New Cell Lines
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 the 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 human neurons by turning on stem cell genes in these cells. Several groups have been able to turn fibroblasts into pluripotent cell lines that resemble embryonic stem cells using viral infection of the differentiated cells. Once we have generated these lines, we can determine whether they serve as better models for human neurological disease than neurons derived from the few human ES cell lines now available for research. If successful, this novel method should allow us, and other researchers, to generate the best possible in vitro models for a wide range of human neurological diseases.
Statement of Benefit to California:
The goal of this study is to develop a novel method 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 and our method could be widely used by researchers throughout California. 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.
Executive Summary This is a proposal to explore the feasibility of generating pluripotent cell lines from post-mitotic neurons using lentiviral transduction with pluripotency genes. This approach is based on the hypothesis that the genomic DNA found in neurons may not be functionally equivalent to the genomic DNA of other cells. The applicant cites literature identifying chromosomal abnormalities within different neuronal cells that lead to DNA deletions or duplication. Such changes have been linked to autism and mental retardation. If irreversible DNA changes exist in neurons, the author argues, effective neuronal replacement will require cells derived directly from the indicated neural sub-types. In addition, the most accurate in vitro models would have to be derived directly from human neurons. As neurons are non-mitotic cells and therefore cannot be expanded for in vitro analyses, the goal here is to generate pluripotent cells from human neurons. Such cell lines would then supply sufficient cells for whole genome analyses and for generating model systems in vitro that could be used to identify biomarkers and test new therapeutic compounds. Three specific aims are proposed. In the first, human embryonic stem cells (hESCs) will be genetically modified and then differentiated toward dopaminergic neurons with the goal of deriving irreversibly labeled neurons. In the second aim the applicant will apply a variation of the induced pluripotent stem cell (iPS cell) approach to human neurons to generate pluripotent cell lines. In the third aim the applicant will utilize human neurons obtained from a stem cell resource center to generate iPS cells. This is an interesting and novel proposal from a well qualified applicant. If the proposed hypothesis is correct, the derivation of cell lines from diseased neurons would be very significant. However, reviewers were unconvinced that the underlying hypothesis was justified by existing data, so the real significance of this work remains unclear. If the hypothesis is incorrect, the proposal loses its relevance. Furthermore, the applicant has no experience with human cells, and many of the proposed, complex techniques have not been tested. Finally, in some places the experimental design is weak or superficial. Overall, reviewers were interested but unconvinced that the approach would generate useful cell lines. The first aim of the proposal is straightforward and seems feasible, and alternatives to the proposed transgenic strategy have been proposed. The second aim is where the bulk of the work will be done, and it is complicated and high-risk. Since neurons are highly differentiated and post-mitotic, the applicant anticipates that these cells are less easily reprogrammed than fibroblasts, and thus proposes a complex iPS generation strategy that is a significant technical challenge. Returning neurons to a neural stem cell state, which might be more compatible with iPS protocols than the fully differentiated state, is proposed. The applicant provides no assurances that this strategy will work. Furthermore, it is unclear exactly which neurons the applicant will use initially in these experiments. In the third aim, the applicant proposes to generate pluripotent cell lines by converting banked human neurons directly into iPS cells. In particular the applicant hopes to use neuronal cells from patients suffering from neurological diseases or harboring genetic susceptibility genes. In this context, the collaboration with the stem cell bank was considered to be important. Development of in vitro models of individual diseases will, however, depend upon satisfactory progress in the prior specific aims. There was disagreement among reviewers regarding whether the applicant has enough expertise to carry out the proposed experiments. The applicant has an excellent background in molecular biology and has worked with mouse cell lines. However, preliminary data are limited and do not include work on human cells. Reviewers commented, for instance, that it is not trivial to generate dopaminergic neurons using the proposed approach, and no preliminary data on this technique is included. In conclusion, the reviewers appreciated the novelty of the proposal, but their reservations about the underlying hypothesis and other criticisms left them unable to recommend this application for funding. Reviewer One Comments Significance: This is an interesting and novel protocol from a well qualified applicant. The essence of the proposal is that while it has been assumed that neurons derived from hESCs will recapitulate normal differentiation and development in vitro, this might not be the case. The applicant cites the literature that there may be chromosomal abnormalities