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RS1-00153-1: Control of neural stem-to-progenitor transitions by Cyclin D family members in ES cells

Recommendation: Recommended if funds available
Scientific Score: 73

First Year Funds Requested: $302,063.00
Total Funds Requested: $636,488.00

Public Abstract (provided by applicant)

Mouse embryonic stem (mES) cells are the earliest pluripotent mammalian cell ever described, capable of generating entire viable mice in vivo and producing most, if not all, cell types in vitro. In combination with this pluripotency, an extensive rate of proliferation confers on mES cells the status of an unlimited renewable source of cells. The use of ES-derived cells as therapeutic agents has been previously shown to be possible in mice. Therefore, the recent isolation of human ES (hES) cells has stimulated interest in their potential clinical value. hES cells have became the subject of intensive studies aimed at cell therapies, creating a great hope for curing many degenerative diseases. Among them, Amyotrophic Lateral Sclerosis (ALS) is a neurological disease that attacks and degenerates the motor neurons (MNs) that control voluntary muscles.Spinal MNs can be generated in vitro from ES cells and used to achieve a functional restoration of motor units after injury in model animal. But the relationship between the stage of neuronal differentiation and the efficiency of in vivo engraftment and differentiation has not been empirically investigated so far. It is very important to extensively study the potentiality of engraftment of cells according to their stage of differentiation, in order to optimize the transplantation process. The MN differentiation pathway is very well characterized, divided in at least three stages, but very little is known about the molecular mechanism regulating the neuronal differentiation process, driving the cells from one stage to an other, or the engraftment capacities and degree of commitment of these different developmental intermediate.Preliminary observations obtained in the lab led us to consider Cyclin Ds, known as positive regulator of the cell cycle, as makers of different stages in the MN differentiation process. In addition, it has been demonstrated that CyclinD1 is able to modify the activity of transcription factors. We thus have hypothesized that the dynamic expression of Cyclin Ds observed in the CNS could be part of the molecular mechanism involved in the regulation of neuronal specification. Preliminary results confirmed this hypothesis and prompted us to ask if, similarly, the modulation of Cyclin Ds level of expression could be a way to influence ES cells along the in vitro MN differentiation process. The goal of this proposal is to test this hypothesis and determine whether it allows amplification of progenitors at different stages in the MN lineage. Preliminary results will be obtained on mES, for the ease of manipulation, but soon similar experiments will be conducted on hES cells. If our hypothesis is confirmed, the efficiency of engraftment will be analyzed for the different populations, hoping that this will leads to optimize the engraftment process, bringing us closer to a cure for many neurodegenerative diseases.

Statement of Benefit to California (provided by applicant)

Amyotrophic lateral sclerosis (ALS), often referred to as “Lou Gehrig’s disease,” is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord, resulting in muscle weakness and atrophy. Nearly 120,000 cases are diagnosed worldwide each year, with a life expectancy after diagnosis as short as 2-4 years. Replacing the degenerating motor neurons using a renewable cell source such as human embryonic stem cells appears to be one of the most promising treatments for ALS, as well as other neurodegenerative diseases. Unfortunately, if the proof of principle for such treatment has already been demonstrated in rodent, long is the way toward such cure for human. Notably, a drastic improvement for the transplantation process is necessary. With this project, we propose to extensively study the potentiality of engraftment of cells according to their stage of differentiation, in order to optimize the transplantation process. If successful, this study will constitute a huge step toward curing ALS patients as well as many patients whom may require neuronal graft. This will also benefit the State of California and its citizens. It is of primary importance to support innovative research as it will maintain a dynamic environment propitious to attract more qualified people. Mastering such cutting edge technology also represents a important advantage for the State of California and its citizens as it may lead to the development of health care program and possibly biotech companies, which represents an advantage for the local economy.

Review

SYNOPSIS: This is a solid proposal to study Cyclin D family regulation of early neural differentiation transitions in mouse embryonic stem (mES) and human embryonic stem (hES) cells. The goal here is to test whether the modulation of Cyclin D family members is important in directing motor neuron differentiation independently of their cell cycle functions, and whether a state more amenable to the differentiation of motor neurons can be found. The aims are to 1) characterize developmental intermediates in mouse then human, 2) modulate Cyclin Ds to look at motor neuron differentiation, and 3) put motor neurons into a spinal cord injury model and see if they reconstruct corticospinal tract.

SIGNIFICANCE AND INNOVATION: This is an important topic, both from a basic understanding of the generation of motor neurons and the molecular control of the process, and also toward the production of these cells for cell therapies, especially in amyotrophic lateral sclerosis (ALS). This is fundamental and significant as the foundation for future work in the field. The applicant will use state-of-the-art techniques to explore the interrelationships of the Cyclin Ds in both mouse and human embryonic stem cells (ESCs) and their role in ESC differentiation along a motor neuron pathway.

STRENGTHS: There is a pressing need for the production of large numbers of human motor neurons for ALS therapy. The Principal Investigator (PI) has an extremely strong track record in the field, and this extremely strong lab, investigator, knowledge, and rigor with proven productivity allows “loose ends” to be somewhat overlooked. The work will be done in a world-class institution.

WEAKNESSES: The in vivo experiments are weaker and much less well-defined and illuminating than earlier experiments in the proposal. It is clear that the earlier aims are entirely within the applicant’s expertise, but the in vivo work is less so and a letter of support from the collaborator named by the applicant is not included. One suggestion is that the applicant work on describing the in vivo experiments better, e.g., why would one want spinal motor neurons in this model of injury? If there are good reasons, then motivate the work in those directions. If the work is not as clearly motivated, then re-direct experiments to best advance fundamental knowledge.

As the applicant will work on both mouse and human ESCs, it is not entirely clear whether all of the first two aims will be done with both. In subsequent work, they will use mESCs but why not go directly to hESCs? Although the applicant states that they will use the data gathered to go onto models of ALS, might it not be possible to go straight to these?

DISCUSSION: This proposal comes from a preeminent developmental biologist and marks a transition from established interests to a new focus on hESC work. The in vivo models proposed were unclear (except for one of the injury models), and while motor neuron replacement is one of the goals the applicant is looking for locomotor effects. Reviewers felt that the proposal would have been better without the in vivo work. Many different neuronal types are damaged following spinal cord injury, and it is unclear why the PI feels that replacement of a single type of neuron would fix the injury. Finally, the proposal could have been presented more carefully; inconsistencies and “loose-ends” in the proposal made it difficult to read.

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:

• Csete, Marie