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RN1-00584-1: Generating pluripotent cell lines from neurons.
Recommendation: Recommended for funding
Scientific Score: 90
First Year Funds Requested: $560,675
Total Funds Requested: $2,803,375
Public Abstract (provided by applicant)
Stem cell research holds great promise for neurological disease. One in three Americans will suffer from diseases of the nervous system ranging from stroke to Alzheimer’s disease to epilepsy. Very few treatments for neurological disease exist, in part because of he lack of suitable in vitro models with which to test therapeutics. In addition, many neuronal disorders, including Parkinson’s disease and ALS, are characterized by loss of important subpopulations of neurons. In affected patients, the only way to restore function may be to provide them with replacement neurons. Many researchers are already working on methods to generate replacement neurons from human embryonic stem cells or to generate accurate in vitro models of neurological diseases. Here, we propose to perform the reverse experiment; we aim to generate pluripotent cell lines directly from neurons, using two novel technologies. The first goal of these experiments is to generate cell lines so that we can compare the chromosomes of neurons with those of neurons derived from ES cells. If differences exist, and are important for the proper function of neurons, it is essential to identify these changes. Similarly, if neurons in diseased patients have DNA changes that cause disease symptoms, it would be better to derive ES cells directly from neurons and then to “re-differentiate ” them into better in vitro models for drug screening. Both of these findings will significantly impact the ~ 30% of CIRM funded grants aimed at curing various diseases of the nervous system.
Statement of Benefit to California (provided by applicant)
The goal of this study is to develop novel techniques to generate stem cell lines directly from neurons, which is currently impossible in humans. Our findings will also allow us to validate or improve current strategies to generate replacement neurons from human embryonic stem cells. Our experiments should suggest new ways to derive patient specific cell lines to treat or study common human neurological diseases such as Alzheimer’s and autism. These findings may lead to relief for patients who suffer from currently untreatable diseases of the nervous system. In addition, our novel methods may foster innovation in the dynamic biotechnology and health-care sectors of the California economy, which would benefit many Californians in by creating jobs and promoting economic growth.
Review
SYNOPSIS: The goal of this application is to use mouse model systems to develop novel methods to generate pluripotent cell lines directly from post-mitotic neurons, either by somatic cell nuclear transfer (SCNT, cloning) or by lentiviral expression of pluripotency genes in neurons. These experiments will also involve a survey of the genomes of individual neurons using state-of–the-art technology to search for unknown irreversible DNA changes that accompany neuronal differentiation or disease. A benefit of this research would be to develop a new method to generate patient/disease-specific cell lines derived directly from neurons, without SCNT. The Principal Investigator (PI) was involved in the previous cloning of mice from specific neurons and has generated important gene-targeted mouse lines to allow the cloning of mice and production of cell lines from three additional types of central nervous system (CNS) neurons.
The applicant proposes to clone mice using the nuclei of single CNS neurons as genetic donors. As a back-up approach/contingency plan, s/he proposes to generate neuronal cell lines from single neurons using the recently-described cocktail of four transcription factors that can convert ordinary fibroblasts into pluripotent stem cells. Armed with these mice or these cell lines, s/he will use state of the art technology to search for evidence of genetic diversity within individual neurons.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The long-term goal of this proposal is to understand how irreversible and reversible genetic changes govern neuronal differentiation and disease. With respect to the level of innovation, scientific impact, and clinical/translational impact, reviewers stated that this grant is outstanding. The studies proposed here have the potential to change the way we think about learning and memory, and to change the way we study learning, memory, and disease. With respect to clinical/translational impact, the applicant notes that a major role of current human stem cell research is to develop novel methods to differentiate stem cells into specific cell types of neurons in vitro. If the underlying premise of the studies proposed here is correct (i.e., that neurons may differ from other somatic cells at the chromosomal level), then much current work in the area of stem cell replacement for neurological disease states will have to be reconsidered.
