Neurological Disorders

Coding Dimension ID: 
303
Coding Dimension path name: 
Neurological Disorders
Funding Type: 
Early Translational I
Grant Number: 
TR1-01245
Investigator: 
Type: 
PI
Institution: 
Type: 
Partner-PI
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.
Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01735-A
Investigator: 
Type: 
PI
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.
Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01735-B
Investigator: 
Type: 
PI
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.
  • 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 no-cost extension (year 4) of CIRM funding, we have addressed both Specific Aims 2 and 3, and have specifically answered the following questions: 1) is the HLA-A haplotype important to match in order to prevent brain stem cell rejection, and 2) what are the transcriptome profiles of mouse vs. human compartments? 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 a new technique utilizing RNA sequencing technology on brain sections from 'humanized' mice engrafted with human NSCs. 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. A manuscript has recently been submitted detailing this work to the Journal of Experimental Medicine.
Funding Type: 
Transplantation Immunology
Grant Number: 
RM1-01720
Investigator: 
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.
Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01107
Investigator: 
Name: 
Type: 
PI
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: 

The ability to target a specific locus in the mouse genome and to alter it in a specific fashion has fundamentally changed experimental design and made mice the preeminent model for studying human diseases . However, pathogenesis in humans have unique pathways that may not be revealed by only using mouse or other animal models. An approach that combines the advantages of mouse models with parallel experiments in human embryonic stem cells (hESCs) offers significant advantages over current methodologies. With the large number of hESC lines available, the ability to grow cells in defined media, the development of drug resistant feeders and the reports of strategies to insert DNA with increasing efficiency into hESC, it would only be a matter of time to obtain homologous recombinants in hESCs.

In order to provide direct clues to pathogenesis in human tissues, we propose to use homologous recombination to establish in vitro human disease models in hESCs. As a proof of principle, we have chosen Lou Gehrig's disease (or amyotrophic lateral sclerosis, ALS). ALS is a disease that progressively and selectively attacks motoneurons in the brain and the spinal cord. It becomes fatal when motoneurons controlling breathing are affected. Approximately 2% of ALS cases have been identified to be caused by mutations of the the Cu-Zn superoxide dismutase (SOD1) gene in an autosomal dominant trait. Animal models have been established and researchers have been able to propose disease mechanisms which led to potential treatments, although no cure has been offered yet. This in part might be due to lack of human cell based models and varied mutant copy numbers in transgenic animals as well as the random nature of their integration into the genome.

Here, we propose to generate hESC lines by gene targeting to harbor point mutations in the SOD1 gene, which recapitulates the genetic defects in SOD1 mutated ALS patients. We will further target these mutations in hESC reporter lines of the two important cell types in ALS: motoneurons and astrocytes. The availability of these SOD1 mutated hESC and hESC reporter lines will allow researchers to obtain purified “diseased” motoneurons and astrocytes, which will facilitate the dissection of ALS pathogenesis. The completion of this proposal will provide (1) a highly efficient protocol for performing homologous recombination in hESCs, (2) a package of motoneuron and astrocyte reporters which are useful for both disease and developmental studies along the neural lineages, and (3) a set of ALS disease platforms of hESC lines to serve as an hESC ALS disease in vitro model, as well as a virtually unlimited source of “diseased” motoneurons and astrocytes. This work not only will provide tools to move pathogenesis research for ALS, but also can be reliably extended into other neural and non-neural lineage diseases, of which genetic defects have been identified, including Huntington's disease (HD) and Parkinson’s disease (PD).

Statement of Benefit to California: 

The overall objectives for this proposal are to create in vitro human neurodegenerative disease models using human embryonic stem cells (hESCs), and as a proof of principle, three point mutations of the SOD1 gene which cause familial amyotrophic lateral sclerosis (FALS) will be tested first. These SOD1 missense mutations, G37R, G85R and G93A, have been identified in FALS patients and widely used in rodent models of FALS. We propose to create SOD1 mutations in hESC lines by gene targeting technology which has been proven to be revolutionary in establishing disease models in animals. In addition, we will use similar protocol to generate motoneuron and astrocyte reporter lines in hESCs, since these two cell types and the interaction between them play the most critical roles in the pathogenesis of ALS. After obtaining the three SOD1 missense mutants in motoneuron and astrocyte reporter lines, we will extend our efforts to characterization of these lines, by examining their growth, survival, cell death and other biochemical properties. We will also perform large scale comparisons for genomic and proteomic profiles of the diseased hESC lines with wild type hESCs, as well as comparing the “diseased” and wild type hESC-derived populations of motoneurons and astrocytes.

These experiments will not only provide direct clues for ALS pathogenesis research but also serve as a proof of principle for general disease research using hESCs as a model system. The protocols and reagents developed in this work will be available for Californian researchers and physicians to use for similar neurodegenerative diseases or diseases of other systems. This work will eventually facilitate the scale-up in establishment of human diseases models using human tissues or human cell culture systems for our colleagues in California and around the world.

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.
Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01021
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$918 000
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 

Human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells have considerable potential as sources of differentiated cells for numerous biomedical applications. The ability to introduce targeted changes into the DNA of these cells – a process known as gene targeting – would have very broad implications. For example, mutations could readily be introduced into genes to study their roles in stem cell propagation and differentiation, to analyze mechanisms of human disease, and to develop disease models to aid in creating new therapies. Unfortunately, gene targeting efficiency in hESCs is very low. To meet this urgent need, we propose to develop new molecular tools and novel technologies for high efficiency gene targeting in hES and iPS cells. Importantly, this approach will be coupled with genome-wide identification and functional analysis of genes involved in the process in dopaminergic neuron development, work with fundamental implications for Parkinson's disease.

