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 DNAchanges 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.
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.
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 with array-based comparative genomic hybridization 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 PI was involved in the previous cloning of mice from olfactory sensory 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 CNS neurons.
As a point of departure for this proposal Dr. Baldwin cites long-standing analogies that have been made between neuronal diversity in the central nervous system and immunoglobin diversity in the immune system. She notes, moreover, genetic expansions and deletions that occur frequently within the neurons of patients afflicted by diseases such as Huntington's and Fragile X. Finally, she notes that small copy number variations in genomes are quite common, nonrandom, and that an increased number of these variations is associated with autism. Based on this evidence she hypothesizes that neurons acquire de novo DNA changes during development or as a consequence of environmental insults.
How can this hypothesis be tested? Whole genome scanning requires more DNA than can be obtained from a single cell. The applicant proposes to clone mice using the nuclei of single CNS neurons as genetic donors. As a back up approach/contingency plan, she 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, she will use array-based comparative genomic hybridization (CGH) and other methods to search for evidence of genetic diversity within individual neurons.
The first aim is to expand the genome of single neurons from the retina, cerebellum, and telencephalon by SCNT. These experiments require that a cloned blastocyst contribute to all tissues of the animal. Since the applicant wishes to investigate DNA alterations that may preclude contribution to all tissues she will use a two-step cloning protocol in which embryonic stem (ES) cell lines are derived directly from cloned blastocysts (called nES cell lines). The PI will be aided by collaborators, Drs. K. Eggan and S. Kupriyanov.
The second specific aim is a back-up strategy. The applicant proposes to expand the genome of individual neurons by using lentiviral transduction of four transcription factors that have recently been shown to be sufficient to convert differentiated fibroblast cells into pluripotent stem cells. In preliminary studies, the applicant has shown that at least two different subtypes of neurons can be induced to divide by this procedure. Terminally differentiated neurons frequently undergo programmed cell death (apoptosis) in response to inappropriate mitogenic cues. To circumvent this problem, the applicant plans to inhibit cell death using RNAi to suppress p53 or expression vectors that encode anti-apoptotic proteins such as bcl-2. The PI will be aided with respect to the construction and use of the lentiviruses by a collaborator, Dr. Anton Maximov.
The final specific aim is to survey the entire genome of ES cells, cloned mice, and neuronal cell lines derived in aims one and two by the method of array CGH. The hypothesis is that structural mutations in neurons are relevant to differentiation and/or disease. This hypothesis garners some support from recent reports that copy number changes are common and that increased copy number polymorphisms are associated with autism-spectrum disorders. The PI will be able to determine whether copy number polymorphisms, DNA rearrangements, etc. are associated with neuronal differentiation, whether there are frequent changes at particular sites, and whether changes are specific for certain neuronal subsets. The PI will be aided by a collaborator, Dr. Ira Hall at Cold Spring Harbor Laboratory; a publication describing the methods and some recent findings are in press by Hall and colleagues in Nature Genetics.
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 merits an "A+". 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, the proposal is an exciting one that potentially will have a very significant impact on the field. The proposal is clearly written and provides some alternative approaches if experiments do not work out. It is a very risky if not a fearless proposal, attempting to break many barriers with respect to ideas about stem cell biology.
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 already claimed that cloning from adult central nervous system neurons is impossible. In preliminary studies (conducted as a postdoctoral fellow at Columbia), Baldwin and her collaborators were able to clone mice from the nuclei of single olfactory neurons. The applicant readily acknowledges special features of olfactory neurons that distinguish them from typical CNS neurons.
One recent paper (from the same authors that claimed previously that CNS neurons cannot be cloned) has claimed success in generating ES 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, Dr Baldwin notes that CNS tissue is heterogeneous and the neuronal origins of the nuclei in the recent success stories was not documented. A key element of Baldwin's study plan is a cre-lox strategy that allows her to irreversibly mark the nuclei of donor neurons.
Aim 1 will test the developmental potential of CNS neurons from the telencephalon, cerebellum and retina by generating nuclear transfer derived ES cells and mice. The question of whether genetic changes occur during neurogenesis is an interesting and important question with significance for therapeutic applications and cancer research.
