Derivation of Novel Late Onset Disease-Specific Stem Cell Lines Using Highly Accurate Single Cell Genotyping Technology

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00682
ICOC Funds Committed: 
$0
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
iPS Cell
Public Abstract: 
The creation, propagation, and free distribution of novel human embryonic stem cell (hESC) lines are urgently needed in order to bring medical researchers one step closer to successfully treating many of the existing debilitating diseases. Although several federally approved embryonic stem cell lines exist, they are limited in their utility for a variety of reasons, most notably lack of genetic diversity. In order to fill this gap, we propose to derive novel hESC lines from embryos with inherited susceptibility to diseases that are relatively common and debilitating, such as cardiomyopathy, type II diabetes, early onset Alzheimer’s disease, and familial early onset breast cancer. Preimplantation genetic diagnosis (PGD) of embryos during in vitro fertilization (IVF) remains the most effective method for derivation of disease-specific hESC lines, and is the focus of this application. A major drawback of current PGD methods is their inability to simultaneously detect both aneuploidy (a change in the number of chromosomes) and multiple genes known to cause a disease. Most PGD screens for embryos with abnormal number of chromosomes to improve implantation rates and reduce spontaneous abortions, and in nearly all other cases, PGD only screens for diseases that develop during early childhood. As a result, none of the PGD screens for diseases that develop during adulthood. We will be able to create that opportunity with our {REDACTED} product, which is the only method that can simultaenously measure both disease-linked genes and aneuploidy in a single cell isolated from an embryo. In collaboration with our key California IVF clinic partners, we plan to first test our PSTM technology with unused embryos, and then apply the technology to screen hundreds to thousands of embryos per year. Given our ability to test for both chromosome abnormalities and genetic disease simultaneously, we will be in a unique position to screen each of these embryos for diseases that are not routinely tested by other PGD methods. With the parents’ consent, embryos that test positive for susceptibility to diseases of interest will be used to derive hESC lines and made available for free distribution to the research community worldwide. If funded, we will derive hESC lines that will make several critical contributions to the field of stem cell research, ranging from empowering scientists to answer fundamental biological questions to facilitating the development of safe targeted therapeutics.
Statement of Benefit to California: 
Our company is headquartered in {REDACTED}. We provide technical, high-salaried employment for dozens of California residents. With Series A venture backing from {REDACTED} we have outlined an ambitious plan for growth, promising to bring even more jobs to the state. Additionally, we have close strategic partnerships with three California-based IVF clinics: {REDACTED} These IVF clinics, and their patients, will be among the first beneficiaries of our groundbreaking technologies. Most importantly, if this grant is funded, our company will, in collaboration with the {REDACTED} Department of Genetics, help bring novel human embryonic stem cell lines to California scientists. These disease-specific lines will be a superb resource for California researchers working to understand, prevent, and cure debilitating genetic diseases. This resource will help keep California at the forefront of stem cell research worldwide, enriching our local economy and earning further prestige for nonprofit research institutions. Local drug companies will benefit from local resources in nonprofit stem cell research. Finally, California residents could eventually benefit from advances in medicine that result from this important local biological resource.
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.

© 2013 California Institute for Regenerative Medicine