Barriers to targeted genetic modification include the effective delivery of gene targeting constructs into cells and the introduction of defined changes into the genome. We have developed a high throughput approach to engineer novel properties into a highly promising, safe, and clinically relevant gene delivery vehicle. For example, we have engineered variants of this vehicle with highly efficient gene delivery to neural stem cells (NSCs), and the resulting vehicles can mediate efficient gene targeting. We now propose to engineer novel gene delivery and targeting vehicles optimized for use in hESCs and iPS cells. One application of such an improved vector system will be to study the mechanism of ESC differentiation into dopaminergic neurons aided by the key transcription factor Lmx1a. We propose to identify target genes that are regulated by Lmx1a during dopaminergic neuron differentiation using the newly developed technique of ChIP-seq, in combination with RNA expression and bioinformatics analysis. This work will identify essential control genes that drive dopaminergic neuron differentiation. Furthermore, our improved gene delivery and targeting system will be used for overexpressing candidate genes, knocking them down via RNA interference, and knocking in reporter genes to analyze gene expression networks during neuronal differentiation.

The generation of efficient targeting technologies, in combination with genome wide analysis of gene regulation networks, will provide a general method for identifying and testing key regulatory genes for stem cell self-renewal and differentiation, as well as generating stem cell-based models of human disease. This blend of bioengineering and cell biology therefore has strong potential to create an important new capability for basic and applied stem cell research.

Statement of Benefit to California: 

This proposal will develop novel molecular tools and methodologies that will strongly enhance the scientific, technological, and economic development of stem cell therapeutics in California. The most important net benefit will be for the treatment of human diseases.

Efficiently introducing specific genetic modifications into a stem cell genome is a greatly enabling technology that would impact many downstream medical applications. This capability will further enable investigations of self-renewal and differentiation, two defining properties of human stem cells. New tools to introduce targeted alterations of ES and iPS cells will also yield key model systems to elucidate mechanisms of human disease, and most importantly enable the generation of mutant cell lines to serve as models of human disease and systems for high throughput screening to develop novel therapies. Finally, the reverse process, the repair of genetic lesions responsible for disease, can in the long run enable the generation of patent-specific stem cell lines for therapeutic application.

Each of these applications will directly benefit biomedical knowledge and human health. Furthermore, this proposal directly addresses several research targets of this RFA – the development and utilization of efficient homologous recombination techniques for gene targeting in human stem cells, the development of safer and more effective viral vectors for gene transduction in human stem cells, and the development and analysis of human embryonic stem cell lines with reporter genes inserted into key loci – indicating that CIRM believes that the proposed capabilities are a priority for California’s stem cell effort. While the potential applications of the proposed technology are broad, we will apply it to a specific and urgent biomedical problem: elucidating mechanisms of ES cell differentiation into dopaminergic neurons, part of a critical path towards developing therapies for Parkinson’s disease. While hESCs clearly have this capacity, the underlying mechanisms are incompletely understood, and the efficiency of this process must be improved. We will elucidate transcriptional networks that underlie this process, and utilize our novel gene targeting system to identify and analyze key components of these networks. This work will lead to a better fundamental understanding of mechanisms regulating stem cell differentiation, as well as enhance our ability to control this complex process for biomedical application.

The co-investigators have a strong record of translating basic science and engineering into practice through interactions with industry, including the founding of biotech companies in California. Finally, this collaborative project will focus diverse research groups with many students on an important interdisciplinary project at the interface of science and engineering, thereby training future employees and contributing to the technological and economic development of California.

Progress Report: 
  • The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.
  • The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.
  • The central goal of this project is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. Harnessing a novel directed evolution technology we have developed to improve the properties of a promising viral vehicle, we have significantly increased its gene delivery efficiency to human embryonic and human induced pluripotent stem cells. Furthermore, this advance resulted in considerable improvements in the efficiency of gene targeting (i.e. editing) in the genomes of these cells.
  • In parallel, we have a strong interest in understanding and elucidating mechanisms of luripotent stem cell differentiation into neurons, with for example implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We attempted chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. Progress in this objective was ultimately hampered by the lack of a suitable antibody against Lmx1a. However, in parallel we have used an analogous approach to investigate mechanisms by which RNA transcription is regulated during the differentiation of embryonic stem cells into neurons, including motor neurons. These basic results can now be applied to enhance the efficiency of neuronal differentiation.
Funding Type: 
New Faculty II
Grant Number: 
RN2-00952
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$2 847 600
Disease Focus: 
Stroke
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 

GOALS We propose to determine the effects of different forms of apoE on the development of induced pluripotent stem (iPS) cells into functional neurons. In Aim 1, iPS cells will be generated from skin cells of adult knock-in (KI) mice expressing different forms of human apoE and in humans with different apoE genotypes. In Aim 2, the development of the iPS cells into functional neurons in culture and in mouse brains will be compared. In Aim 3, the effects of different forms of apoE on the functional recovery of mice with acute brain injury treated with iPS cell–derived neural stem cells (NSCs) will be assessed. RATIONALE AND SIGNIFICANCE The central nervous system (CNS) has limited ability to regenerate and recover after injury. For this reason, recovery from acute and chronic neurological diseases, such as stroke and Alzheimer’s disease (AD), is often incomplete and disability results. Embryonic stem cells have great promise for treating or curing neurological diseases, but their therapeutic use is limited by ethical concerns and by rejection reactions after allogenic transplantation. The generation of iPS cells from somatic cells offers a way to potentially circumvent the ethical issues and to generate patient- and disease-specific stem cells for future therapy. In the CNS, apoE plays important roles in lipid homeostasis and in neuronal maintenance. However, apoE2, apoE3, and apoE4 differ in their ability to accomplish these tasks. ApoE4, the major genetic risk factor for AD, is associated with poor clinical outcome and more rapid progression or greater severity of head trauma, stroke, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis—all potential targets of stem cell therapy. This proposal builds on three novel findings in human apoE-KI mice. (1) NSCs express apoE. (2) ApoE plays a role in cell-fate determination (neuron vs astrocyte) of NSCs. (3) ApoE4 impairs the neuronal development of NSCs. Thus, we hypothesize that transplantation of iPS cells derived from apoE4 carriers (~20% of the general population and ~50% of AD patients) might not be beneficial or even detrimental for patients with neurological diseases. We propose in vitro and in vivo studies to assess the effects of different forms of apoE on the development of iPS cells into functional neurons and on the functional recovery of mice with acute brain injury treated with iPS cell-derived NSCs. These studies will shed light on the regulation of neuronal development of iPS cells and help to “optimize” future iPS cell therapy for neurological diseases. SPECIFIC AIMS Aim 1. To establish adult mouse and human iPS cell lines with different apoE genotypes. Aim 2. To determine the isoform-specific effects of apoE on the development of iPS cells into functional neurons in culture and in mouse brains. Aim 3. To assess the isoform-specific effects of apoE on the functional recovery of mice with acute (stroke) brain injury treated with iPS cell-derived NSCs.