Aim 2 proposes to use an alternative, potentially more amenable, approach to generate pluripotent cells from neurons: transcription factor induced reprogramming using lentiviruses. While it has been shown that fibroblasts can be reprogrammed by lentiviral expression of Oct4, Sox2, c-myc and Klf4, it remains unclear if other cell types can be reprogrammed by the same factors and if postmitotic cells can undergo reprogramming. Recent studies on marker expression during the reprogramming process suggest that reprogramming is a gradual process that requires multiple cell divisions to be complete. Thus, it remains unclear if this approach will work at all. Moreover, there appear to be different stoichiometric requirements for the 4 factors based on the number of individually integrated viral copies. The proposed lentiviral constructs will express two factors per lentivirus, which may be problematic if different levels for the two factors are necessary.
Aim 3 will then analyze if there are chromosomal changes in neuron-derived pluripotent cells by CGH analysis. These studies are of interest although risky in the sense that they depend on the success of the previous aims and because it is possible that chromosomal changes are not present or not detected (e.g., because key epigenetic changes are not detected). One reviewer expressed concern about vector design and the feasibility of the direct reprogramming approach.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Reviewers commented that one of the strengths of the proposal is the PI. Dr. Baldwin has an impressive training with experience gained in two outstanding laboratories as well as fruitful collaborations in the past that are continuing. Although the number of papers that she has published is small, they have been important ones in the field. She is well-equipped to carry out the planned studies and is a very promising investigator in the stem cell field. She clearly is a fearless person with respect to science since she 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 of Cell Biology at the Scripps Research Institute. Her early career development bears an interesting resemblance to that of Nobel laureate Linda Buck. To wit: 1) her predoctoral training is in the field of immunology (PhD with Mark David at Stanford University); 2) she conducted postdoctoral research at Columbia University with Richard Axel for an extended period of time and; 3) her postdoctoral tenure culminated in a single (but seminal) research publication. Dr. Baldwin won several prizes for academic excellence at the undergraduate and pre-doctoral levels, including a Howard Hughes Medical Institute Pre-doctoral Fellowship. She has recently been appointed as a Pew Scholar in the biomedical sciences and she has a grant from the Whitehall Foundation entitled Mapping Fine Scale Olfactory Sensory Representations in the Cortex.
Although the number of papers that she has published is small, they have been important ones in the field. She published two papers during her PhD, both as first author, one in J. Immunology and one in J. Exp. Medicine. She published two papers during her post-doctoral fellowship years, one as a shared first author with K. Eggan in an important study published in Nature involving cloning a mouse from olfactory sensory neurons. She lists no publications since 2004. Her background is not specifically in stem cell research, but she was recently invited to join a local group called Young Investigators in Stem Cell Biology.
In sum, the PI’s scientific credentials are outstanding. She is well-equipped to carry out the planned experiments, key collaborators are in place, and she has developed a realistic plan for her scientific and professional development. The PI’s prospects for future scientific leadership appear to be very strong, and she promises to be a strong investigator in the stem cell field.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The Department Chair has provided a letter describing Dr Baldwin's current situation at Scripps. This letter goes into extensive detail on the space package for Dr. Baldwin (1300 sq. ft of lab space and 10 desk areas) and describes several impressive core facilities that will facilitate her 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 she will be involved in Dr. Baldwin’s mentoring. Ten additional faculty are planned over the next 4 years with at least some of them involved in stem cell research. The environment is outstanding for the PI. In general it appears that the institutional commitment is strong.
The reviewers addressed a few weak points. First, it would appear that Baldwin's salary is only guaranteed for a three-year period of time. In the current climate of austerity at the NIH many other institutions are more generous with the salary “safety net” afforded to their new faculty hires. In addition, the Department Chair describes an unorthodox, flexible timeline to tenure at Scripps. The Chair opines that this flexible timeline might work to the benefit of some junior faculty; however, this same “flexible timeline” to tenure is featured in many clinical departments where some evidence suggests that it works to the disadvantage of women faculty because a certain amount of academic "gamesmanship" is required to initiate the promotion process.
DISCUSSION: Reviewers were captivated by 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 RAG expression 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 and with strong scientific collaborators. She 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.