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.
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.
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.