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RC1-00115-1: Molecular and Cellular Transitions from ES Cells to Mature Functioning Human Neurons

Recommendation: Recommended for funding
Scientific Score: 89

First Year Funds Requested: $702,744.00
Total Funds Requested: $2,879,210.00

Public Abstract (provided by applicant)

Human embryonic stem cells (hESCs) are pluripotent entities, capable of generating a whole-body spectrum of distinct cell types. We have developmental procedures for inducing hESCs to develop into pure populations of human neural stem cells (hNS), a step required for generating authentic mature human neurons. Several protocols have currently been developed to differentiate hESCs to what appear to be differentiated dopaminergic neurons (important in Parkinson’s disease (PD) and cholinergic motor neurons (important in Amyolateral Sclerosis (ALS) in culture dishes. We have developed methods to stably insert new genes in hESC and we have demonstrated that these transgenic cells can become mature neurons in culture dishes. We plan to over express alpha synuclein and other genes associated with PD and superoxide dismutase (a gene mutated in ALS) into hESCs and then differentiate these cells to neurons, and more specifically to dopaminergic neurons and cholinergic neurons using existing protocols. These transgenic cells can be used not only for the discovery of cellular and molecular causes for dopaminergic or cholinergic cell damage and death in these devastating diseases, but also can be used as assays to screen chemical libraries to find novels drugs that may protect against the degenerative process. Until recently the investigation of the differentiation of these human cells has only been observed in culture dishes or during tumor formation. Our recent results show that hESC implanted in the brains of mice can survive and become active functional human neurons that successfully integrate into the adult mouse forebrain. This method of transplantation to generate models of human disease will permit the study of human neural development in a living environment, paving the way for the generation of new models of human neurodegenerative and psychiatric diseases. It also has the potential to speed up the screening process for therapeutic drugs.

Statement of Benefit to California (provided by applicant)

We plan to develop procedures to induce human ES cells into mature functioning neurons that carry genes that cause the debilitating human neurological diseases, Parkinson’s disease and Amyolateral Sclerosis (ALS). We will use the cells to reveal the genes and molecular pathways inside the cells that are responsible for how the mutant genes cause damage to specific types of brain cells. We also will make the cells available to other researchers as well as biotech companies so that other investigators can use these cells to screen small molecule and chemical libraries to discover new drugs that can interfere with the pathology caused by these mutant cells that mimic human disease, in hopes of accelerating the pace of discovery.

Review

SYNOPSIS OF PROPOSAL: This proposal will look at three important “transition points” during the differentiation of human embryonic stem cells (hESCs) into human neural stem cells (hNS), and then from hNS to committed lineage restricted neuronal progenitors (hNP). The final transition point is from hNP to mature functioning human neurons of specific neuronal lineages, and the studies proposed here will focus on dopaminergic (hNda or DA) and cholinergic (hNchat) neurons that are clinically significant for Parkinson’s Disease (PD) and Amyotrophic lateral sclerosis (ALS). Mechanisms associated with cellular changes at these transition points will be studied at the protein and small non-coding RNAs level.

IMPACT AND SIGNIFICANCE: This proposal has impact and significance to the field in several areas. There is a need to develop better cell culture models of human diseases such as PD and ALS, and there is also a need to generate large numbers of transplantable cells that possess the appropriate phenotype (e.g. DA and ChAT); ES cells are appreciated as offering these outcomes become of their extensive proliferative potential and their “blank slate”, making them suitable for controlled differentiation and specific fate choice-inducing protocols. The choice of looking at protein expression and small non-coding RNAs at specific checkpoints of commitment to ultimately DA and ChAT precursor cells is a good one. Once the specific molecular mechanisms and pathways are gleaned to direct these cells to these phenotypes, they can then be used not only for transplantation in animal models of disease, but they can be used for high throughput screening of drugs and factors that underlie or reverse disease processes. In doing this, the cells can be studied for how mutant genes wreak havoc in these and other types of CNS cells. This would be an extremely significant advance to the field of restorative neurology.

The basic understanding of neuronal subtype specification presents one of the highest priorities in hESC work, and it is very clear that the basic molecular knowledge of these early events will have a tremendous impact on our understanding of the normal state of differentiation, as well as the diseased state. In addition, the success of this project will establish powerful models to understand human disease, such as Parkinsons and ALS. These models are going to be different than the ones currently available, as they represent mouse with human neurons in vivo providing the platform for direct study of the disease state in human cells.

This work proposes a very general and broad approach to study neural development and neuronal differentiation in hESCs, and to develop hES-derived hNS models relevant to disease. The use of a number of innovative techniques is proposed, and the anticipated impact is high.

QUALITY OF THE RESEARCH PLAN: This is a proposal of very high quality written by a world specialist in this field. The PI has proven successfully that human neurons can home, integrate, differentiate, and contribute to the circuitry in the context of the mouse CNS.

