Tools and Technologies I
Parkinson's disease is a neurodegenerative disease resulting in the loss of a specialized class of cells in the brain that control movement and coordination. Current pharmaceutical treatments for Parkinson’s disease typically supplement the neurotransmitter produced by the cells, yet unfortunately the disease is very progressive and even more of these cells are lost. A very promising treatment, therefore, would focus on a strategy to protect these cells and prevent the disease from progressing. One way to protect these cells is to use pharmaceutical medicines. But in order to identify candidate drugs, most strategies require growing large number of these cells in a dish and testing compounds that can protect them from the effects of Parkinson’s-causing chemicals or genetic predisposition. To date, insufficient sources of cells have existed, making large-scale screening impossible. Embryonic stem (ES) cells have the marvelous capacity to divide indefinitely, making them an ideal resource to derive large numbers of specialized cells. Yet the differentiation process, by which they are made into specialized cells, varies for each type of cell desired. Previously, a lengthy process was required in order to derive the specialized cells involved in Parkinson’s disease, but recent research has advanced this goal such that a simple differentiation technique may be soon realized. Our strategy takes advantage of previous results to produce two genetically modified human embryonic stem cell lines for use in a drug screening assay to protect Parkinson’s cells from similar toxins that cause the disease. One key advance of our strategy will be the inclusion of recombination sequences in key genes, allowing other researchers to ‘hot swap’ genes and other markers of interest for a variety of purposes. The assay described in this proposal will be the first of its kind in California, and will pave the way towards the development of large-scale pharmaceutical screens. The proposed research provides long-term benefits to California State citizens through design of a novel in vitro neuroprotective assay that could lead to novel and safer drugs for Parkinson's patients, significant academic research tools to aid in the understanding of the biology Parkinson's disease, intellectual property gains fostering commercial development, increase in the biotechnology strength in the state, generate jobs, generate sales of reagents and supplies from California companies, and bring in revenues from companies outside the state via corporate partnering.
Statement of Benefit to California:
By approving Proposition 71, voters in California agreed to fund ground breaking stem cell research in regenerative medicine to improve the lives of California citizens, and to benefit the State economically. Our proposal is designed to combat Parkinson's disease, which affects an atypically large percentage of California State citizens compared to most states. Parkinson's disease is a neurodegenerative disease resulting in the loss of a specialized class of dopamine-producing neurons. Although there are genetic origins of Parkinson's disease, the large majority of cases are idiopathic. According to many reports, pesticides such as paraquat and dieldrin used by California's significant agricultural community may be linked to many of these cases. The successful completion of the goals proposed here will result in two genetically modified stem cell lines for use in a drug screening assay to protect these neurons from toxic insults. Further insights into Parkinson’s disease may also alter pesticide application and will translate into better protection for California State citizens. As the first of its kind in California, this assay will provide long-term benefits leading to new preclinical pharmaceutical development for environmental and genetic Parkinson's disease, improving immeasurably the lives of thousands of California State citizens and reducing associated health care expenditures. As well, it is anticipated that the cell lines generated from this project will serve as significant research tools for improving the differentiation of dopaminergic neurons for clinical purposes, the novel in vitro neuroprotective assay developed with this funding could lead to novel and safer drugs for Parkinson's patients, significant academic research tools to aid in the understanding of the biology Parkinson's disease, intellectual property gains fostering commercial development, increase in the biotechnology strength in the state, generate jobs, generate sales of reagents and supplies from California companies, and bring in revenues from companies outside the state via corporate partnering.
This proposal focuses on the development of methods for the differentiation and selection of midbrain dopaminergic (DA) neurons from human embryonic stem cells (hESCs) for the purpose of testing neuroprotective compounds against chemicals that cause Parkinson’s disease (PD). In Aim 1 the applicants plan to accomplish their objectives by first producing a genetically engineered hESC marker line with a fluorescent reporter gene under control of a DA-neuron specific promoter. The applicant proposes that the DA-reporter hESC line will allow more efficient differentiation of dopaminergic neurons to greater heterogeneity than currently possible without the reporter line. In Aim 2, the engineered hESCs from Aim 1 will be differentiated into DA neurons using a novel transcription factor-based differentiation technique which will further enhance the dopaminergic differentiation efficiency and purity. Finally, Aim 3 studies are focused on confirmation that the generated dopaminergic neurons are functional in a classic neuroprotective assay. Reviewers agreed that generation of pure populations of functional dopaminergic neurons from hESC, if successful, would have a significant impact on the ability perform in vitro testing of novel protective therapeuties for Parkinson’s Disease. However, they expressed major concerns about the feasibility of the particular approaches, and felt that the research design was overly ambitious for a 2-year project and lacked sufficient preliminary data to suggest that the project would be complete in a short funding period. Reviewers felt that the PI may not have sufficient expertise to drive the project but acknowledged that some of the collaborators were recognized experts in the area and could overcome the relative inexperience of the PI in this area. Overall, in terms of impact, reviewers agreed that the generation of hESC lines along with novel techniques for DA neuron differentiation would have a broad impact in the field. Nonetheless, reviewers felt that significant progress in this area is being may by other teams, who have achieved very high efficiency of DA neuron production, based on the expression of two other transcription factors, Lmx1 and Msx1. Given these reports in the literature, reviewers found the absence of these transcription factors in the proposed differentiation protocol somewhat puzzling. As a consequence, one reviewer thought that the applicant did not offer a convincing case that the differentiation methods represented a significant advantage over existing technologies. In this regard, reviewers questioned the novelty of the proposal, given the number of researchers working on similar approaches to the problem. Reviewers had major concerns about the feasibility of the proposal. First, the work as described is a significant and ambitious undertaking and success cannot be guaranteed in the 24 months allotted. The research plan includes the identification and validation of a progenitor cell specific gene with an appropriate expression pattern, which has to be accomplished quickly, since this reagent is required for all the subsequent studies. Another reviewer thought that the proposal would be much stronger if the applicant developed a protocol that reliably distinguished between mesocortical and nigrostriatal DA neurons, since these neuronal subtypes have quite different functions and projections in the CNS and they can be discriminated quite easily by immunocytochemistry. Reviewers also expressed concern about the validity of an in vitro model for testing neuroprotective compounds. Specifically, they wondered whether healthy DA cells grown in vitro would adequately mimic diseased Parkinsonian cells for the purposes of neuroprotective assays. While reviewers praised the proposal for being well written and clearly presented, more questions were raised about the research design. One reviewer thought that insufficient detail was provided about the proposed strategy to promote DA neuron differentiation. Another reviewer wondered what the applicant would do if the data showed cell cultures to be homogeneous for DA neurons, but heterogeneous for DA neuron subtypes. All of these reviewers would have appreciated a section discussing pitfalls. In terms of the research team, reviewers praised the principal investigator’s (PI) training and outstanding collaborators. However, some concerns were expressed about a lack of in-house expertise for day-to-day consultation with the PI. One reviewer would have liked to see either original peer-reviewed publications by the PI or compelling data in this particular area of research. Reviewers’ concerns were amplified by extremely low levels of effort listed for the external collaborators (1%, 1% and 2%). The budget seems appropriate and justified.