Statement of Benefit to California:
This proposal targets the characterization and function description of human neural crest cells (NCs) derived from human embryonic stem cells. The IP has recently demonstrated a procedure that allows rapid different of hESCs into neural precursor cells (NPCs), and proposes to isolate and characterize a subpopulation of these cells that are neural crest. The proposal has two specific aims. The first is to use in situ hybridization, immunostaining, prospective isolation of candidate NC cells by FACS and in vitro differentiation analysis to determine the vitro fates of candidate human NC cells. The second specific aim will determine the in vivo fates of candidate human NC cells by transplanting genetically labeled hES-NPCs and their subpopulations into the early chick embryo and following their fates during chick development. Due to the use of chimeric approaches, the NIH cannot fund this grant. SIGNIFICANCE AND INNOVATION: Neural crest cells are a transient population that are derived from the border of the neural plate during very early development. They contribute to main structure of the adult organism, including but not limited to the interior cranio-facial structure, and thus have never been isolated from human embryos. As human NC cells have never been isolated, our knowledge regarding them is null, and is solely based on information obtained from model systems. This proposal will fill this gap, and thus enjoys an extremely high significance. The grant is also very innovative in that it uses cross-species chimeras to functionally test human NC cells in the context of chimeric chick embryos. This proposal is significant as it may lead to the development of new diagnostic and therapeutic strategies. hESC-derived neural crest possibly represents a unique model of early human development. STRENGTHS: First reviewer: The strengths of this proposal lie on many levels. One is the fact that the PI has been a leader in producing neural precursor cells from hESCs. The second is that the project does not limit itself to a simple in vitro differentiation of NPCs into NCs. It rather takes the in vivo approach based on the transplantation of these cells into early chick embryos. Another strength is the fact that the in vivo experiments will be done in collaboration of Dr. Bonner-Fraser, a world leader in neural crest and NC biology, with an enthusiastic letter of collaboration attached. The approach is a major strength of this proposal. The introduction of hESCs into the developing chick embryo is an elegant procedure that may lead to significant results. Second Reviewer: The PI and his team have the necessary expertise and the Burnham Institute has excellent facilities and support to carry out the proposed work. The experiments seem feasible and the preliminary data demonstrates that the researchers have a good chance of success. WEAKNESSES: The main weakness of the proposal is that currently there is no direct evidence that hESC NPCs can actually differentiate in vitro into NCs. But as the hypothesis of this proposal rests on the resolution of this issue, the proposal cannot be criticized on this ground. The second limitation regards the in vivo chimeric experiment with the chick. As the timing of NC differentiation in humans and chick are completely out of sync (the chick moving much faster than humans), the PI does not address how this timing differential of timing might affect the process. A more cohesive and carefully written proposal with a more developed hypothesis would have been better received. DISCUSSION: This proposal goes all the way in studying the problem of interest - here, neural crest cells. Neural crest cells have unique biology relative to peripheral nerves, craniofacial cells and other nerve types. The proposal is very significant, and innovative in its use of cross-species chimeras to test the in-vivo function of the human cells. There is no evidence currently that neural precursor cells can differentiate into neural crest cell and this proposal will test this hypothesis. The differential differentiation timing of chick and human cells may pose problems in the in-vivo studies, as one reviewer pointed out that based on previous work the human cells may take on the expression timing of the chimeric host. The PI is a leader in the stem cell field, and has produced neural precursor cells. The inclusion of Dr. Bonner-Fraser is a key strength.