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RS1-00215-1: Identifying small molecules that stimulate the differentiation of hESCs into dopamine-producing neurons

Recommendation: Recommended if funds available
Scientific Score: 87

First Year Funds Requested: $279,099.00
Total Funds Requested: $564,309.00

Public Abstract (provided by applicant)

In this application, we propose to identify small molecule compounds that can stimulate human embryonic stem cells to become dopamine-producing neurons. These neurons degenerate in Parkinson’s disease, and currently have very limited availability, thus hindering the cell replacement therapy for treating Parkinson’s disease.

Our proposed research, if successful, will lead to the identification of small molecule compounds that can not only stimulate cultured human embryonic stem cells to become DA neurons, but may also stimulate endogenous brain stem cells to regenerate, since the small molecule compounds can be made readily available to the brain due to their ability to cross the blood-brain barrier. In addition, these small molecule compounds may serve as important research tools, which can tell us the fundamental biology of the human embryonic stem cells.

Statement of Benefit to California (provided by applicant)

The proposed research will potentially lead to a cure for the devastating neurodegenerative, movement disorder, Parkinson’s disease. The proposed research will potentially provide important research tools to better understand hESCs. Such improved understanding of hESCs may lead to better treatments for a variety of diseases, in which a stem-cell based therapy could make a difference.

Review

SYNOPSIS: The goal of this proposal is to identify small molecules that will stimulate human embryonic stem cells (hESCs) to differentiate into dopaminergic (DA) neurons. Because of the large variability of DA neuron production of different batches of cultured hESC, the Principal Investigator (PI) plans to use an engineered model system that he/she has established as an in vivo assay for a chemical screen of 5,000 small molecules. Initially, the PI will identify small molecules that affect the induction of DA neurons or their later differentiation and survival in the engineered in vivo model. Effects of the small molecules on the model will be observed in a 96 well format under a fluorescent microscope. The morphology and the state of reporter neurons will be examined. Where alterations of reporter patterns are observed, those organisms will be processed for tyrosine hydroxylase immunolabeling to further validate the alterations of DA neurons. Following these studies, the PI will take the small molecule hits identified and test them in hESC.

INNOVATION AND SIGNIFICANCE: The reviewers found there to be a tremendous amount of innovation and significance in this proposal. At present, there are difficulties obtaining a large number of homogeneous dopaminergic neurons that are pure and free of xenobiotic products, such as feeder cells. In addition, the transcription factors and the pathways that are important in the differentiation and maintenance of these cells are poorly defined. Therefore, this proposal is significant since it may lead to knowledge and to the efficient generation of dopaminergic differentiated cells that may impact on the treatment of Parkinson’s disease. The small molecules that may be identified in the planned studies may have a use in direct treatment of patients as they may cross the blood-brain-barrier and may slow down DA neuronal death, or they may stimulate regeneration and thus avoid transplantation into a delicate region of the brain.

The proposal is very innovative since it uses an engineered in vivo model system to carry out a chemical genetic screen. Although this approach has been used before for chemical screens in other conditions, it remains very novel. In fact, the PI was responsible for generating the engineered model that make the proposal possible by allowing for the direct visualization of the state of DA neurons. The model provides a potential number of advantages over in vitro studies, especially since cells that differentiate in vitro may not maintain their differentiated state when transplanted, i.e., it may be valuable to screen for molecules that lead to differentiation of ESCs in an organism rather than in vitro.

STRENGTHS: This is an elegantly interwoven set of two experimental aims to glean small molecules able to induce DA neurogenesis in the model system and later in a hESC culture setting. The availability of the engineered model that was generated by the PI is a potentially powerful tool. The approach that the PI is taking involving the use of the model with its consistent, small and easily quantifiable numbers of DA neurons to identify a molecule that can then be tested in hESC is a valuable and clever screening approach. The model has been previously used to screen chemical libraries, i.e., there is precedence for this approach but not in this specific manner. The use of an in vivo assay to look for small molecules important in the differentiation of DA neurons is clearly more physiologically relevant than an in vitro assay – as suggested by the fact that in vitro differentiation sometimes disappears after placement of cells within the CNS. Also, an ongoing grant in the PI’s lab looking at DA neuron development in the model system is highly complementary to the studies proposed here.

The institutional small molecule center is a valuable resource for the planned studies. The PI is productive with publications in excellent journals on the topic of DA neurons in model systems as well as a publication related to studies of toxins in Parkinson’s disease. Finally, the collaboration with an expert in hESC is a valuable one.

WEAKNESSES: The weakest point of this proposal is the possibility that the PI is fishing for active compounds that affect DA precursor gene expression, and DA neuron generation is clearly risky and might not prove successful. For example, the drugs that affect DA neuron differentiation in the model system may be species-specific and not function in hESC. Five thousand compounds is a relatively small library for screening. The model is likely limiting for high thoughput screening – thus the focus on a five thousand compound library. If no hits are found in this library, there is a growing list of small molecule/drug libraries potentially available to screen in this elegant model so lack of hits in the proposed library might not be a daunting issue. The PI certainly has thought about the issue of no hits and proposed to try combinations of small molecules (simultaneously or sequentially) if the single small molecule attempts fail to translate from the model system to the hESC culture approach.

There was a lack of clarity as to the endpoint and what constituted a positive hit. It appeared that the PI is screening for small molecules by examining the general morphology as well as the state of the reporter neurons. One reviewer was not quite certain what this meant. Does the PI expect that new DA neurons will form, that DA neurons will form earlier, that DA neurons will grow larger than normal, that DA neurons will have an increase in fluorescence? Are there examples where model neurons change their neurotransmitter characteristics? These are important questions that should have been more clearly explained in the proposal. Also, it is not clear how the PI will follow up with small molecules that have a negative effect on the reporter neurons. Might these also be of interest?

Finally, initial studies involving the in vivo screen could be carried out with federal grant money. There was also some surprise that the PI only listed 10% effort on this proposal.

DISCUSSION: A creative and important aspect of the proposal is the use of the engineered model for screening. There was discussion about how the screen will work, such as is it automated, is there quantitative fluorescence used, and what was the endpoint – what constitutes a “positive” hit. There was a discussion as to the timing of the exposure with respect to whether would be observing a direct or an indirect effect. Finally, the biotechnology aspect of the proposal was very attractive including the potential for industry interest in a successful outcome.

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, Marcie
• Lansing, Sherry
• Sheehy, Jeff