Overall, this is an exciting, clearly-written proposal that has the potential to have a very significant impact on the field. It is a very risky if not a fearless proposal, attempting to break many barriers with respect to ideas about stem cell biology. Preliminary data support the feasibility of some of the risky experiments, and the PI provides some alternative approaches if experiments do not work out
The major weakness of the study plan is the high risk/high gain character of the work proposed. Feasibility of the attack is presently hard to assess. On one hand, multiple investigators (publishing in respectable journals) have claimed that cloning from adult central nervous system neurons is impossible. However, in recent work the applicant and collaborators were able to clone mice from the nuclei of single neurons. The applicant readily acknowledges special features of these neurons that distinguish them from typical CNS neurons. Another recent paper (from the same authors that claimed previously that CNS neurons cannot be cloned) has claimed success in generating embryonic stem cells and live mice from the nuclei of adult cortical or CNS neurons. While this later paper is actually encouraging from the perspective of the applicant’s study plan, the applicant notes that CNS tissue is heterogeneous and the neuronal origins of the nuclei in the recent success stories was not documented. S/he proposes an innovative strategy to overcome this limitation. Reviewers pointed out some specific shortcomings, e.g. some of the proposed studies depend on the success of the earlier aims, and it is possible that the hypothesized chromosomal changes either are not present or will not be detected.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Reviewers commented that one of the strengths of the proposal is the PI. The applicant has impressive training in immunology and neurobiology with experience gained in two outstanding laboratories, as well as fruitful collaborations in the past that are continuing. The applicant’s background is not specifically in stem cell research, but s/he was recently invited to join a local Stem Cell Biology group. Although the number of papers that s/he has published is small, they have been important ones in the field. S/he is well-equipped to carry out the planned studies and is a very promising investigator in the stem cell field. The applicant clearly is a fearless person with respect to science since s/he has tackled a very risky study. Reviewers commented that this drive to carry out risky but high impact research may be the reason that there are relatively few publications.
The applicant is an Assistant Professor at a top institution in California. The applicant won several prizes for academic excellence at the undergraduate and pre-doctoral levels, including a Howard Hughes Medical Institute Pre-doctoral Fellowship, and has recently received professional recognition through awards.
In sum, the PI’s scientific credentials are outstanding. S/he is well-equipped to carry out the planned experiments, key collaborators are in place, and s/he has developed a realistic plan for his/her scientific and professional development. The PI’s prospects for future scientific leadership appear to be very strong, and s/he promises to be a strong investigator in the stem cell field.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: In general it appears that the institutional commitment is strong. The Department Chair has provided a letter describing the applicant’s current situation at the institution. This letter goes into extensive detail on the space package for this investigator and describes several impressive core facilities that will facilitate the proposed work. The mouse genetics core will have SCNT capability soon added to the core. The PI is said to have had a generous start-up package, although details are not supplied. The Chair suggests that s/he will be involved in the applicant’s mentoring. Ten additional faculty are planned over the next few years with at least some of them involved in stem cell research. The environment is outstanding for the PI.
DISCUSSION: Reviewers were enthusiastic about the provocative hypothesis and the bold, imaginary approach described in this proposal. They commented that it investigates fundamental processes of how we learn and remember, suggesting that it might be similar to how we make antibodies (through DNA rearrangement). Panelists revisited the history of the concept of DNA rearrangement in neurons and the possible parallels between immune and neural cells. When expression of a gene required for DNA rearrangement in immune cells was first observed in the nervous system it was hypothesized that neurons may rearrange DNA, but no evidence for recombination was discovered. This remains an open question, and the work described in this proposal might put the notion that recombination occurs in the CNS to rest. Reviewers felt that this would be a successful outcome of the proposed work
Reviewers noted that the feasibility of this project is hard to assess. For instance, it is not clear that using post-mitotic cells for SCNT will be possible. However, the work is being conducted by a high-potential PI in an excellent scientific environment with strong scientific collaborators. S/he also has several backup strategies. The panel felt that this is the sort of high-impact, high-risk project that the CIRM should be funding.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- Wagers, Professor Amy
- Orkin, Stuart