Statement of Benefit to California: 

CONTRIBUTION TO THE CALFORNIA ECONOMY: A major goal of regenerative medicine is to repair damaged cells or tissue. My research focuses on (1) understanding the role of neuronal regeneration in central nervous system function and (2) developing stem cell therapy for acute and chronic neurological diseases, including stroke and Alzheimer's disease. Stroke and Alzheimer's disease are the leading causes of disability and dementia and are the fastest growing form of neurological diseases in California, in the USA, and worldwide. My research could benefit the California economy by creating jobs in the biomedical sector. Ultimately, this study could help reduce the adverse impact of neurological diseases. Thereby, I hope to increase the productivity and enhance the quality of life for Californians. The results of my studies will also help develop new technology that could contribute to the California biotechnology industry. The studies will characterize multiple lines of induced pluripotent stem (iPS) cells carrying apoE3, a protein protective to the brain, or apoE4, which is detrimental to the brain and is associated with increased risk of Alzheimer’s disease and other neurodegenerative disorders. These cell lines could be valuable for biotechnology companies and researchers who are screening for drug compounds targeting different neurological diseases. CONTRIBUTION TO THE HEALTH OF CALFORNIANS: The most important contribution of the studies will be to improve the health of Californians. Diseases that are the target of regenerative medicine, such as stroke and Alzheimer’s disease, are major causes of mortality and morbidity, resulting in billions of dollars in healthcare costs and lost productivity. As we continue our efforts in medical research, we hope to one day unlock the secrets of brain development and repair. This knowledge will help medical researchers develop beneficial therapies beyond what is currently available and potentially improve the quality of life and life expectancy of patients with neurological diseases, such as stroke and Alzheimer’s disease.

Progress Report: 
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in Aims 1 and 2.
  • First, we further demonstrated that neural stem cells (NSCs) express apoE. ApoE-KO mice had significantly less hippocampal neurogenesis, but significantly more astrogenesis, than wildtype mice due to decreased Noggin expression in NSCs. In contrast, neuronal maturation in apoE4 knock-in (apoE4-KI) mice was impaired due to reduced survival and function of GABAergic interneurons in the hilus of the hippocampus, and a GABAA receptor potentiator rescued the apoE4-associated decrease in hippocampal neurogenesis. Thus, apoE plays an important role in hippocampal neurogenesis, and the apoE4 isoform impairs GABAergic input to newborn neurons, leading to decreased neurogenesis. A paper describing these data was published in Cell Stem Cell (Li G. et al. 2009, 5:634-645), which evidently is the 400th publication of CIRM-funded projects.
  • Second, we established mouse iPS cell lines from adult mouse fibroblasts of wildtype, apoE knockout (apoE-KO), human apoE2-KI, human apoE3-KI, and human apoE4-KI mice.
  • Finally, we developed NSC lines from mouse iPS cells with different apoE genotypes (wildtype mouse apoE, apoE-KO, apoE2, apoE3, and apoE4). These cell lines will be used to study the effects of apoE isoforms on neuronal development in vitro in culture and in vivo in mouse models.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in the past year, as summarized below.
  • First, We developed human iPS cells from skin fibroblasts of individuals with different apoE genotypes. We are fully characterizing these human iPS cell lines.
  • Second, We are establishing neural stem cell (NSC) lines from human iPS cells with different apoE genotypes. Some of the NSCs have been maintained in monolayer cultures for many generations. These NSCs will be used to study the effects of apoE isoforms on neuronal development in vitro in cultures and in vivo in mice.
  • Finally, we demonstrated that mouse apoE4-NSCs generated significantly fewer total neurons and fewer GABAergic interneurons than mouse apoE3-NSCs in culture. Thus, the detrimental effects of apoE4 on neurogenesis and GABAergic interneuron survival, as we observed in vivo in apoE4 knock-in mice (Li G. et al. Cell Stem Cell, 2009, 5:634-645), are recapitulated in cultures of mouse iPS cell–derived NSCs in vitro.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We have fully characterized two apoE3/3-hiPS cell lines and two apoE4/4-iPS cell lines.
  • 2) We have established NSC lines from human iPS cells with an apoE3/3 or apoE4/4 genotype. The hNSCs have been maintained in suspension or monolayer culture for multiple passages.
  • 3) We demonstrated that apoE4-hNSCs generated ~50% fewer GABAergic interneurons than apoE3-hNSCs in culture. Thus, the detrimental effects of apoE4 on GABAergic interneuron survival, as we observed in vivo in apoE4 knock-in mice (Li G. et al. Cell Stem Cell, 2009, 5:634-645), are recapitulated in cultures of human iPS cell-derived NSCs in vitro.
  • 4) We established protocols in our lab to differentiate human iPS cell-derived NSCs into different types of neurons in cultures.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of induced pluripotent stem (iPS) cells into functional neurons both in vitro and in mice. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We demonstrated that apoE4-miPSC-derived mNSCs had a greater “age-dependent (passage-dependent)” decrease in generation and/or survival of MAP2-positive neurons in cultures.
  • 2) We also demonstrated that apoE4-miPSC-derived mNSCs had an even greater “age-dependent (passage-dependent)” decrease in generation and/or survival of GAD67-positive GABAergic neurons, as seen in vivo in apoE4 knock-in mice (Li et al., Cell Stem Cell, 2009, 5:634–645).
  • 3) We expanded the pilot study reported last year and confirmed the detrimental effect of apoE4 on GABAergic interneuron development/survival of hiPS cell-derived hNSCs. ApoE4 also increased tau phosphorylation, one of the pathological hallmarks of Alzheimer’s disease, in neurons derived from apoE4-hiPS cells.
  • 4) We established a protocol to transplant apoE-miPS cell-derived mNSCs into mouse brains. The transplanted apoE-mNSCs developed into neurons and astrocytes and integrated into the neural circuitry.
  • The goal of this proposal is to determine the isoform-specific effects of apolipoprotein (apo) E on the development of pluripotent stem cells into functional neurons in vitro in culture and in vivo in mice for potential cell replacement therapy. Toward this goal, we have made significant progress in all three aims in the past year, as summarized below.
  • 1) We demonstrated that mouse GABAergic progenitors transplanted into the hilus of apoE3-KI and apoE4-KI mice developed into mature interneurons and functionally integrated into the hippocampal circuitry.
  • 2) We also demonstrated that transplantation of mouse GABAergic progenitors into the hilus of apoE4-KI mice rescued learning and memory deficits.
  • 3) Transplantation of mouse GABAergic progenitors into the hilus of hippocampus also rescued learning and memory deficits in apoE4-KI mice expressing Alzheimer’s disease-causing APP mutations.
Funding Type: 
New Faculty II
Grant Number: 
RN2-00919
Investigator: 
ICOC Funds Committed: 
$2 259 092
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Neuropathy
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 

