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Developing a transplantation therapy for Parkinson's disease using hESC-derived dopaminergic neurons.

Funding Type: 
New Faculty I
Grant Number: 
Funds requested: 
$3 004 020
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Parkinson’s disease is a devastating neurodegenerative disorder for which there is no cure. Several foundations including the Michael J. Fox Foundation have identified cell therapy as one possible avenue for research as cell therapy using fetal tissue-derived cells has been shown to be successful in multiple transplant studies. Work in the field had been limited however, due to the limited availability of cells for transplantation as cells from 6-10 human fetuses of 6-10 weeks post-conception is required for a single patient. Human embryonic stem cells offer a potentially unlimited source of the right kind of cell required for cell replacement therapy due to their remarkable ability to self-renew (they can divide indefinitely in culture) and to be trained to become any cell type of the body. In this proposal we seek to understand how human embryonic stem cells differentiate into authentic dopaminergic neurons using a culture method that we have tested. We believe that this differentiation can be broken into several stages each of which is regulated by growth factors and other molecules. We also believe that these stages can be understood by selecting cells at each stage and comparing their properties using molecular tools and by examining their behavior after transplanting the cells into a mouse disease model. In addition, we will need to develop a noninvasive method of following cells after transplantation and we propose to develop a TH-ferritin (that can be detected by magnetic resonance imaging, MRI) reporter line based on previous successful work in mice to monitor dopaminergic neurons in animal models and possibly in future clinical trials. We believe that these experiments are critical to enhancing our understanding of the disease and providing the tools that will be necessary to move cell therapy to the clinic.
Statement of Benefit to California: 
We have proposed three aims in this proposal to develop markers that can distinguish stages in the process of development, to develop reporter lines that allow us to isolates cells at each specific stage of differentiation, and to identify the optimal cells for therapy using animal models that mimic the human disease. We have also proposed to assess the long-term integration and differentiation of transplanted cells using a magnetic resonance imaging (MRI)-based labeling system. We believe these experiments not only provide a blueprint for moving towards the clinic for Parkinson’s disease sufferers but also a generalized plan for how cell therapy will need to be developed for treatment of multiple disorders including motor neuron diseases and spinal cord injury. The tools and reagents that we develop will be made widely available to Californian researchers and we will select California-based companies for commercialization of such therapies. We will hope that it is California-based physicians who will be at the forefront of developing this promising avenue of research. We expect that the money expended on this research will benefit the Californian research community, and the tools and reagents we develop will help accelerate the research of our colleagues in both California and worldwide.
Review Summary: 
SYNOPSIS: The overall goal of this proposal is to develop effective transplantation of dopaminergic (DA) cells derived from human embryonic stem cells (hESCs) for the treatment of Parkinson’s disease (PD). In order to carry this out, the PI will assess the process of dopaminergic differentiation in hESCs and compare the efficacy of transplantation of cells purified at different stages of differentiation in an animal model of PD. To achieve the goal, the applicant has proposed to: develop reporter lines that allow isolation of cells at each specific stage of differentiation; identify the optimal cells for therapy using animal models that mimic the human disease; and assess long-term integration and differentiation of transplanted cells using a magnetic resonance imaging (MRI)-based labeling system. The first specific aim is to assess the process/stages of dopaminergic differentiation in hESCs by stage-specific expression of markers known to be associated with dopaminergic development. Markers for undifferentiated hESCs, NSCs, neurons, DA neurons, and contaminating non-DA markers will be used to determine the time of harvest for cells to be assessed by immunohistochemistry, RT-PCR, quantitative RT-PCR, and a focused microarray that includes neural stem cell genes and 36 DA genes. DA neuronal maturation will also be assessed by measuring neurophysiological properties and dopamine production. In order to enrich for DA neurons, FACS will be used to at different stages using cell surface markers. This aim will allow the PI to determine a temporal profile of dopaminergic differentiation, identify a core set of markers that can be used to assess the process of differentiation, and determine the optimal stage(s) at which to sort cells. In the second specific aim, the PI will develop stage-specific knock-in hESC GFP reporter lines via homologous recombination that can distinguish different stages of differentiation. Cells from the generated mice will be sorted by the reporter and used to identify novel stage-specific regulators and markers by comparing global gene expression profiles of purified cells at different stages. Some of the genes identified in these experiments will be tested as stage-specific markers. The PI will also generate a knock-in hESC line for magnetic resonance imaging (MRI) studies (specific aim 3). Since the PI is aware of potential difficulties with homologous recombination, an alternative approach was described using promoter-reporter constructs. In the third specific aim, the PI will transplant cells purified at different stages of development into a mouse model of PD (where 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) is used to simulate Parkinson’s disease in mice). Grafts of cells from different stages of development will be assessed for survival, integration, and differentiation as well as for teratoma formation. Transplanted animals will then undergo a panel of behavioral tests including open field analysis of motor activities, specialized rotarod, inverted wire screen, and horizontal beam tests which are standard methods in the field. Transplantation will also be carried out using DA populations that express an MRI label so that hESC-derived dopaminergic neurons can be monitored non-invasively in living animals using MRI. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The proposed studies are important ones for better understanding aspects that are involved in the success of cell transplants in PD, and the proposal is logical and clearly written. The main concern with this proposal is the fact that the aims are highly interdependent and the experimental design lacks depth and is not likely to provide substantive insight into the problem of hES dopamine derivation. The experiments for the most part use established approaches and techniques and the PI takes few risks, making the project less exciting and cutting-edge. One reviewer commented that the literature is replete with successful and efficient DA derivation protocols. The main obstacle is long term in vivo survival of hES derived DA neurons and understanding the mechanisms underlying poor survival. Teratoma formation has been reported by some investigators, but it is clearly due to the persistence of undifferentiated ESCs within the cell mix. Such cells are readily identifiable by their expression of highly specific markers The applicant approaches the problem of in vivo survival in a tangential manner. While the strategy is theoretically technically feasible, the biological justification is lacking. Why would selecting cells at different stages of differentiation result in enhanced in vivo survival? More specifically the applicant defines the "stages" of differentiation very superficially and focuses on the well-recognized stages of neural precursors, young neurons, and dopamine neurons. The markers being used are not likely to define a novel stage, e.g. the poorly-defined dopamine neuron precursor. If the goal is to select the DA neurons, then a mature marker is sufficient. Selecting for the identified marker will not result in a midbrain dopamine neuron fraction but rather in any cell expressing this marker (e.g. GABA neurons, peripheral neurons or forebrain neurons, noradrenergic neurons or even transient marker-expressing cells). A more stringent approach would include co-expression of other specific markers within the marker+ population. The other markers proposed by the PI are similarly non-specific. This is particulalry concerning since the PI plans to use homologous recombination strategies, a highly challenging endeavor in hES cells which is presented fairly casually by the applicant. There was a low degree of enthusiasm for Aim 1, except for analysis of a specific cell population, identified by putative markers of maturing neurons. Although this is a key part of the proposal, it is unfortunate that preliminary data is not shown to support it and therefore the usefulness of these markers for such a purpose is not assured. In addition, it is not specifically stated whether they will sort double-positive cells or single-positive cells. It is unlikely that the investigators will be successful in generating knock-in reporter lines, and if they are unable to efficiently purify cells aim 3 may not yield interpretable results. In discussing Aim 2, reviewers noted that stage-specific knockin into hESC reporter lines via homologous recombination for neural stem cells, neural progenitors, and mature dopaminergic neurons using the indicated markers has been conceived and attempted by many. Efficient homologous recombination in hESCs for these and other loci have not been readily achieved. The investigator does not propose any novel methods to produce these targeted hESC clones and has not yet established these tools. On the other hand, reviewers did comment that, although these studies are technically difficult, the PI presents preliminary data demonstrating the generation of one marker-GFP knock-in hESC line. The last aspect of the proposal, which involves producing, transplanting, and imaging hESCs carrying an MRI label, is the most novel and innovative part of the application. The PI has experience in these kinds of studies and previously reported the first transplantation of hESC-derived DA populations into another PD model, 6-Hydroxydopamine (6-OHDA)-treated rats. The investigator has shown that they can adequately image neurons derived from the substantia nigra from labelled transgenic mice after transplantation into wild-type animals. Again, this concept is enticing and innovative. There were flaws to this aim, however. Reviewers were concerned that label-expressing hESCs were obtained by a collaborator, not the PI on this application. Thus, the in vivo aim is credible by its association with the collaborator. One reviewer questioned why the more hazardous (to the investigator) MPTP mouse model is necessary, vs. the easier 6-OHDA model. Further, transplanting within 7 days of MPTP administration is a bit too early and might expose the neurons to a more toxic environment. Another reviewer was concerned with the need to use immunosuppression in the PD model. One reviewer noted that the murine model is a non-immunodeficient model, and immunosuppression may confound the results by either reducing function or differentiation and/or integration, as cyclosporine is a known neurotoxin. Finally, although the PI clearly proposes to optimize survival and integration following transplantation, the rationale is not clear for the hypothesis that engraftment/integration and function in vivo in this model depends on the stage of the cells transplanted. In general, while the investigator does have experience with differentiating hESCs to neural lineage and in particular generating cultures with marker-positive