Different ES and more committed precursor cell types (all five of them) will be generated, and there is a focus on (hESC, hNS, hNP,hNda, and hNchat). Specifically, the applicant proposes to use commercially available technologies for gene identification, and for characterization of interesting small non-coding RNAs. Validation of molecular targets will be performed in tissue culture. Differential molecular profiles in cellular models for PD and ALS will be assessed. The molecular phenotype of mutant and wild type cells will also be compared. Chimeras will be generated, following grafting of the cells into mouse embryos, for the study of cell-autonomous versus cell-non-autonomous effects of disease genes. This is a very reasonable interconnected series of goals that will provide novel and important findings on ES cell manipulation for studying and potentially treating PD and ALS. The timetable for performing these studies is appropriate.

The research plan is logical and well lined out – though in very big strokes. Many of the proposed experiments are interdependent, and it would have been valuable to provide more detail for many of the planned experiments. For example, selection criteria for identification of candidates and tools for certain experiments were not described in any detail. While there is a considerable lack of detail and preliminary data, the outstanding record of the PI in the field can overcome many of these concerns.

The biggest concern however relates to transplantation studies as proposed. They would most certainly not lead to the robust integration of desired cells in the spinal cord as motoneurons to model of ALS, or in the midbrain as dopamine neurons to model of PD. In both cases all host motoneurons or dopamine neurons have been already generated (postmitotic), and it seems very unlikely that the signals would be still available to instruct undifferentiated hESCs rapidly into the appropriate phenotype.

In addition there are obviously a lot of other issues regarding experimental variability and differentiation into unexpected phenotypes that will make it very unlikely to yield a robust human disease model. It would be extremely surprising if these experiments work as proposed. There is also no discussion of alternative strategies in the event that the proposed experiments are unsuccessful.

STRENGTHS: This is a very strong investigator and the experiments are logical, well-described, and with the proper insights for translating these new findings to reagents for the field as well as for therapeutic applications (a major goal of CIRM) in motor neuron disease and Parkinson’s. The experiments are complex and time consuming, but there is a great deal of enthusiasm for this investigator who continually provides the field with high quality science in the field of stem cells.

Small non-coding microRNAs and other approaches used here to discover ways to control the fate of ES and adult neural stem cells into dopaminergic and cholinergic neuronal phenotypes will without question provide invaluable information to the field.

A series of very innovative techniques are proposed as an integral part of this proposal and the experiments are proposed in a logical progression. Also a strength is the acknowledgment that cell sorting may be necessary for these studies.

Appreciation of the state of transitions during cell fate specification is a strength. This is an embryological concept mostly neglected by non-developmental biologists approaching the hESC field. Another strength of this proposal is that it combines basic research with direct potential clinical applications, not only unraveling the molecular aspect of differentiation, but also establishing a platform for modeling disease. Finally, and perhaps more importantly, the use of in vivo assays in chimeric mouse to study the behavior of the cell types in their natural in vivo milieu is a strength, again an area neglected in the hESC field.

WEAKNESSES: There are really very few if any weaknesses with this very nice proposal. Some of the proposed studies are certainly ambitious, considering the massive number of small RNAs that will present themselves; the productivity and expertise of this PI and his group reduces concerns of the amount of work involved, and the potential payoff in new insights and reagents allay any concern.

The PI will focus on determining consequences of mutants of a known gene at different transition points as cells are induced to express the desired neuronal phenotype. Certainly the known gene is not the only one involved, and even though it is reasonable to focus on this for the current model studies, there are other genes and mutations that might be more interesting and potentially important in the hESC models.

Scarce preliminary data is available: no HB9 data, no differentiation, characterization and promoter driven isolation of phenotypes, and the experiments are interdependent. If the first ones fail, little useful information will be gained.

The description of the approach to cell purification is vague, and limited information is offered on how the PI will use the results of proposed screens.

There is little discussion of potential pitfalls throughout the proposal.

The generation of disease specific models as proposed seems problematic for specific technical reasons relating to timing of the birth of specific types of neurons and grafting into the lateral ventricle. Such a study may require early intraparenchymal grafts and should be supported by preliminary data in the spinal cord and midbrain.

This grant can be qualified as overly ambitious by regular funding agencies. However the track record and productivity of the PI is tremendously high. Therefore, this should not impact this grant negatively.

DISCUSSION: The proposal offers a compelling approach to using non-coding RNAs to probe the transition points of differentiation of hESC into mature functioning dopaminergic and cholinergic neurons. There was agreement that the proposal is ambitious and concern that the workload was perhaps a bit unrealistic, but the research team was determined to be likely to handle the challenges based on previous achievements.

There was also concern that some experimental details were either vague or significantly lacking.

The discussants were troubled by the transplantation experiments as proposed. There was agreement that the chimera experiments may be unlikely to work given that precise timing is needed in integration of appropriate local signals for differentiation of neurons. The reviewers felt that transplantation may need to be done at a much earlier stage than that proposed in order to achieve the proposed goal.

There were several suggestions offered to improve the proposal. Additional preliminary data would strengthen the proposal, as would a better description of technical details for screening and selection criteria. Discussion of alternative strategies should be included throughout the proposal. The chimera experiments should be re-designed and appropriate preliminary data should be provided.

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:

• Rothstein, Jeffrey