An important class of neurological diseases predominantly affects spinal motor neurons, the neurons that control muscle movement. The most well known of these motor neuronopathies is Amyotrophic Lateral Sclerosis (ALS), commonly referred to as Lou Gehrig’s disease for the famous Yankee first baseman who died of the disease. The first symptoms of ALS are usually increasing difficulty walking or speaking clearly. People with ALS progressively lose their ability to initate and control movements, and may become totally paralyzed during the late stages of the disease. There are no cures or effective treatments for these diseases. Riluzole (Rilutek), the only FDA approved medication for ALS, only modestly slows disease progression. Consequently, ALS is usually fatal within one to five years from onset, with half dying within eighteen months. Although genetic studies have identified many mutations that cause these diseases, it is not understood why these mutations kill motor neurons. This lack of understanding about the root causes of motor neuron diseases currently hinders the development of effective treatments. We seek to study motor neurons carrying these mutations in cell culture dishes to understand how these diseases sicken and kill these cells. To generate these motor neurons, we will use embryonic stem cells. Embryonic stem cells can become any cell in our body, including motor neurons. We have developed a new technology that allows us to quickly replace healthy genes with mutant genes in mouse embryonic stem cells. We will use this technology to insert both normal and disease-associated versions of genes into embryonic stem cells. Study of the healthy and mutant mutant motor neurons derived from these embryonic stem cells will shed light on the ways in which the mutations cause harm. The development of cell based models of human diseases is likely to have additional benefits as well. For example, diseased motor neurons grown in cell culture dishes can be quickly and efficiently screened with potential drugs to discover agents that slow, halt or reverse the cellular damage. It is our hope that these experiments will both deepen our understanding of important neurodegenerative disorders, and lead to new directions for the development of effective therapies.

Statement of Benefit to California: 

Over 6,000 Americans are diagnosed each year with motor neuronopathies, about the same as are diagnosed with multiple sclerosis. One form of this illness, ALS, is responsible for about one in every 800 deaths, and cause many lengthy and costly hospital admissions. We propose using stem cells to model these diseases so that we can gain a deeper understanding of their root causes. It is our expectation that this deeper understanding will lead to new and better approaches to the treatment of these disorders. In addition, our technology for developing embryonic stem cell-based models of human diseases is likely to have applications in the biotechnology sector. Although our technology is most applicable for modeling simple dominant genetic diseases, it can be adapted to model recessive and complex disorders. Beyond increasing our understanding of human diseases, these cellular models represent useful screening tools for testing novel pharmacological treatments. Identification and development of these new therapies may support new companies or new products for existing companies. We hope that using stem cells to model neurodegenerative disorders will lead to progress in the fight against these diseases, as well as provide the tools and examples for those in academia and industry who hope to create stem cell models of other clinically important disorders.

