Statement of Benefit to California:
SYNOPSIS: This proposal aims to test the hypothesis that hES cells can be used to study human embryonic development in vitro – in particular, fate decisions of embryonic ectoderm as it chooses to become neural or epidermal. Specific Aim 1 will generate new GFP, RFP, and YFP reporter lines under control of known neural or epidermal lineage promoters, and following differentiation, FACS and immunophenotyping will be used to determine if these new reporter lines accurately reflect the differentiation state. Aim 2 will apply gene expression profiling on the lines generated in Aim 1 to identify the succession of genes required for neural versus epidermal fate choice, paying particular attention to genes shared amongst the two. Finally, Aim 3 will examine known murine equivalent genes and novel genes identified in Aims 1 and 2 and overexpress them in human embryoid bodies (hEB) to affect growth and differentiation of the hEBs. These cells will then be transplanted in immunocompromised mice to follow their fate in vivo. INNOVATION AND SIGNIFICANCE: The proposed work is highly innovative. The generation of the described reporter cell lines will be extremely useful in developing models of human embryonic development, in particular in comparison of a neural versus epidermal fate. The data generated could have significance toward an understanding of mechanisms involved in transdifferentiation. STRENGTHS: This is an innovative, cutting-edge proposal. hESC and hEB studies offer numerous advantages to studying human embryonic development that cannot easily be studied in human embryos and fetuses, and the choice point to be studied here (ectoderm to neural vs epidermal) is excellent for establishing this model. Recent studies showing transdifferentiation potential of skin cells to brain cells adds credence to the proposed studies attempting to determine such fate choices in hESCs. The choice of trying to generate dopamine neurons vs keratinocytes also seems like a good one to study, based on the extensive body of literature already available for fate choice determining factors of these two populations. This is a gifted young investigator, fresh out of a very productive postdoctoral and graduate training experience. He is working in an excellent academic setting with good collaborations. He has most of the tools in place and experience with working with cells in developmental biology paradigms like this to assure successful completion of these interesting studies. WEAKNESSES: While the initial parts of the grant proposal are well-written and thoughtful, the Research Design section has no structure and would be an easier read if it were separated out into Specific Aims. There is also a lack of detail in some places in the method descriptions. For example, in the neural transplantation section, to simply cite references on the techniques to be used is insufficient – despite the applicant's acknowledgement of relying on Dr. Kornblum here. Another noted weakness is that the studies of Aim 3 aimed at expression of the murine equivalent in mouse ectoderm to see if the candidate genes discovered in Aim 2 will be expressed during the transition of ectoderm to neural or epidermal tissue is interesting, but this potentially time-consuming and maybe-not-simple-to-interpret study does not completely fit with the other proposed studies. DISCUSSION: This PI is exactly the type of young investigator that the RFA intended to attract, although it was noted that the applicant needs to develop better grant writing skills. Reviewers agreed that the development of hESC reporter lines is simply not fundable by NIH, and once created these cells will be in high demand. The reviewers predict success from these studies given the extensive literature available for studying fate choice in dopamine neurons, and the tools and experience already in place. This is a fresh perspective on the development of dopaminergic cells.