embryonic stem cell-derived neural stem cells into brains of both healthy and Parkinson's diseased mice. We will then detect: 1) abundance of brain immune cell infiltrates, 2) production of immune molecules, and 3) numbers of brain-engrafted embryonic stem cells. Establishing this important system would allow for a predictive model of human stem cell transplant rejection based on immune system matching. We would then know how similar HLAs need to be in order to allow for acceptance stem cell grafts.
Statement of Benefit to California: 
In this project, we propose to focus on the role of the human immune system in human embryonic stem cell transplant rejection. Specifically, we aim to develop cutting-edge experimental mouse models that possess human immune systems. This will allow us to determine whether immune system match versus mismatch enables embryonic stem cell brain transplant acceptance versus rejection. Further, we will explore this key problem in stem cell transplant biology both in the context of the healthy and diseased brain. Regarding neurological disease, we will focus on neural stem cell transplants for Parkinson's disease, which is one of the most common neurodegenerative diseases, second only to Alzheimer's disease. If successful, our work will pave the way toward embryonic stem cell-based treatment for this devastating neurological disorder for Californians and others. In order to accomplish these goals, we will utilize two of the most common embryonic stem cell types, known as WiCell H1 and WiCell H9 cells. It should be noted that these particular stem cells will likely not be reauthorized for funding by the federal government due to ethical considerations. This makes our research even more important to the State of California, which would not only benefit from our work but is also in a unique position to offer funding outside of the federal government to continue studies such as these on these two important types of human embryonic stem cells.
Progress Report: 
  • In order for the promise of stem cell transplantation therapy to treat or cure human disease to be realized, the key problem of stem cell transplant rejection must be solved. Yet, despite over three decades of research in human embryonic stem cells, little is known about the factors governing immune system tolerance to grafts derived from these cells.
  • The goal of our CIRM Stem Cell Transplantation Immunology Award is to overcome this formidable hurdle by generating pre-clinical mouse models that have human immune systems. This next-generation model system will provide a testing platform to evaluate the importance of matching immune system components known as human leukocyte antigens (HLAs). Because mouse and human immune systems are fundamentally different, these cutting-edge ‘humanized’ mice are currently the only animal models within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human umbilical cord blood stem cells (HSCs). These mice go on to develop a human immune system, complete with HLAs, and can subsequently be used to engraft embryonic stem cell-derived brain cells that are either HLA matched or mismatched and to monitor for graft acceptance vs. rejection.
  • During this first year of CIRM funding, we have accomplished three main goals leading to completion of Specific Aim 1: To establish mouse models with human immune systems (year 1). Firstly, we have increased purity of HSCs from 75% to 93%. This has enabled us to complete our second goal of generating 10 mice bearing 50% or more human immune cells. Thirdly, we have characterized the human adaptive immune systems of these mice and have found presence of 40-60% of human T lymphocytes in lymphoid organs of ‘humanized’ mice.
  • For the promise of stem cell transplantation therapy to treat or cure human disease to be realized, the key problem of stem cell transplant rejection must be solved. Yet, despite over three decades of research in human embryonic stem cells, little is known about the factors involved in immune system tolerance to grafts derived from embryonic stem cells.
  • The goal of our CIRM Stem Cell Transplantation Immunology Award is to overcome this formidable hurdle by generating pre-clinical mouse models that have human immune systems. This cutting-edge model system will provide a testing platform to evaluate the importance of matching immune system components, known as human leukocyte antigens (HLAs), between the human embryonic stem (hES) cell-derived neural stem cell (NSC) graft and the patient. Because mouse and human immune systems are fundamentally different, these next-generation ‘humanized’ mice are currently the only animal models within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human umbilical cord blood stem cells (HSCs). These mice go on to develop a human immune system, complete with HLAs, and can subsequently be used to engraft embryonic stem cell-derived brain cells that are either HLA matched or mismatched and to monitor for graft acceptance vs. rejection.
  • During this second year of CIRM funding, we have accomplished three main goals leading to completion of Specific Aim 2, which is designed to perform HLA haplotype ‘mix and match’ experiments using hES cell-derived NSCs as donors and ‘humanized’ mice as recipients (year 2). Firstly, we have now successfully generated ‘humanized’ mice that have 50% or more engraftment of human immune cells in lymphoid organs, defined as percentage of human immune cells within the mouse. Secondly, we have successfully HLA haplotyped these human donor CD34+ HSCs, and have additionally transplanted hES cell-derived NSCs with known HLA haplotypes. Finally, we have ‘mixed and matched’ HLA haplotypes in adoptive transfer experiments using human HSC reconstituted mice as recipients and human NSCs as donors. This critically important new tool will allow for a predictive model of human stem cell transplant acceptance vs. rejection.