Progress Report: 
  • We have been developing new tools for the genetic modification of embryonic stem cells (ESCs). Part of the potential for use of ESCs in treatments or as models of disease depends on the ability to change genes within ESCs. We have developed a novel system, which we call the Floxin system, that allows for the more efficient modification of genes within mouse ESCs than has been historically feasible. We have used this system to insert mutations that cause human diseases into mouse ESCs. Introducing human mutations into ESCs has allowed us to study the function of these mutations in the context of stem cell function and gain insight into how these mutations cause human disease.
  • We are interested in extending our findings by modeling an important class of neurological diseases that predominantly affect spinal motor neurons, the neurons that control muscle movement. The most well known of these motor neuronopathies is Amyotrophic Lateral Sclerosis (ALS), commonly referred to as Lou Gehrig’s disease, but there are a number of other motor neuronopathies including Hereditary Motor Neuronopathy and Spinal Muscular Atrophy.
  • Human genetic studies have identified many mutations that cause these diseases, but it is not understood why these mutations kill motor neurons. This lack of understanding about the root causes of motor neuron diseases currently hinders the development of effective treatments. We are currently using the Floxin system to introduce human motor neuronopathy-associated mutations into mouse ESCs. We have introduced mutations into two disease-associated genes, and are deriving motor neurons from these modified ESCs to study how the mutations kill these cells.
  • The development of cell-based models of human diseases is likely to have additional benefits as well. For example, diseased motor neurons grown in cell culture dishes can be quickly and efficiently screened with potential drugs to discover agents that slow, halt or reverse the cellular damage. It is our hope that these experiments will both deepen our understanding of important neurodegenerative disorders, and lead to new directions for the development of effective therapies.
  • We have made the resource of Floxin vectors and the greater than 24,000 characterized Floxin compatible ESC lines available to the research community. Application of the Floxin technology to this resource will allow genetic modification of more than 4,500 genes in ESCs. Furthermore, we are adapting the Floxin technology for use in human ESCs which may allow for tractable genetic engineering in these cells. We anticipate that this technology will allow many researchers to create cellular models of human disease and other genetic modifications that will facilitate the use of stem cells in fighting diverse diseases.
  • We have developed new tools for the genetic modification of embryonic stem cells (ESCs) and are using these tools to model human diseases. Part of the potential for use of ESCs in treatments or as models of disease depends on the ability to change genes within ESCs. We have developed a novel system, which we call the Floxin system, that allows for the more efficient modification of genes within mouse ESCs than has been historically feasible. We use this system to insert mutations that cause human diseases into mouse ESCs. Introducing human mutations into ESCs has allowed us to study the function of these mutations in the context of stem cell function and gain insight into how these mutations cause human disease. To date, we have investigated an inherited congenital malformation syndrome called Orofaciodigital syndrome and elucidated that the underlying birth defects are caused by misregulation of cilia and centrioles, structures within all cells. We have also used our system to investigate how genes are regulated by Polycomb-like proteins and to reveal how cilia control ESC differentiation into motor neurons, findings that shed light on the control of motor neuron production from ESCs.
  • We are extending our findings by modeling an important class of neurological diseases that predominantly affect spinal motor neurons, the neurons that control muscle movement. The most well known of these motor neuronopathies is Amyotrophic Lateral Sclerosis (ALS), commonly referred to as Lou Gehrig’s disease, but there are a number of other motor neuronopathies including Hereditary Motor Neuronopathy and Spinal Muscular Atrophy. Human genetic studies have identified many mutations that cause these diseases, but it is not understood why these mutations kill motor neurons. This lack of understanding about the root causes of motor neuron diseases currently hinders the development of effective treatments.
  • We have used the Floxin system to introduce human motor neuronopathy-associated mutations into mouse ESCs. We have introduced mutations into two disease-associated genes, and have derived motor neurons from these modified ESCs to study how the mutations kill these cells. The development of cell-based models of human diseases is likely to have additional benefits as well. For example, diseased motor neurons grown in cell culture dishes can be quickly and efficiently screened with potential drugs to discover agents that slow, halt or reverse the cellular damage. It is our hope that these experiments will both deepen our understanding of important neurodegenerative disorders, and lead to new directions for the development of effective therapies.
  • We have made the resource of Floxin vectors and the greater than 24,000 characterized Floxin compatible ESC lines available to the research community. Application of the Floxin technology to this resource will allow genetic modification of more than 4,500 genes in ESCs. Furthermore, we are hoping to adapt the Floxin technology for use in human ESCs which may allow for tractable genetic engineering in these cells. We anticipate that this technology will allow many researchers to create cellular models of human disease and other genetic modifications that will facilitate the use of stem cells in fighting diverse diseases.
  • An important class of neurological diseases predominantly affects spinal motor neurons, the neurons that control muscle movement. The most well known of these motor neuronopathies is Amyotrophic Lateral Sclerosis (ALS), commonly referred to as Lou Gehrig’s disease for the famous Yankee first baseman who died of the disease. The first symptoms of ALS are usually increasing difficulty walking or speaking clearly. People with ALS progressively lose their ability to initate and control movements, and may become totally paralyzed during the late stages of the disease. There are no cures or effective treatments for these diseases. Riluzole (Rilutek), the only FDA approved medication for ALS, only modestly slows disease progression. Consequently, ALS is usually fatal within one to five years from onset, with half dying within eighteen months.
  • Although genetic studies have identified many mutations that cause these diseases, it is not understood why these mutations kill motor neurons. This lack of understanding about the root causes of motor neuron diseases currently hinders the development of effective treatments. We seek to study motor neurons carrying these mutations in cell culture dishes to understand how these diseases sicken and kill these cells.
  • To generate these motor neurons, we are using embryonic stem cells. Embryonic stem cells can become any cell in our body, including motor neurons. We have developed a new technology that allows us to quickly replace healthy genes with mutant genes in mouse embryonic stem cells. We are using this technology to insert both normal and disease-associated versions of genes into embryonic stem cells. Study of the healthy and mutant mutant motor neurons derived from these embryonic stem cells will shed light on the ways in which the mutations cause harm.
  • We have been using the mutant embryonic stem cells to assay leading hypotheses about how diseases like ALS begin. In addition, we are using the embryonic stem cells to create new animal models of ALS. Finally, we are adapting our technology to be able to create more faithful models of disease using embryonic stem cells in order to expedite understanding into the origins of these diseases.
  • Neurodegenerative diseases, including Alzheimer disease, Parkinson disease, and Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig’s disease), affect an increasing proportion of our population as the median age increases. There are no cures for any of these disorders. One reason for the absence of cures has been the absence of good models to understand how neurodegeneration happens.
  • Genetic studies have identified many of the genes involved in neurodegeneration. To understand how these mutations lead to motor neuron degeneration in ALS, we have creased embryonic stem cells (ESCs) that contain the human ALS-associated mutations. We have also created mice that express these human ALS-associated mutations. We are studying motor neurons derived from the ESCs and the mutant mice to understand how motor neurons die in ALS. We are defining the proteins and RNAs that interact with normal and disease-associated proteins, and following the mutant neurons over time to examine how they die. Currently, we are testing the hypothesis that disease mutations alter the gene product’s normal interactions, leading to a tonic increase in cell death rate. After several decades of life, the loss of neurons surpasses compensatory mechanisms, leading to the emergence of symptoms.
  • Neurodegenerative diseases, including Alzheimer disease, Parkinson disease, and Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig’s disease), affect an increasing proportion of our population as the median age increases. There are no cures for any of these disorders. One reason for the absence of cures has been the absence of good models to understand how neurodegeneration happens.
  • Genetic studies have identified many of the genes involved in neurodegeneration. To understand how these mutations lead to motor neuron degeneration in ALS, we have creased embryonic stem cells (ESCs) that contain the human ALS-associated mutations. We have also created mice that express these human ALS-associated mutations. We studied motor neurons derived from the ESCs and the mutant mice and found that motor neurons with ALS-associated mutations die at increased rates. We identified proteins that interact with normal and disease-associated proteins. We identified that mutant proteins showed different interactions than normal proteins. After several decades of life, the loss of neurons surpasses compensatory mechanisms, leading to the emergence of symptoms.
Funding Type: 
New Faculty II
Grant Number: 
RN2-00915
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$2 994 328
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 