neurons, the remainder of the preliminary data, while supportive, indicate that some of the proposed experiments have not been developed sufficiently to determine whether they are feasible. The methodologies involved are not clearly described, particularly newer methodologies to overcome current roadblocks in efficiently targeting these loci in hESCs and in deriving clonal populations. In fact, the laboratory has not yet made the targeting constructs but the PI is proposing to do so. Without these reagents in hand, it appears premature to propose experiments using these lines. The applicant would benefit from a better understanding of the biological questions at hand, in order to better focus the aims of the research. A final weakness is that the overall structure of the project is linear with somewhat interrelated aims, and the outcome appears to be highly dependent on efficient purification, which in turn is dependent on successful generation of reporter lines, which as discussed is not yet worked out. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The applicant is clearly an extremely well-trained, promising and productive investigator committed to stem cell research. S/he obtained a PhD at a foreign institution, completed two postdoctoral fellowships (in molecular immunology and stem cell biology and neuroscience) at an American institution, and has worked with established experts in stem cell biology. The PI has an excellent background gained while a post-doctoral fellow, and has demonstrated productivity. S/he has a good publication record and has obtained extramural support to study the role of transcription factors in dopaminergic neuron differentiation from hESCs. S/he lists more than 20 publications, including 5 with the applicant as first author in a single recent year, and approximately 10 papers (6 first- or last-author papers) since 2006. These publications deal primarily with stem cells, especially related to DA neuron development. There appears to be good synergy between other faculty at the institution, with an investigator with experience in mouse models of PD, and with a leading expert in neural differentiation from embryonic stem cells. The PI has been an Assistant Professor since 2005. S/he is a principal investigator in a CIRM Shared Research Laboratory and an NIH\NCRR Interdisciplinary Research Consortium, indicating his/her expertise in stem cell biology. S/he also has funding as a co-PI on a CIRM grant, a Stem Cell Techniques Course grant, and a start-up grant from a private research foundation. The career development plan is acceptable. The proposed mentors are good scientists, although perhaps the PI could benefit from the input of a mentor with more specific expertise in neuroscience or development, i.e. someone who would highlight the biology as opposed to technlogy alone, in addressing questions of DA neuron development. The applicant’s career goals focus on acquiring additional experience in rodent Parkinson disease models, as well as with other federally-restricted and GMP-grade hESC lines. S/he also proposes to gain an understanding of working with other mouse models including those of stroke, Alzheimer’s, Huntington’s disease and ALS so that hESC derivatives can be tested in these model systems. Finally, s/he proposes to obtain additional didactic and course training in a variety of courses and workshops offered at the institution. In conclusion, the PI is talented, well trained and productive. The career development plan is acceptable. The applicant’s mentors are good scientists, but the applicant could benefit from the input of a mentor with more specific expertise in the neuroscience or development in addressing questions of DA neuron development. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The PI has been an Assistant Professor at the present institution since 2005. S/he received a start-up package of ~$1.8 million. S/he presently has a lab space of 400 sq. feet, which will soon be increased to 800 sq. feet. The PI has a dedicated hESC culture room and access to a FACS, and the description of facilities and faculty suggest a positive environment. There are a number of Core facilities including genomics, proteomics, transgenic technology, and morphology/imaging Cores. The primary mentor will be the institute’s Director of the program in stem cell and regenerative medicine, whose expertise is in adult neurogenesis in stroke and neurodegenerative disease. The secondary mentor will be an expert on hESCs and is the PI's former mentor. The PI has planned informal meetings as well as regular formal meetings with the mentors; a formal annual report will be made by the mentors. There are institutional performance reviews at the end of 5 years with the possibility of extensions of the candidate’s term. The supportive letters are quite good, as are the development plans detailed by the institution’s director. Recruitment of 10 new investigators is planned over the next 3 yeas with 2-3 in the stem cell program. DISCUSSION: This is a well-trained, talented investigator with a reasonable publication record who is committed to stem cell research. S/he has a good institutional environment and a satisfactory career development plan. Unfortunately, reviewers felt that the research proposal itself was logical but neither novel or exciting. It consists of three linear, interrelated aims that aren’t very innovative and do not address the important questions in the field. Reviewers agreed that the most exciting part of the application is the development of the novel reporter lines which could be used to non-invasively trace cells using MRI, but it is not clear that this aim will be successful. They were concerned at the lack of preliminary data on GFP reporter lines – generating these lines is a difficult process and the PI does not even appear to have the required vectors. Markers being used to identify dopaminergic cells and precursors are non-specific or not stage-specific, and therefore it is not clear that these markers will identify the approproiate dopaminergic precursors.

© 2013 California Institute for Regenerative Medicine