Role of HLA in neural stem cell rejection using humanized mice

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01735-B
ICOC Funds Committed: 
$1 472 634
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
One of the key issues in stem cell transplant biology is solving the problem of transplant rejection. Despite over three decades of research in human embryonic stem cells, little is known about the factors governing immune system tolerance to grafts derived from these cells. In order for the promise of embryonic stem cell transplantation for treatment of diseases to be realized, focused efforts must be made to overcome this formidable hurdle. Our proposal will directly address this critically important issue by investigating the importance of matching immune system components known as human leukocyte antigens (HLA). Because mouse and human immune systems are fundamentally different, we will establish cutting-edge mouse models that have human immune systems as suitable hosts within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human blood-derived stem cells. These mice go on to develop a human immune system, complete with HLAs, and can subsequently be used to engraft embryonic stem cell-derived brain cells that are either HLA matched or mismatched. Due to our collective expertise in the central nervous system and animal transplantation studies for Parkinson’s disease, our specific focus will be on transplanting embryonic stem cell-derived neural stem cells into brains of both healthy and Parkinson's diseased mice. We will then detect: 1) abundance of brain immune cell infiltrates, 2) production of immune molecules, and 3) numbers of brain-engrafted embryonic stem cells. Establishing this important system would allow for a predictive model of human stem cell transplant rejection based on immune system matching. We would then know how similar HLAs need to be in order to allow for acceptance stem cell grafts.
Statement of Benefit to California: 
In this project, we propose to focus on the role of the human immune system in human embryonic stem cell transplant rejection. Specifically, we aim to develop cutting-edge experimental mouse models that possess human immune systems. This will allow us to determine whether immune system match versus mismatch enables embryonic stem cell brain transplant acceptance versus rejection. Further, we will explore this key problem in stem cell transplant biology both in the context of the healthy and diseased brain. Regarding neurological disease, we will focus on neural stem cell transplants for Parkinson's disease, which is one of the most common neurodegenerative diseases, second only to Alzheimer's disease. If successful, our work will pave the way toward embryonic stem cell-based treatment for this devastating neurological disorder for Californians and others. In order to accomplish these goals, we will utilize two of the most common embryonic stem cell types, known as WiCell H1 and WiCell H9 cells. It should be noted that these particular stem cells will likely not be reauthorized for funding by the federal government due to ethical considerations. This makes our research even more important to the State of California, which would not only benefit from our work but is also in a unique position to offer funding outside of the federal government to continue studies such as these on these two important types of human embryonic stem cells.
Progress Report: 
  • For the promise of stem cell transplantation therapy to treat or cure human disease to be realized, the key problem of stem cell transplant rejection must be solved. Yet, despite over three decades of research in human embryonic stem cells, little is known about the factors involved in immune system tolerance to grafts derived from embryonic stem cells.
  • The goal of our CIRM Stem Cell Transplantation Immunology Award is to overcome this formidable hurdle by generating pre-clinical mouse models that have human immune systems. This cutting-edge model system will provide a testing platform to evaluate the importance of matching immune system components, known as human leukocyte antigens (HLAs), between the human embryonic stem (hES) cell-derived neural stem cell (NSC) graft and the patient. Because mouse and human immune systems are fundamentally different, these next-generation ‘humanized’ mice are currently the only animal models within which to conduct our stem cell brain transplant experiments. Such models rely on immunocompromised mice as recipients for human umbilical cord blood stem cells (HSCs). These mice go on to develop a human immune system, complete with HLAs, and can subsequently be used to engraft embryonic stem cell-derived brain cells that are either HLA matched or mismatched and to monitor for graft acceptance vs. rejection.
  • During the third year of CIRM funding, we have addressed two specific questions that have arisen during the completion of Specific Aim 2: 1) which component of the HLA haplotype is most important to match in order to prevent brain stem cell rejection, and 2) can we expand blood stem cells obtained from a single umbilical cord blood sample? In response to question 1, we have determined that HLA-A is expressed at significantly higher levels in NSCs than the other HLA components, which makes this HLA type the critical player in immune system acceptance-rejection. As evidence of this, ‘humanized’ mice transplanted with NSCs expressing completely mismatched HLA-A elicited an immune response. Regarding question 2, we were able to accomplish ex vivo expansion of HSCs while maintaining their ‘stem-ness’ properties, which allows us to coordinate between the birth of mouse pups and the isolation of HSCs from umbilical cord blood samples, and also to significantly increase cell numbers to generate more ‘humanized’ mice. Additionally, in collaboration with Dr. George Liu from Cedars-Sinai Medical Center, we utilized ‘humanized’ mice to successfully model another disease that has become a threat to Californians’ health: skin infection by Staphylococcus aureus. While mice are generally not susceptible to this ‘human selective’ disease, ‘humanized’ mice did respond to the infection, closely mimicking the skin lesions observed in humans.