Buried deep inside the brain are cells known as choroid plexus epithelial (CPe) cells. Although not as famous as other cells in the nervous system, CPe cells perform a large number of important jobs that keep the brain and spinal cord healthy. They produce the fluid (known as cerebrospinal fluid, or CSF) that bathes the brain and spinal cord with many nourishing chemicals, which promote normal nervous system health and function, learning and memory, and neural repair following injury. In addition, CPe cells protect the brain and spinal cord from toxins – such as heavy metals and the amyloid-beta peptide associated with Alzheimer’s disease – by absorbing them or preventing them from entering the nervous system altogether by forming the so-called blood-CSF barrier. Accordingly, as CPe functions diminish during normal aging or in accelerated fashion in certain diseases, memory loss, Alzheimer’s disease, and a number of other neurologic and neuropsychiatric disorders may ensue or become worse. The ability to grow and make CPe cells should therefore enable many clinical applications, such as CPe cell replacements, transplants, and pharmaceutical studies to identify beneficial drugs that can pass through the blood-CSF barrier. However, all of these potential applications are limited by the current inability to make and expand CPe cells in culture. Our published and preliminary studies suggest that it should be feasible to generate CPe cells in culture. Our broad goals are to study how CPe cells form during normal development, then use this information to make human CPe cells for clinical applications. To achieve this goal, our approach will be to use mice to study how the CPe develops normally, then use both mouse and human stem cells to make CPe cells in culture. Our published and preliminary studies have defined one critical factor for this process (known as Bmp4) and identify candidate factors that work with Bmp4 to regulate whether or not CPe cells are formed. In Aim 1, we test whether a molecule known as Fgf8 provides CPe “competency” – i.e. whether Fgf8 allows cells to become CPe cells when exposed to Bmp4. In Aim 2, we test whether a gene known as Lhx2 prevents cortical cells from becoming CPe cells in response to Bmp4. In Aim 3, we manipulate Bmp4, Fgf8, and Lhx2 in hESC cultures to make human CPe cells. If successful, this proposal should greatly improve our understanding of normal CPe development and enable a number of CPe-based clinical applications with significant potential to improve human health.

Statement of Benefit to California: 

Our proposal to study choroid plexus epithelial (CPe) cell development and to make CPe cells in culture for clinical applications should benefit the State of California and its citizens in a number of ways. In the short term, this project will provide employment, education and training in stem cell research for a handful of California residents, and will support California-based companies that provide supplies for the stem cell and biomedical research communities. In the longer term, success in making CPe cells in culture should enable many new CPe-based clinical applications, stimulate CPe studies and applications by stem cell companies, and enable screens to identify agents that allow for passage of therapeutics across the blood-CSF barrier, which remains a significant roadblock to the development of pharmaceuticals for neurological and neuropsychiatric disorders. Such outcomes would ultimately stimulate investment in California-based companies and benefit the health of many California citizens, which may reduce the economic burden of health care in the state.

