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RC1-00338-1: Converting Human Embryonic Stem Cells to Dopamine Neurons for Parkinson's Disease

Recommendation: Not recommended for funding 

Public Abstract (provided by applicant)

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 (provided by applicant)

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.

Review

SYNOPSIS: This proposal will generate new 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 rodent and large animal models 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 no cure. There is a great hope that cell-based therapies could replace the dying and dead dopamine (DA) neurons as a treatment modality. 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 ability to conduct human DA neuron transplants to treat PD has been limited by, among 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 use of large animal 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 DA 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 DA cells that express fluorescent reporters for later analysis. The plan is 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 DA neurons. Aim 2 will optimize each stage of differentiation of mouse and human ESC differentiation to DA 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. Aim 3 will generate transgenic mice with fluorescent labeling of DA neurons. The animals created in this aim will establish whether DA 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 hESC-derived DA neurons generated 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 a large animal 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 hESC-derived precursors, as well as phenotype all of the new lines both in vitro and in vivo (i.e. following grafting).

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: The PI and collaborators are clear experts in the PD grafting field. One of them has 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 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 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 proposal is heavily dependent upon the intense participation of several collaborators one of whom indicates that (s)he plans to spend 50% of his/her time in the laboratory over 4 years, by combining both the sabbatical and leave of absence from his/her position as Professor and Head of the Division of the Department. This is an extremely unusual statement and probably needs to be verified since without this individual’s participation, funding for this proposal should not be provided. Furthermore, should it be funded, one reviewer expected mandatory confirmation that the collaborator is spending 50% of his/her time, in this state, rather then just taking advantage of California State money dedicated to stem cell research.

WEAKNESSES: Unfortunately, this proposal is almost entirely based on use of mouse cells with 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 what the investigators learn in mice will be easily translatable to humans. Moreover, the proposed 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.

One reviewer noted 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.

Another reviewer concurred, pointing out that 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 mice with labeled 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. Like Aim 2, Aim 4 does not propose truly innovative approaches to enhance DA neuron survival in vivo.

Preliminary data was presented and seems that some of this work is already underway in the out-of-state collaborator’s laboratory. One reviewer wanted additional information on what special diffusible factors might be involved in producing this preliminary observation that could so much influence the course of the studies proposed here.

Another reviewer felt that preliminary data on human homologous recombination (HR) would be required for the current proposal. Alternatively, 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.

It was also noted that there is a need to not just focus on DA neuron cell replacement; both rodent and large animal 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.

The exact contribution of the PI to the project is unclear. Most of the work seems to rest on the expertise and area of interest of one of the collaborators, who proposes to move 50% of his/her time to California for a sabbatical during the grant period. A second out-of-state collaborator includes a letter indicating that (s)he plans to spend 50% of his/her time in the laboratory in San Francisco over 4 years, by combining both the sabbatical and leave of absence from his/her current position as Professor and Head of the Division of his/her Department. This is an extremely unusual statement and probably needs to be verified since without their participation funding for this project should not be provided. Should it be funded, one reviewer would expect mandatory confirmation that the fact that this collaborator is spending 50% of his/her time, in this state, rather then just taking advantage of California State money.

In summary, 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, there is a lack of preliminary data for human cells, and a lack of novel strategies that could make HR more efficient in hESCs. Aim 2& 4 are too limited in scope to make a major contribution to the field.

DISCUSSION: This is an important issue and the investigative team includes experts in therapeutic application of cells in a large animal 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 one of the out-of-state collaborators who says (s)he’ll be in California 50% of the time for the 4 year granting period. The reviewers thought this is unlikely.

There are many concerns with the science. The PI will be using 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.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• Feit, Marcy
• Lansing, Sherry
• Sheehy, Jeff