with different cells, leading to a DNA deletion or duplication, so-called copy number variation (CNV). Such changes have linked to CNVs in autism and mental retardation. If irreversible DNA changes exist in neurons then the most accurate in vitro models would be derived directly from neurons. As neurons are non-mitotic cells, the goal here is to generate pluripotent cells from human neurons. Such cell lines would then supply sufficient cells for whole genome analyses and for generating model systems in vitro that could be used to identify biomarkers and test new therapeutic compounds. Success in all of this would be very significant. Feasibility: The applicant proposes three Specific Aims. In the first aim the goal is straightforward in that they want to produce irreversibly labeled post-mitotic human neurons. The technique to do this as described seems relatively straightforward. They will use a Cre-lox transgene approach to mark the cells derived. Then they will differentiate the cells toward a dopaminergic neuronal sub-type. Cells expressing tyrosine hydroxylase (TH) will then express the mCherry reporter and can then presumably be sorted and enriched based on this. Alternatives to this transgenic strategy if it fails have been proposed. The second aim is where the bulk of the work will be done. The key to successful iPS reprogramming with survival of the neurons on being forced to enter the cell cycle, will be to prevent their death by apoptosis. This is where things become complicated and high risk. The approach that they describe as a “staggered and combinatorial iPS strategy” is a significant technical challenge and we have no assurance that it will work. They do propose a variety of anti-apoptotic genes that could be used however. Maintaining the cells as neural stem cells which might be more compatible to iPS protocols, also remains to be tested. It was not clear exactly which neurons they will use initially here – they say that they will use the marked neurons if the original work fails. Testing the pluripotency of the cell lines will be carried out appropriately. The connection with neural stem cell bank at CHOC could be of great interest. Development of in vitro models of individual diseases will of course depend upon satisfactory progress in the prior specific aims. The applicant is well qualified to carry out the proposed studies. Much of her background comes from the eight years spent in R. Axel’s at Columbia. During that time she was co-first author on the Nature paper on cloning mice from olfactory neurons by SCNT. Her publication record is not extensive and most of her support comes from a previous CIRM grant. She appears to have access to the appropriate space and equipment. Her collaborators are important. Jeanne Loring is an expert in culturing hESCs and Karl Willert will provide important support and facilities in the UCSD hESC Shared Research Lab (CIRM funded). Collaboration with the Human Neural Stem Cell Resource at CHOC is important. Preliminary data to support this application is somewhat limited. Much of the applicant’s experience is from her work with mouse cell lines. She acknowledges that moving to human carries a degree of risk. She does include data on lentiviral transduction of mouse neurons with GFP. Nothing is included on human cells and this is a weakness of the proposal. Responsiveness to RFA: I think it will succeed in producing truly pluripotent cells based on the successes of others. Their plans to test this are appropriate as are their plans to share the cells. Reviewer Two Comments Significance: The overall aim of this application is to develop novel methods to generate human iPS cell lines from post mitotic neurons using lentiviral expression of pluripotency genes. The authors speculate that post mitotic neurons derived from individuals with neurodegenerative disorders may harbor neuronal specific genetic alterations which would not be picked up using non-neuronal somatic cells, and would thus provide a better cell source for potentially understanding the pathogenesis of neurodegenerative and genetic forms of brain disease. Feasibility: The applicants first propose to design methods that allow for the generation of highly enriched labeled post mitotic neurons from human embryonic stem cells and once having done this, to use this as a means of enriching neuronal cells from brain tissue. 1) The author intends to use genetic engineering to generate neural cells which can be selected using antibiotic selection. 2) The applicant proposes that terminally differentiated neurons can be induced to re-enter cell cycle. This section of the grant is very vague in terms of which cell populations would be used and how this fits in with the rest of the application. 3) The author proposes to generate pluripotent cell lines by using IPS technology to convert banked human neurons directly into iPS cells. In particular the authors hope to use neuronal cells from patients suffering from neurological diseases or harboring genetic susceptibility genes. In some places the experimental design is weak or superficial. There would seem to be potential overlap with other funded work in the PI’s lab. (Staff note: during the review meeting, reviewers were reminded that scientific overlap will be considered during administrative review, should this application be funded, and should not be considered during the scientific review.) Responsiveness to RFA: The applicant devotes little if any attention to the characterization of putative pluripotent cells derived from neurons. In terms of dissemination of the cells to other researchers, they claim that they will follow CIRM guidelines for distributing the cells to other research groups.