Progress Report: 
  • Our project goals are to define the factors involved in choroid plexus epithelial (CPe) cell development in mice, then apply this information to generate CPe cells from mouse and human embryonic stem cells (ESCs) for clinical applications. The first Aim is to determine whether a factor known as Fgf8 promotes CPe fate, the second Aim addresses whether the Lhx2 transcription factor inhibits CPe, and the third Aim is to generate human CPe cells in culture. Significant progress on these Aims has been made during this first year of the grant. Most importantly, multiple lines of evidence for CPe differentiation from both mouse and human ESCs have been obtained. In addition, the genetically-engineered mESC lines needed for the Lhx2 studies in Aim 2 have been successfully generated and validated. Our major goals for the next year are to further replicate, confirm, and optimize the generation of CPe cells in our mouse and human ESC cultures, and to perform the initial experiments that should determine whether manipulating Fgf8 and Lhx2 in the ESC cultures will enhance CPe generation in culture.
  • Our goal is to define the factors involved in choroid plexus epithelial (CPe) cell development in mice, then to apply this knowledge to generate CPe cells from mouse and human embryonic stem cells (ESCs) for clinical applications. The first two Aims examine Fgf8 and Lhx2 as promoter and inhibitor, respectively, of CPe fate, and the third Aim is to generate human CPe cells in culture. Unexpectedly, we obtained significant evidence for CPe differentiation from both mouse and human ESCs during year 1 of the award. Our aims for year 2 were therefore modified to accelerate the translation of our findings towards a CPe-based regenerative medicine. This year, we developed a second cell culture system for deriving mouse CPe cells, and established a functional assay for CPe cells in culture, which we used to confirm the function of our derived mouse CPe cells. To sort and purify CPe cells for clinical applications, we began characterizing CPe cell complexity, size, and mitochondrial content by flow cytometry, obtained a mouse line with fluorescent CPe cells, and identified three antibodies that may be useful for sorting human CPe cells. A stereotaxic injection system was built, and institutional approvals were obtained, to establish methods for replacing or transplanting CPe cells in the mouse brain.
  • The goal of this project is to define the factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. The first two Aims used mice to examine a potential promoter and inhibitor, respectively, of CPEC fate, and the third Aim is to generate human CPECs in culture. Unexpectedly early success in CPEC derivation from human ESCs has allowed us to accelerate Aim 3 and the pursuit of translational goals this year. We further optimized our existing human CPEC derivation method and developed a second method (a combined suspension-adherent system) that may prove to be much more efficient. Several new GMP-compliant human ESC lines were approved and obtained. To facilitate the translational efforts, we made many new mouse ESC lines that were designed to fluoresce when CPECs are produced, and this was confirmed using the first of these lines. A crude CPEC purification strategy was also developed, and using this strategy, transplantation of partially-purified CPECs into mice was established in the lab this year. Remarkably, we found that transplanted mESC-derived CPECs, on their own, can integrate into endogenous choroid plexus with relatively high efficiency. This opens up several new and exciting therapeutic possibilities. To further enhance choroid plexus engraftment, a mouse CPEC ablation approach is currently being tested. A collaboration was initiated to profile all of the genes expressed by the purified mouse ESC-derived CPECs, and to compare this profile to those expressed by the choroid plexus in developing mice and humans. Industry partnerships and non-provisional patenting were also pursued to enhance the prospects for human CPEC applications in drug screening and treating patients with a wide range of neurodegenerative and other nervous system disorders.
  • The goal of this project is to define factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. Unexpected early success in generating ESC-derived CPECs (dCPECs) allowed us to accelerate and focus on the more translational goals of the project this year. We tested two new culture systems, with promising results from a more controllable and scalable monolayer culture system that will facilitate the improvement of dCPEC generation efficiency. New transcriptome profiling studies allowed us to better define highly-expressed genes for cell surface proteins, which will be targeted to purify dCPECs for downstream applications. New double-labelling and whole mount preparations of mouse choroid plexus have been devised to facilitate ongoing efforts to improve dCPEC engraftment of host choroid plexus after injection, and a new functional assay for dCPEC barrier formation and regulation has been established to complement an already-existing functional secretion assay in the lab. Efforts are also now underway to generate fluorescent and luminescent CPEC reporter hESC lines that should greatly facilitate dCPEC process development (derivation and purification). During this past year, new industry partners were recruited, an initial paper describing the dCPEC technology was published, and an initial patent application on the dCPEC technology was filed.
  • The goal of this project is to define factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. Unexpected early success in generating ESC-derived CPECs (dCPECs) allowed us to accelerate and focus on the more translational goals of the project this year. We further developed two culture systems - a more controllable monolayer system and more scalable rotational aggregate system - that will facilitate the dCPEC work. After several disappointments, improvements in dCPEC differentiation efficiency were obtained with two pharmacologic agents. With help from transcriptome profiling studies, we identified cell surface proteins that could be utilized for dCPEC enrichment, with initial promising results for one candidate surface antigen. A robust whole mount choroid plexus culture system was newly developed to facilitate efforts to improve dCPEC engraftment of host choroid plexus, and methods surrounding the stereotactic injection of dCPECs have been improved. After some difficulties, human TTR BAC constructs that express fluorescent and luminescent reporters were created and validated; these will be used to generate new CPEC reporter mouse lines for endpoint and longitudinal studies, and for in vivo drug testing of compounds that enhance TTR production and CPEC secretion. The initial patent application on the dCPEC technology was reviewed by the US PTO, and a revision was submitted.
Funding Type: 
New Cell Lines
Grant Number: 
RL1-00682
Investigator: 
ICOC Funds Committed: 
$1 589 760
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 

Parkinson's disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.

The mechanisms of PD progression are currently unknown. However, genetic studies have identified that mutations (changes) in seven genes, including ?-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases.

One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).

In this study, we propose to create stem cell lines that possess PD-associated mutations in two causative genes, PINK1 and parkin, using either rejected early stage embryos or cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that PD-associated abnormal parkin or PINK1 genes cause degeneration of stem cell-derived dopaminergic neurons, and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which harbor abnormal or mutant parkin or PINK1 genes; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.

Statement of Benefit to California: 

Parkinson's disease (PD) is the second leading neurodegenerative disease with no current cure available. Compared to other states, California is the highest in the incidence of this particular disease. First, California growers use approximately 250 million pounds of pesticides annually, about a quarter of all pesticides used in the US (Cal Pesticide use reporting system). A commonly used herbicide, paraquat, has been shown to induce parkinsonism in both animals and human. Other pesticides are also proposed as potential causative agents for PD. Studies have shown increased PD-caused mortality in agricultural pesticide-use counties in comparison to those non-use counties in California. Second, California has the largest Hispanic population. Studies suggest that incidence of PD is the highest among Hispanics (Van Den Eeden et al, American Journal of Epidemiology, Vol 157, pages 1015-1022, 2003). Thus, finding effective treatments of PD will significantly benefit citizens in California.