Induction of immune tolerance after spinal grafting of human ES-derived neural precursors

Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01720
ICOC Funds Committed: 
$1 387 800
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Previous clinical studies have shown that grafting of human fetal brain tissue into the CNS of adult recipients can be associated with long-term (more then 10 years) graft survival even after immunosuppression is terminated. These clinical data represent in part the scientific base for the CNS to be designated as an immune privilege site, i.e., immune response toward grafted cells is much less pronounced. With rapidly advancing cell sorting technologies which permit effective isolation and expansion of neuronal precursors from human embryonic stem cells, these cells are becoming an attractive source for cell replacement therapies. Accordingly, there is great need to develop drug therapies or other therapeutic manipulations which would permit an effective engraftment of such derived cells with only transient or no immunosuppression. Accordingly, the primary goal in our studies is to test engraftment of 3 different neuronal precursors cell lines of human origin once grafted into spinal cord in transiently immunosuppressed minipigs. In addition, because the degree of cell engraftment can differ if cells are grafted into injured CNS tissue, the survival of cells once grafted into previously injured spinal cord will also be tested. Second, we will test the engraftment of neuronal cells generated from pig skin cells (fibroblasts) after genetic reprogramming (i.e., inducible pluripotent stem cells, iPS). Because these cells will be transplanted back to the fibroblast donor, we expect stable and effective engraftment in the absence of immunosuppression. Jointly by testing the above technologies (transient immunosuppression and iPS-derived neural precursors), our goal is to define the optimal neuronal precursor cell line(s) as well as immunosuppressive (or no) treatment which will lead to stable and permanent engraftment of spinally transplanted cells.
Statement of Benefit to California: 
Brain or spinal cord neurodegenerative disorders, including stroke, amyotrophic lateral sclerosis, multiple sclerosis or spinal trauma, affect many Californians. In the absence of a functionally effective cure, the cost of caring for patients with such diseases is high, in addition to a major personal and family impact. Our major goal is to develop therapeutic manipulations which are well tolerated by patients and which will lead to stable survival of previously spinal cord-grafted cells generated from human embryonic stem cells. If successful, this advance can serve as a guidance tool for CNS cell replacement therapies in general as it will define the optimal immune tolerance-inducing protocols. In addition, the development of this type of therapeutic approach (pharmacological or cell-replacement based) in California will serve as an important proof of principle and stimulate the formation of businesses that seek to develop these types of therapies (providing banks of inducible pluripotent stem cells) in California with consequent economic benefit.
Progress Report: 
  • The use of autologous, induced pluripotent stem cell-derived cell lines in replacement therapies holds great promise in future clinical use. No need for immunosuppression, otherwise required to prevent transplanted cell rejection, would represent a substantial advance in the current clinical utilization of cell replacement therapies. In our recently completed studies we have found that autologous porcine iPSC-derived neural precursors (NPCs) grafted back to the donor animal spinal cord in the absence of immunosuppression was associated with a poor cell survival and extensive inflammation at cell-grafted sites. Our data raises immunological concerns on the use of autologous iPS-cell derivatives for future regenerative medicine in humans.
  • The use of autologous, induced pluripotent stem cell-derived cell lines in replacement therapies holds great promise in future clinical use. No need for immunosuppression, otherwise required to prevent transplanted cell rejection, would represent a substantial advance in the current clinical utilization of cell replacement therapies. In our recently completed studies we have found that autologous porcine iPSC-derived neural precursors (NPCs) grafted back to the donor animal spinal cord in the absence of immunosuppression was associated with a poor cell survival and extensive inflammation at cell-grafted sites. In more recent study we have determined that the same cell population of iPS-NPCs survive and mature once grafted spinally in immunosupressed pigs.The mechanism of the immunogenicity of iPS-NPCs is being currently determined.
  • The use of autologous, induced pluripotent stem cell-derived cell lines in replacement therapies holds great promise in future clinical use. No need for immunosuppression, otherwise required to prevent transplanted cell rejection, would represent a substantial advance in the current clinical utilization of cell replacement therapies. In our recently completed studies, we have found that autologous porcine iPSC-derived neural precursors (NPCs) trigger a positive T-cell mediated reaction in vitro and that this response is not present if autologous T-cells are co-cultured with autologous fibroblasts. These data show that the reprogramming step induces a potent immunogenicity and that extensive screening of clonally-derived iPS-NPCs will be needed to identify clones of autologous NPCs with acceptable immunogenicity profile. Identification of differences in gene activity in differentially derived iPS-NPCs is currently in progress.

