$2 937 237
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 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.
SYNOPSIS: The Principal Investigator (PI) is an Assistant Professor at the Buck Institute of Age Research and the focus of this proposal is on dopaminergic differentiation of human embryonic stem cells (hESCs). He proposes to assess the process and stages of dopaminergic differentiation in hESCs through markers, to sort the cells at different stages of differentiation, to develop stage-specific reporter lines, to identify novel regulators and markers using global gene expression profiling, and then to transplant the cells at different stages in an MPTP mouse model of Parkinson's disease. IMPACT & SIGNIFICANCE: Many cell transplant studies have been carried out in Parkinson's disease (PD), including transplantation of human fetal dopaminergic cells into brains of people with the disease. The results of the transplants have been quite controversial and it is widely acknowledged that the cells may accelerate the onset of on-off syndrome. Goldman, et al. (2006) reported successful use of dopaminergic hESCs to treat a rat 6-OHDA model of PD but also observed continued proliferation of the cells and possible tumorigenic potential of such growth. It is not clear that this study will contribute very much more to the field. It is possible that transplanting hESC's at various stages of differentiation towards dopaminergic cells may avoid some of the problems of transplanting fetal cells in the brain. However, it is not clear why this should be so. That said, hESCs represent a potential cell source for cell replacement therapy of PD. The selective degeneration of a specific cell type, the A9 ventral mesencephalic dopaminergic (DA) neurons, being the direct cause of this common and devastating disease, makes PD an important and ideal target disease for stem cell therapy. With the long-term goal to assess the potential of hESC-derived DA neurons for cell replacement therapy of PD, this application proposes to achieve a comprehensive understanding of human DA development, to establish optimal techniques to enrich appropriate matured DA cell populations, and to assess functional and behavior improvements after transplantation of hESC-derived DA cells in PD animal models. Thus, this proposal in general contains logical steps and approaches for successful hESC-based cell therapy of PD and is expected to significantly advance the field and to establish important bases for therapeutic application. PD and DA neuron differentiation are important areas of research. FACS based on cell surface markers is a potentially powerful strategy to make cell DA neuron grafting safer. Technology development is quite strong in this proposal with the help of Dr. Rao. However, there are concerns that the key limitations of current hESC DA neuron approaches are not addressed. QUALITY OF THE RESEARCH PLAN: The research plan appears to be quite feasible and the applicant appears to quite capable of producing hESC reporter lines by homologous recombination and doing large-scale genomic analysis of the hESC reporter lines. The techniques are well-described. The applicant discusses the use of cyclosporin to prevent immune-rejection of the transplanted human cells started one day before transplantation. He will use ferritin-labeled cells (by the way, the ferritin may be taken up by other cells and therefore is not a particularly long-term marker). He also shows preliminary data on hESC cultures and cells at different stages of differentiation towards dopaminergic cells. The proposed work is divided into 3 specific aims. In aim 1, the PI proposes to assess the process/stages of DA differentiation in hESCs by monitoring specific marker expression. First, the PI will examine gene expression associated with DA development at different stages of hESC culture and differentiation using PA6-conditioned medium and other signals, by several techniques such as immunocytochemistry, RT-PCR, qPCR, and focused microarrays. This is a tremendous effort to provide a snapshot of DA-associated gene profiles at different stages. For instance, the PI plans to perform microarray analysis for more than 15 time points along the 30 day protocol (Table 1). Second, the PI will assess the maturation of the DA neurons by electrophysiology and DA production. HPLC analyses will be used to measure DA and its metabolites at different stages of development. Thirdly, the PI will attempt to purify and/or enrich DA cell populations by FACS using cell surface markers such as PSA-NCAM, TrkB, and p75. Using each antibody, FACS will be used to isolate differentiating cells and their viability determined. It is highly important to define a core set of stage-specific markers for our comprehensive understanding of DA differentiation. Toward this goal, the proposed experiments will provide useful information. However, there are some serious flaws and concerns in the proposed approaches according to one reviewer. First, although this reviewer absolutely agrees with the PI that a comprehensive understanding of human DA development is critical for successful cell replacement therapy of PD, s/he is not convinced that DA differentiation by PA6-CM indeed represents the natural course of differentiation. Second, considering the heterogeneous cell populations at each stage, the tremendous effort described in experiment 1.1. (including more than 15 time points of microarray analyses) is not well-justified. Ironically, the PI mentions that “further purification of cells will be required prior to any transplant or gene expression studies” in experiment 1.3. In fact, this reviewer is doubtful if this extensive effort using very heterogeneous cell populations at multiple time points could provide additional insights about stage-specific markers of DA differentiation. In aim 2, the PI will develop stage-specific reporter hESC lines using site-specific integration and homologous recombination. The PI’s rationale is to determine a temporal profile of DA differentiation and to identify a core set of markers, and to determine the optimal stages to sort cells. In contrast, it seems that the PI already planned to use Sox1, Ta1, and TH genes to make reporter lines specific for neural stem cells, neuronal progenitors, and mature DA neurons, respectively. The PI will also generate TH-ferritin reporter hESC line for noninvasive investigation by MRI. These are very innovative ideas and will generate very useful resources for stem cell research in relation to PD. A reviewer points out that Aim 2 is not well focused because competing strategies are used to achieve the same goal. Stable genetic modification in hESC are difficult to achieve, particularly via homologous recombination. It is unclear why the applicant proposes two strategies for the same goal instead of focusing on the most