Progress Report: 
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • The mechanism of PD progression are currently unknown. However, genetic studies have identified that mutations (changes) in multiple genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that possess PD-associated mutations in two causative genes, PINK1 and parkin, using either rejected early stage embryos or cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that PD-associated abnormal parkin or PINK1 genes cause degeneration of stem cell-derived dopaminergic neurons, and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which harbor abnormal or mutant parkin or PINK1 genes; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last year, we have successfully generated primary skin fibroblast cultures from PD patients harboring mutations of parkin, PINK1, and DJ-1 genes, as well as sporadic PD patients and normal individuals. By using these cells, we have already generated four induced stem cell lines expressing multiple pluripotent markers (two from PD patients and two from normal individuals. These lines can also form teratomas with cells from three germ layers using mouse as host. These findings suggest that the induced pluripotent cell lines generated in the lab are likely PD patient specific stem cells.
  • During the next report year, we will continue to generate more PD patient-specific induced pluripotent stem cells. We will carefully characterize all lines generated in the lab as proposed. Furthermore, we will adapt protocols to differentiate the new lines into dopaminergic neurons.
  • Public Summary of Scientific Progress
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder affecting approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately, there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • Genetic studies have identified that mutations (changes) in multiple genes cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes in PD-affected dopamine-secretion neurons may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that possess PD-associated mutations in two causative genes, PINK1 and parkin, using either rejected early stage embryos or cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that PD-associated abnormal parkin or PINK1 genes cause degeneration of stem cell-derived dopaminergic neurons, and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which harbor abnormal or mutant parkin or PINK1 genes; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last year, we have successfully obtained more primary skin fibroblast cultures from PD patients harboring mutations of parkin, PINK1, DJ-1 and PLA2G6 genes, as well as sporadic PD patients and normal control individuals. By using these cells, we have already generated 9 induced stem cell lines expressing multiple pluripotent markers (7 from PD patients and 2 from normal individuals). These lines can also form teratomas with cells from three germ layers using mouse as host. These findings suggest that the induced pluripotent cell lines generated in the lab are likely PD patient specific stem cells.
  • During the next report year, we will continue to generate more PD patient-specific induced pluripotent stem cells. We will carefully characterize all lines generated in the lab as proposed. Furthermore, we will adapt protocols to differentiate the new lines into dopaminergic neurons.
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • The mechanism of PD progression is currently unknown. However, genetic studies have identified that mutations (changes) in multiple genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that either have the genetic background of sporadic PD patients or possess PD-associated mutations using cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that the degeneration of stem cell-derived dopaminergic neurons and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which either have the genetic background of sporadic PD patients or harbor PD specific gene mutantions; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last year, we have finished to develop 15 lines of iPSCs. These include 5 lines from normal control individuals, 5 lines from sporadic Parkinson disease patients, and 5 lines from Parkinson disease patients harboring disease related mutations of PINK1, DJ-1 and PLA2G6 genes. These lines provide an unique opportunity to systematically study comparative pathophysiology of Parkinson disease using sporadic and genetic cases. Moreover, we indeed spent more than a year in optimizing the condition for differentiation of these lines. It is recognized that iPSCs are more difficult to differentiate than the hESCs. We are now able to finalize the protocols to have all lines be differentiated in vitro. Therefore, we will be able to compare differences among the controls, sporadic PD and genetic PD at the level of cell biology and molecular biology.
  • During the next report year, we will differentiate all lines into DA neurons and carefully the functional changes of these cells. We hope that the results will reveal some molecular basis of PD pathogenesis from these human neurons.
  • Parkinson’s disease (PD) is currently the most common neurodegenerative movement disorder, severely debilitating approximately 1-2% of the US population. The disease is caused by a selective loss of dopamine-producing neurons located in a specific region of the brain. This loss leads to significant motor function impairment and age-dependent tremors. Unfortunately there is currently no cure for PD, however a synthetic dopamine treatment (L-DOPA), temporarily alleviates symptoms.
  • The mechanism of PD progression is currently unknown. However, genetic studies have identified that mutations (changes) in multiple genes, including α-synuclein, LRRK2, uchL1, parkin, PINK1, DJ-1 and ATP13A2 cause familial PD. Although the familial form of PD only affects a small portion of PD cases, uncovering the function of these genes may provide insight into the mechanisms that lead to the majority of PD cases.
  • One of the best strategies to study PD mechanisms is to generate experimental models that mimic the initiation and progression of PD. A number of cellular and animal models have been developed for PD research. However, a model, which closely resembles the human degeneration process of PD, is currently not available because human neurons are unable to continuously propagate (grow) in culture. Human stem cells provide an opportunity to fulfill this task because these cells can grow and be programmed to generate dopamine nerve cells (the neurons under assault in PD patients).
  • In this study, we propose to create stem cell lines that either have the genetic background of sporadic PD patients or possess PD-associated mutations using cultured patient fibroblasts. These cell lines will in effect, represent a model of human PD degeneration of dopaminergic neurons. Our working hypothesis is that the degeneration of stem cell-derived dopaminergic neurons and dopaminergic neurons in vivo via the same mechanism. We will fulfill three tasks in this study; 1/ To generate the PD-stem cell (PD-SCs) line which either have the genetic background of sporadic PD patients or harbor PD specific gene mutantions; 2/ To determine the whether the PD-SCs cell lines can form into midbrain dopaminergic nerve cells; 3/ To determine whether mutations in parkin and PINK1 effect the survival of dopaminergic neurons which are derived from the PD-SCs cells. Successful completion of this study will yield novel cellular models for studying the mechanisms involved in PD initiation and progression, and further screening remedies for PD treatment.
  • During last four years, we have finished to develop 15 lines of iPSCs. These include 5 lines from normal control individuals, 5 lines from sporadic Parkinson disease patients, and 5 lines from Parkinson disease patients harboring disease related mutations of PINK1, DJ-1 and PLA2G6 genes. These iPS lines are shown to have biochemical and genomic characteristics of human ES cells. These lines provide an unique opportunity to systematically study comparative pathophysiology of Parkinson disease using sporadic and genetic cases. Using these lines, we have identified a group of genes differentially expressed and differentially methylated between iPS cells derived from PD patients and iPS cells derived from normal control individuals. However, we recognize that iPSCs are more difficult to differentiate than the hESCs. We are yet to finalize the protocols to have all lines be differentiated in vitro. Our goal is to compare differences among the controls, sporadic PD and genetic PD at the level of cell biology and molecular biology.

Pages