Parkinson's disease is caused by the death of a small number of brain cells that produce an important chemical called dopamine. Without dopamine, patients literally cannot move. Most people with Parkinson's disease take drugs such as L-dopa to help replace the chemical deficiency and make it possible to walk and all the other aspects of normal life. The problem with the drug L-dopa is that it must be taken every few hours. Before drugs, patients cannot move. Within an hour, movement is possible but those may have become exaggerated and abnormal. That cycle of "off" and "on" characterizes the daily life of people with Parkinson's. To replace the dead cells, fetal dopamine cells have been transplanted into the brains of humans with Parkinson's disease since 1988. The transplanted cells survive in the brain without immunosuppression and can supply the brain with dopamine. In some patients, the transplants can replace the need for L-dopa. Because recovery of dopamine cells from aborted fetal tissue fragments is difficult, only 300 patients in the world have had transplants. To improve cell transplantation and make it available to the tens of thousands of patients who could benefit, a new source of cells must be developed. Human embryonic stem cells are likely to be that source. My laboratory has been doing research on Parkinson's disease for many years. I have developed and evaluated new treatments for the disease including brain transplantation of dopamine cells. With colleagues who share experience in neurotransplantation for Parkinson's disease and in stem cell research, we will produce dopamine neurons from human embryonic stem cells and transplant those cells into animals which have a condition similar to Parkinson's disease. If these human cells successfully treat animals, these same cells could be used to treat people with the disease. Under current regulations, federal government funds can be used only for research on a few embryonic cell lines developed before August 2001. All of those cell lines are contaminated by contact with mouse cells and are unsuitable for human therapy. Funding by the California stem cell initiative will make it possible to work with new cell lines that could ultimately be used for treating people. CIRM funding is essential to accomplish our goals.
Statement of Benefit to California:
Parkinson's disease cripples about one million people in the United States and at least 100,000 in California. The cost of the medication and doctor visits averages about $12,000 per patient per year. Even with effective drugs, patients have daily fluctuations in condition, sometimes they cannot walk at all and sometimes their arms and legs are flailing out of control. Human embryonic stem cells can be converted into the dopamine neurons that Parkinson patients need. If our research can show that transplants of these cells can improve Parkinson's disease in animals, then the same cells could be used to treat patients in California and around the world. The need for drugs could be eliminated. The improved quality of life and the savings in drug costs could be substantial. The biotechnology involved in generating these cells may provide additional economic benefit since cells will be manufactured in sophisticated laboratories.
SYNOPSIS: This proposal will generate new rodent and primate dopamine (DA) neuronal precursors from both mouse and human embryonic stem cells (hESCs). The Principal Investigator (PI) plans to label them and use the dopaminizing morphogenetic genes to make new transgenic animals and models. Gene-profiling studies will compare ES-derived versus the indigenous midbrain DA neurons, and transplant studies in both rodents and primates will be assessed for their best response in DA-depleted animals. IMPACT & SIGNIFICANCE: Parkinson’s disease (PD)is a devastating neurologic disease; there are existing medical treatments to managing the disease symptoms but certainly no cure. There is a great hope that cell-based therapies could replace the dying and dead dopamine neurons as a treatment modality. This approach has been used previously with fetal neurons with limited success. Therefore the generation of potentially suitable human dopamine neurons is an important goal. The ability to conduct human dopamine neuron transplants to treat PD has been limited by, amongst other things, limited sources of DA neuronal precursors and lack of optimization of cell transplant methodologies. hESCs are a logical source of such cells, and numerous labs around the world have tried to optimize cell culture conditions to favor the selective expansion and differentiation of midbrain-like ES-derived DA neuronal precursor cells. Since PD treatments seem to focus on replacement of the DA axis and tone in the basal ganglia, the present study that proposes to focus on development and exploitation of primate and other models of cell replacement for PD is needed for this field. The methodologies used in this proposal are largely established - transplantation and differentiation methodologies. Unfortunately this field and the proposal has suffered a setback with recent studies published by the Goldman lab (Nature Med) demonstrating one the important methodologies for generating hESC-derived dopamine neurons. Importantly, this study showed that these cells have a clear tendency to form tumors, thus limiting their potential use in humans. Overall this is an ambitious proposal; to define conditions necessary to differentiate mouse ESCs into dopamine neurons and the hope of using the same methodology for hESCs. Once the differentiation is accomplished, the authors plan to generate transgenic animals based on mouse cell-derived