Generation of disease models for neurodegenerative disorders in hESCs by gene targeting

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01107
ICOC Funds Committed: 
$869 262
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • The overall objectives for this proposal are to create in vitro human neurodegenerative disease models and to elucidate pathogenesis of amyotrophic lateral sclerosis (ALS), an adult onset fatal motoneuron disease. Using gene targeting and reprogramming technology, we have created ALS disease models in human pluripotent stem cells and are generating neural lineage reporters which will facilitate the downstream efforts on systemic characterization of these diseased cell lines, at undifferentiated stage and after induced lineage differentiation toward motoneurons and astrocytes. These experiments will not only provide direct clues for ALS pathogenesis but also serve as a proof of principle for general disease research using human pluripotent stem cells as a model system. We also aim to provide optimized protocols for easy to access gene targeting which eventually facilitate the development of personalized medicine, the future of regenerative medicine. The novel targeting protocol combined with our experience on directed differentiation along the neural lineage will not only will make tools to move the pathogenesis research for ALS, but also can be reliably extended to other neural and non-neural diseases, of which genetic defects have been identified, including Huntington's disease and Parkinson’s disease.
  • The overall objectives for this proposal are to create in vitro human neurodegenerative disease models for amyotrophic lateral sclerosis (ALS), an adult onset fatal motoneuron disease. Using gene targeting, site-specific integration and reprogramming technology, we have created ALS disease models in human pluripotent stem cells and generated neural lineage reporters which will facilitate the downstream efforts on systemic characterization of these diseased cell lines, at undifferentiated stage and after forced lineage differentiation toward motoneurons and astrocytes. We have optimized protocols for gene targeting using homologous recombination and site-specific integration and insertion. The novel targeting protocol combined with our experience on directed differentiation along the neural lineage are useful tools to pathogenesis research for ALS, as well as to other neural and non-neural diseases, including Huntington's disease and Parkinson’s disease.

Directed Evolution of Novel AAV Variants for Enhanced Gene Targeting in Pluripotent Human Stem Cells and Investigation of Dopaminergic Neuron Differentiation

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01021
ICOC Funds Committed: 
$918 000
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.
  • The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.
  • The central goal of this project is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. Harnessing a novel directed evolution technology we have developed to improve the properties of a promising viral vehicle, we have significantly increased its gene delivery efficiency to human embryonic and human induced pluripotent stem cells. Furthermore, this advance resulted in considerable improvements in the efficiency of gene targeting (i.e. editing) in the genomes of these cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of luripotent stem cell differentiation into neurons, with for example implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We attempted chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. Progress in this objective was ultimately hampered by the lack of a suitable antibody against Lmx1a. However, in parallel we have used an analogous approach to investigate mechanisms by which RNA transcription is regulated during the differentiation of embryonic stem cells into neurons, including motor neurons. These basic results can now be applied to enhance the efficiency of neuronal differentiation.