promising approach. The use of interesting technology such as illumina bead sorting for gene expression analysis is proposed. However, there is no clear discussion on why this technology will be superior for the biological problem in question. The preliminary data is considered quite strong in some parts of the grant; but preliminary data on cell surface marker sorting need to be presented in order to validate that approach. The preliminary data on DA neuron differentiation are limited to TH expression by immunocytochemistry. TH is strongly expressed in peripheral neurons and could be a confounding factor. It is essential to show co-labeling of TH with midbrain markers. Gene expression analysis cannot compensate for the lack of co-labeling studies. Some of the markers proposed such as p75 will likely pull out neural crest stem progenitors in addition to any other presumptive ventral neural cell types. STRENGTHS: The idea of transplanting hNSCs at different stages of differentiation into animal PD models is a good one. The applicant shows preliminary data that they can indeed get stage-specific reporter cells, purify them, and transplant them. A number of very innovative technologies are proposed, and the experiments may yield some interesting data. The strengths of this application include the expertise, productivity, and enthusiasm of the PI and establishment of excellent collaborators. Dr. Zeng is an extremely talented and promising young scientist with strong background and expertise in hESC studies. Many of Dr. Zeng’s publications are directly related to characterization of differentiation properties of several lines of hESC lines using molecular, cellular, microarray, and transplantation studies. The PI’s collaborators, Dr. Rao and Dr. Andersen, are excellent scientists and leaders in their respective field of research (stem cell research and PD research). Their collaboration will significantly strengthen the feasibility of the proposed experiments. Another strength of this application is the significance and potential impact of the research area for the future treatment of one of the most common neurodegenerative disorders, PD. Assessment and comprehensive understanding of the process/stages of DA differentiation of hESCs are prerequisite for advancing cell replacement therapy of PD. The PI proposes several logical approaches that will likely generate very useful information for the stem cell field WEAKNESSES: It is not clear that this approach will solve the clinical problem of Parkinson's disease where the beneficial effects of transplanting fetal dopaminergic cells have been quite controversial and it is not clear that this experiment will yield results that are much more informative or successful than other studies. The enthusiasm of a reviewer is somewhat dampened by several weaknesses of this proposal. First of all, there is a flaw in the design of experiment 1.1 in aim 1. The PI plans to perform microarray analyses on more than 15 time points during in vitro DA differentiation of the BG01 hESC line. They will have to contend with highly heterogeneous cell populations and it is doubtful that meaningful candidates can be identified from this extensive effort. It is very likely that hundreds, if not thousands, of possible candidates will be identified at different stages. In fact, the PI states that “further purification of cells will be required prior to any transplant or gene expression studies” in experiment 1.3. Second, it was not made clear in the application why the PI proposes to establish hESC reporter lines by phi C31 integrase while this method is not yet established. Instead, homologous recombination seems to be more promising as judged by the preliminary results. Third, reviewers generally agree that the transplantation studies in aim 3 are poorly designed without appropriate and systematic considerations. Behavioral paradigms are not specific and their interpretations and statistical analyses are not carefully designed. Overall, although the proposed experiments are important and worthwhile in general, they lack clear rationale and logical designs in some parts. Additional weaknesses were identified by reviewers. There is not enough emphasis on the need to combine markers for proper midbrain DA neuron identification. No details on sorting strategy are provided which makes it impossible for the reviewer to properly assess the merit of this approach. No justification is provided as to why knock-in and site-specific integration is used for the very same application. There is also confusion about the type of fluorophores to be used (inconsistently listed throughout the application). Also, there is limited justification for some of the technology to be used. Discussion of appropriate controls is weak; NT2 derived DA neurons or human fetal DA neurons are of limited use if they are not matched for developmental stages. The in vivo study does not address the main current limitation in hES DA neuron grafting which is poor survival and maintenance of phenotype; without evidence of surviving TH cells in vivo, the TH-ferritin approach appears premature. Finally, no physiologist is identified for carrying out these studies Reviewers recommended several changes to the proposal. There should be a more in depth discussion of the controversial role of dopaminergic cell transplants for PD and why this approach with hESC would circumvent the problems associated with fetal dopaminergic cell transplants. Aim 1 should be better developed with clear description and rationale of any strategies proposed that may improve DA neuron differentiation. Some preliminary data are required on surface marker/ ACS approach. Aim 2 should be focused on one technology platform (e.g. site directed integration) so that the efforts can be better focused. Aim 3 needs to take into account the current limitations of hESC DA neuron grafts and the use of Ferritin reporter should be proposed only if appropriate preliminary data on TH survival are available. DISCUSSION: Reviewers enthusiastically agreed that a general strength of this application is the PI, who has strong potential and many good recent publications (if not in the highest impact journals). The proposal seems generally logical and reasonable, but when examined carefully, there are many deep concerns about the study design. Additional strengths mentioned included good collaborators, and a good discussion of the differences with the animal models. There is an issue with the poor characterization of the cells and there is no preliminary data on the markers used to purify the cells, and one marker (p75) is a classical neural crest marker but is not purely for DA cells. The methods proposed to establish reporter lines, phi C31 integrase and homologous recombination, are both technically challenging with the former even less established than the latter.