dopamine cells that express fluorescent reporters for later analysis. They plan to phenotype these cells after selection and finally to transplant these cells into rats. The hope is that these steps which are largely mouse-based will provide clues for the use of human stem cells. QUALITY OF RESEARCH PLAN: This is a very ambitious proposal. Four specific aims are proposed. Aim 1 will facilitate the generation of hESC-derived DA neurons for fluorescent labeling to select pure populations of dopamine neurons and their precursors using homologous recombination in mouse and human ESCs, to be inserted downstream from genes marking midbrain dopamine neurons (i.e. en1,lmx1a, nurr1, ptx3, and DAT). Specific Aim 2 will optimize each stage of differentiation of mouse and human ESC differentiation to dopamine neurons. Using the human and mouse ESCs developed in the previous aim, ESCs will be differentiated to labeled progenitors, midbrain neurons,and mesencephalic dopamine neurons using selected differentiation factors such as SHH, FGF8, wnt1 and noggin. Aim 3 will generate transgenic mice with fluorescent labeling of dopamine neurons. The animals created in this aim will establish whether dopamine neurons derived in vitro are the same as mesencephalic DA neurons derived in vivo, using gene microarray phenotyping approaches. Finally, Aim 4 will study transplanted hESC-derived DA neurons in rats. Putative pure populations of human dopamine neurons from Aim 2 will be transplanted into immunosuppressed 6OHDA-lesioned-rats, and both tissue and behavioral analyses will follow. The PI claims that all of these studies can be conducted in the 4 year funding period, but the PI also points out, “…It is possible that by year 4, pilot studies of transplants of purified, hESC-derived dopamine neurons could be transplanted into the rhesus monkey model of Parkinson’s disease. Because the proposed budget is not adequate to pay for such studies, additional grant funds would have to be sought….” It is possible that both additional time, effort, and funds would have to be available to be able to create new transgenic animals in addition to generating the new hES-derived precursors, as well as phenotype all of the new lines both in vitro and in vivo (i.e. following grafting). There are concerns about feasibility for Aim 1; it is overambitious with regard to the generation of human lines, while the availability/use for mouse lines is much less critical for the field. Aim 2 is vague and does not provide any details on the actual conditions to be used for optimizing DA neuron differentiation. Aim 3 does not address the ultimate goal of hESC DA neuronal differentiation and rather serves more as a confirmatory study for the mouse ES lines generated in Aim 1. Aim 4 is rather limited in scope and does not properly address the main limitations in DA neuron grafting which is poor survival/maintenance of DA neuron progeny. It is unlikely that the trophic factor pretreatments proposed will resolve the current limitations in this area. In summary, this approach has been used previously with fetal neurons with limited success. Therefore the generation of potentially suitable human DA neurons, is an important goal. The methodologies used in this proposal are largely established (transplantation differentiation methodologies). This field and the grant has suffered a setback with recent studies by the Goldman lab as mentioned above. Overall this is an ambitious proposal. STRENGTHS: Both Drs. Bankiewicz and Freed are clear experts in the PD grafting field. Dr Freed has already significant experience in the hESC DA neuron field. They make a good team for grafting the best candidate DA neuron precursors from either rodent or human ESCs. The generation of new transgenic mice reporter lines, and developing more intensive fluorophores (e.g. TDT) to label transcription factors that might have a low level of expression could be quite useful. There is preliminary data on the generation of DA neurons, and some limited data on in vivo survival. However, the knock-in data are limited to mouse cells and to a single gene not very relevant for DA neuron purification/grafting strategies. The PI mentions that microarray experiments could lead to the identification of novel surface markers on indigenous A9 DA neuron precursors (both mouse and human) that could facilitate separation and enrichment protocols for all of the ESC studies proposed here. This would be an important contribution to the field, but in itself a very all-encompassing project. This grant is heavily depended upon, the intense participation of Dr. Hoffer. Dr. Hoffer includes a letter indicating that he plans to spend 50% of his time in the laboratory in San Francisco over 4 years, by combining both the sabbatical and leave of absence from his position as Professor and Head of the Division of his Department. This is an extremely unusual statement and probably needs to be verified since without Dr. Hoffer’s participation this grant should not be provided or furthermore, should it be funded, this reviewer would expect mandatory confirmation that Dr. Hoffer is spending 50% of his time, in this state, rather then just taking advantage of the large amount of California State money dedicated to stem cell research. WEAKNESSES: Purification of DA neuronal progeny is one of the limiting factors and the knock-in lines proposed here could be very valuable to the field. However, there is little acknowledgment of the difficulties in generating human knock-in lines, lack of preliminary data for human cells, and lack of novel strategies that could make homologous recombination more efficient in hESCs. Aim 2& 4 are too