North Bay CIRM Shared Research Laboratory for Stem Cells and Aging

Funding Type: 
Shared Labs
Grant Number: 
CL1-00501-1.2
ICOC Funds Committed: 
$5 893 682
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Work in this field has been limited however, due to the limited availability of cells for transplantation. For example, cells from 6-10 human fetuses obtained 6-10 weeks post-conception are required for one patient with Parkinson’s disease to undergo transplantation. Human embryonic stem cells (hESCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. Supported by this facility, we have in the past year successfully developed a process for the production of functional dopaminergic neurons from hESCs that are suitable for potential clinical uses, e.g., in treating Parkinson’s disease (Parkinson’s disease is caused by the death of dopaminergic neurons). Our system provides a path to a scalable Good Manufacture Practice (GMP)-applicable process of generation of dopaminergic neurons from hESCs for therapeutic applications, and a ready source of large numbers of neurons for potential drug screening applications. In addition, we have developed a screening strategy that allows us to rapidly identify clinically approved drugs for use in GMP protocol that can be safely used to deplete unwanted contaminating precursor cells from dopaminergic neurons, a target for cell therapy.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 10 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 7 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 10 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.
  • Age-related diseases of the nervous system are major challenges for biomedicine in the 21st century. These disorders, which include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis and stroke, cause loss of neural tissue and functional impairment. Currently, there is no cure for these devastating neurological disorders. A promising approach to the treatment of age-related neurological disorders is cell therapy, i.e., transplantation of nerve cells into the brain or spinal cord to replace lost cells and restore function. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) offer a potentially unlimited source of any cell type that may be required for cell replacement therapy, due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to develop into any cell type in the body.
  • Funded by CIRM, we have built out approximately 3400 square feet of shared laboratory space within our existing research facility for hESC research, as well as approximately 2400 square feet for classroom facilities dedicated to training in hESC culture and manipulation. In the past year, the facility has supported over a dozen regional investigators seeking expertise in ESC/iPSC techniques. The Shared Lab maintains an average of 10 hESC and/or iPSC lines for investigators both inside and outside the Buck Institute. The facility also routinely generates neural stem cells (NSCs) from both the hESC and iPSC lines and the NSC lines have been used by many of the investigators for differentiation studies. In addition, the Shared Lab has created several genetically modified hESC lines (e.g., GFP-labeled cells) and developed techniques for efficient transfection of hESCs and their differentiated derivatives. These lines and techniques are made available for all investigators and have been used by several of them for studies of aging-related process.
  • Before a hESC-based therapy can be developed, it is essential to train scientists to efficiently grow, maintain and manipulate these cells. We have taught two types of hands-on training courses in the past year with more than 30 scientists across California participated: a basic 5-day hESC culture course and an advanced 5-day hESC culture course, to meet the diverse needs of California scientists. These courses provided scientists with an understanding of hESC biology and enabled them to set up and conduct hESC research after completion of training.

