Right Column

RS1-00193-1: Retinoic Acid-FGF Antagonism during Motor Neuron Differentiation of Human ES Cells

Recommendation: Recommended if funds available
Scientific Score: 75

First Year Funds Requested: $379,500.00
Total Funds Requested: $759,000.00

Public Abstract (provided by applicant)

Retinoic acid is a metabolic derivative of vitamin A that has recently been shown to stimulate differentiation of human embryonic stem cells into motor neurons. However, almost nothing is known about how retinoic acid may perform this function. The recent discovery that retinoic acid antagonizes the action of fibroblast growth factor suggests a possible mechanism for retinoic acid function during motor neuron differentiation. We plan to use our knowledge of retinoic acid-fibroblast growth factor interaction to understand how retinoic acid stimulates human embryonic stem cells to go down the motor neuron lineage. Such knowledge will allow us to devise rational strategies for optimal use of retinoic with other reagents to reliably differentiate human embryonic stem cells into motor neurons. Our studies will contribute to the development of cell-replacement therapies for motor neuron loss in patients with amyotrophic lateral sclerosis or spinal cord injury. We plan to study the effect of retinoic acid on differentiation of human embryonic stem cell lines that are ineligible for federal funding. Because all of the human embryonic stem cell lines approved for federal funding were generated using methods that involved mixing human cells with mouse feeder cells, they are poorly suited for clinical use and are likely to have undergone modifications with unpredictable consequences. Given the scarcity of federally-approved human embryonic stem cell lines, and recognizing that cells passaged for long periods of time in culture tend to become genetically unstable, it is necessary to have the means of characterizing new human embryonic stem cell lines. The studies proposed here on human embryonic stem cell lines that are ineligible for federal funding will provide new insight into how retinoic acid and fibroblast growth factor can best be used to generate motor neurons for therapeutic purposes.

Statement of Benefit to California (provided by applicant)

The studies we perform should benefit the state of California in several ways:(1) We hope to increase the ability to generate motor neurons that can be used in cell-replacement therapies for motor neuron loss in patients with amyotrophic lateral sclerosis or spinal cord injury. This will directly benefit patients in California and elsewhere.(2) The human embryonic stem cell research we perform may bring new biotechnology jobs to California, thus increasing the state’s visibility as a leader in stem cell technology.(3) New therapeutic methods developed using our discoveries could bring revenues to California due to the ability of the state to obtain licensing fees on technology generated using CIRM funds.

Review

SYNOPSIS OF PROPOSAL: The Principal Investigator (PI) seeks to optimize the development of spinal cord motor neurons by manipulating two known pathways during hES cell culture. The idea is based on previous evidence for the function of a known molecule during mouse embryo development. Two aims are proposed that will test if this pathway has relevance during hESC differentiation and if this is important for stimulating neural differentiation.

INNOVATION AND SIGNIFICANCE: The project has the potential to optimize procedures for generating motor neurons, which could impact treatments for patients with motor neuron loss (eg. ALS). It is innovative in taking what is known from established mouse models and applying this information to the hESC system. The proposal thus appears mechanistic, although it does not appear likely to generate new information regarding regulation by these molecules of motor neuron differentiation. It is logical to predict that more motor neurons might be differentiated from human ESCs by modulating a signalling pathway. However, based on previously described pathway interactions, it is questionable whether the proposed studies will significantly increase the production of motor neurons, a major aim of the study.

STRENGTHS OF PROPOSAL:A strength is the application of established normal developmental pathways and regulatory mechanisms to a defined hESC differentiation program. In this regard the proposal is very well focused. It is also very well written and addresses many of the potential pitfalls. The Principal Investigator (PI) is a strength given his expertise in the role relevant factors during normal neural development. The laboratory publishes very well, although there is no previous experience in ES cell models. A local expert has been recruited to collaborate. While the project is relatively limited in scope, the strength is its focus and the likelihood that these pathways will be key toward manipulating lineage outcome.

WEAKNESSES OF THE PROPOSAL: The pathways under study will regulate many developmental programs in the context of the developing embryoid bodies so the project may underestimate the amount of heterogeneity that will be derived. There was little mention of considering different amounts of the facotrs or altering the timing of their delivery, which might be key to optimizing the targeted development of desired lineages. Since the PI has no previous experience, significant effort will be needed to first recapitulate the previously documented baseline differentiation protocol.

The experimental design depends on a well-established motor neuron differentiation model to be in place, which was not available in the PI’s lab. Major weakness is that the proposed study is unlikely to add new information regarding the role of known pathways in motor neurons specification or to significantly increase the production of motor neurons.

DISCUSSION: While this proposal is very clear and well-written and the experiments are well-described, there was general agreement that little new information is expected from this work regarding the specification of motor neurons beyond what is already known in mouse and chick models. A weakness is that since the factor of interest does a lot of things, the timing of application and dose is important, and a reviewer was surprised that the applicant had not addressed this more carefully since the applicant has experience with this kind of study. One reviewer felt that this approach is unlikely to work given the precision needed for pathway modulation.

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:

• None