limited in scope to make a major contribution to the field. Unfortunately, this proposal is largely based on use of mouse cells with an essentially little data that this can be carried out in hESCs. The investigators have provided some preliminary work in mice, but it is not at all clear that this can be translated to humans. This approach is completely offset by recent studies by Goldman who has already differentiated hESCs into DA neurons. It would be far more favorable to utilize the information garnished from the Goldman studies to revise this grant proposal taking to account the new studies available rather to initiate new steps that are likely to fail. Another weakness is the fact that this grant is almost entirely mouse-based with only a loose link and hope that what the investigators learn in mice will be easily translatable to humans. One reviewer noted that there is a need to not just focus on DA neuron cell replacement; both rodent and primate models should be established that favor optimization of cell/drug protection and replacement protocols for all of the neuronal groups (e.g. vagal, brainstem reticular, raphe, and cortex) at risk in PD. Aim 1 is too ambitious with regard to hESC gene knock-in. Preliminary data with hESCs would be required to make such a plan viable. World-wide there are very few karyotypically normal hESC knock-in lines, and it is not clear what technological improvement should increase the efficiency to the levels proposed here. Aim 2 does not propose truly innovative approaches to enhance DA neuron differentiation. The studies proposed in Aim 3 will generate tg mice with fluorescent labeling of DA neurons with the aim to establish whether DA neurons expanded and differentiated in vitro are phenotypically similar to midbrain DA neurons generated in vivo, using microarray profiling experiments. Though virtuous in intent, this is not as easy to do as it sounds, and could occupy the bulk of work on this proposal. There is no reason to assume that outlier genes will provide obvious insights into the generation of a midbrain DA neuron fingerprint that will facilitate in vivo repair approaches. Like Aim 2, Aim 4 does not propose truly innovative approaches to enhance DA neuron survival in vivo. It seems that the data presented in Figures 4 and 5 represent some of the most crucial of this proposal. However, it also seems that this work is already underway in Colorado, and also in need of additional information on what striatal diffusible factors might be involved in this preliminary observation that could so much influence the course of the studies proposed here. One reviewer felt that preliminary data on human homologous recombination would be required for the current proposal. Alteratively, Aim 1 should be scaled back with a stronger focus on Aims 2& 4. This reviewer also recommended making aim 1 less open-ended. Aims 2 & 4 will require more innovative approaches than those proposed to properly address the current limitations in the field. The contribution of Dr. Bankiewicz to the project is unclear. Most of the work seems to be the expertise and area of interest of Dr. Freed, who proposes to move for 50% of his time from Colorado to California for a sabbatical during the grant period. Dr. Hoffer includes a letter indicating that he plans to spend 50% of his time in the laboratory in San Francisco over 4 years, by combining both the sabbatical and leave of absence from his position as Professor and Head of the Division of his Department. This is an extremely unusual statement and probably needs to be verified since without Dr. Hoffer’s participation this grant should not be provided or furthermore, should it be funded, this reviewer would expect mandatory confirmation that Dr. Hoffer is spending 50% of his time, in this state, rather then just taking advantage of California State money. DISCUSSION: This is an important issue and both investigators are experts in therapeutic application of cells in a primate model and in humans, and so could certainly make a contribution. The proposal, however is very open-ended. The team is good, but there is a huge amount of work to be done by the collaborator in Colorado who says he’ll be in California 50% of the time. The reviewers thought this is unlikely. There are many concerns with the science. The PI will be using homologous recombination (HR) to insert many genes upstream and downstream of all trendy dopaminergic pathway genes. A lot of cell lines are already being generated by others. This is an open ended generation of lines for producing new transgenics which is a huge amount of work similar to that being done in many other labs. It would be hard to justify unless it is a much better approach. Generating the GFP knock-ins by HR is the main approach here. This is very challenging in a single line let alone in 5 or 6 lines; this is one of the main weaknesses and there is no preliminary data presented. Moreover the PI is not an expert in HR and may not be able to overcome technical challenges. The PI has expertise in the applications of the cells, and since hESC-derived DA neuronal populations may make teratomas, the purification steps proposed for these cells could be interesting. However, here they are using published protocols for differentiation, and one reviewer was concerned about the studies in Aim 3 to maintain the DA neuronal phenotype using growth factors. Also, in the end this proposal looks more like the PI will be creating DA neurons in the mouse, and will perhaps do the work in humans later. There are better protocols for the dervation of DA neurons from hESC, and the proposed plan is not a better approach. Enthusiasm was not high.