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

Funding Type: 
New Faculty II
Grant Number: 
RN2-00952
ICOC Funds Committed: 
$2 847 600
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in Aims 1 and 2.
  • First, we further demonstrated that neural stem cells (NSCs) express apoE. ApoE-KO mice had significantly less hippocampal neurogenesis, but significantly more astrogenesis, than wildtype mice due to decreased Noggin expression in NSCs. In contrast, neuronal maturation in apoE4 knock-in (apoE4-KI) mice was impaired due to reduced survival and function of GABAergic interneurons in the hilus of the hippocampus, and a GABAA receptor potentiator rescued the apoE4-associated decrease in hippocampal neurogenesis. Thus, apoE plays an important role in hippocampal neurogenesis, and the apoE4 isoform impairs GABAergic input to newborn neurons, leading to decreased neurogenesis. A paper describing these data was published in Cell Stem Cell (Li G. et al. 2009, 5:634-645), which evidently is the 400th publication of CIRM-funded projects.
  • Second, we established mouse iPS cell lines from adult mouse fibroblasts of wildtype, apoE knockout (apoE-KO), human apoE2-KI, human apoE3-KI, and human apoE4-KI mice.
  • Finally, we developed NSC lines from mouse iPS cells with different apoE genotypes (wildtype mouse apoE, apoE-KO, apoE2, apoE3, and apoE4). These cell lines will be used to study the effects of apoE isoforms on neuronal development in vitro in culture and in vivo in mouse models.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in the past year, as summarized below.
  • First, We developed human iPS cells from skin fibroblasts of individuals with different apoE genotypes. We are fully characterizing these human iPS cell lines.
  • Second, We are establishing neural stem cell (NSC) lines from human iPS cells with different apoE genotypes. Some of the NSCs have been maintained in monolayer cultures for many generations. These NSCs will be used to study the effects of apoE isoforms on neuronal development in vitro in cultures and in vivo in mice.
  • Finally, we demonstrated that mouse apoE4-NSCs generated significantly fewer total neurons and fewer GABAergic interneurons than mouse apoE3-NSCs in culture. Thus, the detrimental effects of apoE4 on neurogenesis and GABAergic interneuron survival, as we observed in vivo in apoE4 knock-in mice (Li G. et al. Cell Stem Cell, 2009, 5:634-645), are recapitulated in cultures of mouse iPS cell–derived NSCs in vitro.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We have fully characterized two apoE3/3-hiPS cell lines and two apoE4/4-iPS cell lines.
  • 2) We have established NSC lines from human iPS cells with an apoE3/3 or apoE4/4 genotype. The hNSCs have been maintained in suspension or monolayer culture for multiple passages.
  • 3) We demonstrated that apoE4-hNSCs generated ~50% fewer GABAergic interneurons than apoE3-hNSCs in culture. Thus, the detrimental effects of apoE4 on GABAergic interneuron survival, as we observed in vivo in apoE4 knock-in mice (Li G. et al. Cell Stem Cell, 2009, 5:634-645), are recapitulated in cultures of human iPS cell-derived NSCs in vitro.
  • 4) We established protocols in our lab to differentiate human iPS cell-derived NSCs into different types of neurons in cultures.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We demonstrated that apoE4-miPSC-derived mNSCs had a greater “age-dependent (passage-dependent)” decrease in generation and/or survival of MAP2-positive neurons in cultures.
  • 2) We also demonstrated that apoE4-miPSC-derived mNSCs had an even greater “age-dependent (passage-dependent)” decrease in generation and/or survival of GAD67-positive GABAergic neurons, as seen in vivo in apoE4 knock-in mice (Li et al., Cell Stem Cell, 2009, 5:634–645).
  • 3) We expanded the pilot study reported last year and confirmed the detrimental effect of apoE4 on GABAergic interneuron development/survival of hiPS cell-derived hNSCs. ApoE4 also increased tau phosphorylation, one of the pathological hallmarks of Alzheimer’s disease, in neurons derived from apoE4-hiPS cells.
  • 4) We established a protocol to transplant apoE-miPS cell-derived mNSCs into mouse brains. The transplanted apoE-mNSCs developed into neurons and astrocytes and integrated into the neural circuitry.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of pluripotent stem cells into functional neurons in vitro in culture and in vivo in mice for potential cell replacement therapy. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We demonstrated that mouse GABAergic progenitors transplanted into the hilus of apoE3-KI and apoE4-KI mice developed into mature interneurons and functionally integrated into the hippocampal circuitry.
  • 2) We also demonstrated that transplantation of mouse GABAergic progenitors into the hilus of apoE4-KI mice rescued learning and memory deficits.
  • 3) Transplantation of mouse GABAergic progenitors into the hilus of hippocampus also rescued learning and memory deficits in apoE4-KI mice expressing Alzheimer’s disease-causing APP mutations.

High throughput modeling of human neurodegenerative diseases in embryonic stem cells

