Recommended if funds allow
Statement of Benefit to California:
SYNOPSIS: The PI seeks to optimize the development of spinal cord motor neurons by manipulating the RA and FGF pathways during hES cell culture. The idea is based on previous evidence for the function of RA during mouse embryo development. RA is not required for specification of neural ectoderm, but for patterning along the A/P axis and also for directing subsequent development. Mesodermal derived RA activates expression of Pax6 and olig2, while also inhibiting expression of FGF8. The combined effect leads to commitment towards motor neuron differentiation. Two aims are proposed that will test if this pathway has relevance during hES cell differentiation, namely if RA antagonizes the FGF pathway, and if this is important for stimulating neural differentiation. Several hES cell lines will first be grown or adapted to serum free (FGF2-dependent) media, followed by establishment of a neural differentiation protocol, based on making EBs as described by Zhang and colleagues (Nature Biotech 2005). SIGNIFICANCE AND INNOVATION: 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 hES system. The proposed study will confirm whether retinoic acid- FGF antagonism, which is well delineated in motor neuron specification from animal studies, occurs in motor neuron differentiation from human ESCs. The proposal thus appears more mechanistic, although it does not appear likely to generate new information regarding RA-FGF regulation of motor neuron differentiation. It is logical to predict that more motor neurons might be differentiated from human ESCs by inhibiting FGF signaling. However, since RA treatment, which is required for motor neuron differentiation, antagonizes FGF signaling, it is questionable whether additional block of FGF will significantly increase the production of motor neurons, a major aim of the study. STRENGTHS: A strength is the application of established normal developmental pathways and regulatory mechanisms to a defined hES 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 PI is a strength given his expertise in the role of RA during normal neural development in mouse models. The laboratory publishes very well, although there is no previous experience in ES cell models. Dr. Loring is the local expert and provides 5% effort to the project. 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. Strength of the proposal is the tremendous experience of the PI in studies of retinoic signaling and the necessary tools for analyzing RA-FGF signaling. The experiments are also logically laid out and study is less descriptive than many of the stem cell proposals. WEAKNESSES: RA and FGF will regulate many developmental programs in the context of the developing EBs so the project may underestimate the amount of heterogeneity that will be derived. There was little mention of considering different amounts of RA or altering the timing of RA delivery, which might be key to optimizing the targeted development of MN 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 RA-FGF role in motor neurons specification and to significantly increase the production of motor neurons by further blocking FGF signaling. 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 retinoids do 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 great experience with FGF and retinoids in early development. The specification of new motor neurons is dependent on retinoid signalling by the inhibition of FGF; one objective is to understand whether further inhibition of FGF will lead to motor neurons, but one reviewer felt that this approach is unlikely to work given the precision needed for FGF modulation.