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

Mechanisms in Choroid Plexus Epithelial Development

Funding Type: 
New Faculty II
Grant Number: 
RN2-00915
ICOC Funds Committed: 
$2 994 328
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 
  • Our project goals are to define the factors involved in choroid plexus epithelial (CPe) cell development in mice, then apply this information to generate CPe cells from mouse and human embryonic stem cells (ESCs) for clinical applications. The first Aim is to determine whether a factor known as Fgf8 promotes CPe fate, the second Aim addresses whether the Lhx2 transcription factor inhibits CPe, and the third Aim is to generate human CPe cells in culture. Significant progress on these Aims has been made during this first year of the grant. Most importantly, multiple lines of evidence for CPe differentiation from both mouse and human ESCs have been obtained. In addition, the genetically-engineered mESC lines needed for the Lhx2 studies in Aim 2 have been successfully generated and validated. Our major goals for the next year are to further replicate, confirm, and optimize the generation of CPe cells in our mouse and human ESC cultures, and to perform the initial experiments that should determine whether manipulating Fgf8 and Lhx2 in the ESC cultures will enhance CPe generation in culture.
  • Our goal is to define the factors involved in choroid plexus epithelial (CPe) cell development in mice, then to apply this knowledge to generate CPe cells from mouse and human embryonic stem cells (ESCs) for clinical applications. The first two Aims examine Fgf8 and Lhx2 as promoter and inhibitor, respectively, of CPe fate, and the third Aim is to generate human CPe cells in culture. Unexpectedly, we obtained significant evidence for CPe differentiation from both mouse and human ESCs during year 1 of the award. Our aims for year 2 were therefore modified to accelerate the translation of our findings towards a CPe-based regenerative medicine. This year, we developed a second cell culture system for deriving mouse CPe cells, and established a functional assay for CPe cells in culture, which we used to confirm the function of our derived mouse CPe cells. To sort and purify CPe cells for clinical applications, we began characterizing CPe cell complexity, size, and mitochondrial content by flow cytometry, obtained a mouse line with fluorescent CPe cells, and identified three antibodies that may be useful for sorting human CPe cells. A stereotaxic injection system was built, and institutional approvals were obtained, to establish methods for replacing or transplanting CPe cells in the mouse brain.
  • The goal of this project is to define the factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. The first two Aims used mice to examine a potential promoter and inhibitor, respectively, of CPEC fate, and the third Aim is to generate human CPECs in culture. Unexpectedly early success in CPEC derivation from human ESCs has allowed us to accelerate Aim 3 and the pursuit of translational goals this year. We further optimized our existing human CPEC derivation method and developed a second method (a combined suspension-adherent system) that may prove to be much more efficient. Several new GMP-compliant human ESC lines were approved and obtained. To facilitate the translational efforts, we made many new mouse ESC lines that were designed to fluoresce when CPECs are produced, and this was confirmed using the first of these lines. A crude CPEC purification strategy was also developed, and using this strategy, transplantation of partially-purified CPECs into mice was established in the lab this year. Remarkably, we found that transplanted mESC-derived CPECs, on their own, can integrate into endogenous choroid plexus with relatively high efficiency. This opens up several new and exciting therapeutic possibilities. To further enhance choroid plexus engraftment, a mouse CPEC ablation approach is currently being tested. A collaboration was initiated to profile all of the genes expressed by the purified mouse ESC-derived CPECs, and to compare this profile to those expressed by the choroid plexus in developing mice and humans. Industry partnerships and non-provisional patenting were also pursued to enhance the prospects for human CPEC applications in drug screening and treating patients with a wide range of neurodegenerative and other nervous system disorders.
  • The goal of this project is to define factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. Unexpected early success in generating ESC-derived CPECs (dCPECs) allowed us to accelerate and focus on the more translational goals of the project this year. We tested two new culture systems, with promising results from a more controllable and scalable monolayer culture system that will facilitate the improvement of dCPEC generation efficiency. New transcriptome profiling studies allowed us to better define highly-expressed genes for cell surface proteins, which will be targeted to purify dCPECs for downstream applications. New double-labelling and whole mount preparations of mouse choroid plexus have been devised to facilitate ongoing efforts to improve dCPEC engraftment of host choroid plexus after injection, and a new functional assay for dCPEC barrier formation and regulation has been established to complement an already-existing functional secretion assay in the lab. Efforts are also now underway to generate fluorescent and luminescent CPEC reporter hESC lines that should greatly facilitate dCPEC process development (derivation and purification). During this past year, new industry partners were recruited, an initial paper describing the dCPEC technology was published, and an initial patent application on the dCPEC technology was filed.
  • The goal of this project is to define factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. Unexpected early success in generating ESC-derived CPECs (dCPECs) allowed us to accelerate and focus on the more translational goals of the project this year. We further developed two culture systems - a more controllable monolayer system and more scalable rotational aggregate system - that will facilitate the dCPEC work. After several disappointments, improvements in dCPEC differentiation efficiency were obtained with two pharmacologic agents. With help from transcriptome profiling studies, we identified cell surface proteins that could be utilized for dCPEC enrichment, with initial promising results for one candidate surface antigen. A robust whole mount choroid plexus culture system was newly developed to facilitate efforts to improve dCPEC engraftment of host choroid plexus, and methods surrounding the stereotactic injection of dCPECs have been improved. After some difficulties, human TTR BAC constructs that express fluorescent and luminescent reporters were created and validated; these will be used to generate new CPEC reporter mouse lines for endpoint and longitudinal studies, and for in vivo drug testing of compounds that enhance TTR production and CPEC secretion. The initial patent application on the dCPEC technology was reviewed by the US PTO, and a revision was submitted.

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