Funding opportunities

Analysis of Candidate Neural Crest Cells Derived from Human ES Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00466
Principle Investigator: 
Funds requested: 
$759 000
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Little is known about human Neurla Crest (NC) cells, a transient population of cells briefly present during very early human development; the reason why these cells are extremely difficult to obtain and study. In the model organism NC cells generate an amazing array of tissues, including peripheral and enteric nervous systems, cranial bones and cartilage, some cardiac muscle and virtually all pigmented cells in the body. Abnormalities in NC cells involved in numerous human pathologies including various skeletal syndromes (e.g. Apert syndrome), diseases of nervous system (e.g. Hirschsprung’s disease) and pigment disorders (e.g. Waardenburg syndrome).The lack of fundamental knowledge about human NC impedes technological advancements. Human NC cells have never been isolated and characterized on cellular and molecular levels. The goal of this proposal is to fill this gap in our knowledge. The PI laboratory has recently developed an efficient procedure for the rapid differentiation of human ES cells into uniform neural precursors (hES-NPCs), which was hitherto unachievable. In culture, hES-NPCs become functional neurons and oligodendrocytes. Intriguingly, our preliminary data show that many markers associated with NC cells are upregulated in hES-NPCs or during their derivation process. For instance, genes previously implicated in NC specification and maintenance were clearly detected. Moreover, a subset of hES-NPCs stained positive for cell surface antigen transiently associated with migrating NC cells in chick. Our hypothesis is that human NC cells are present in hES-NPCs, established in our laboratory. To prove (or disprove) this hypothesis we will rigorously examine the molecular and cellular fates of candidate NC cells from hES-NPCs cultures both in vitro and in vivo. To pursue Aim1 we will 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. However, the in vitro differentiation conditions for all potential human NC cell fates are unknown. In Aim2 we 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. To assure the expertise and skills in the NC field (new for this PI) we have established collaboration with {REDACTED} the world’s-leading expert in the NC filed. Our analysis will unequivocally determine the in vitro and in vivo fates of hES cell-derived cells with the neural crest cell markers present in our cultures. If our hypothesis is correct, hES cell-derived neural precursors will be a rich source for neural crest cells, thus allowing for the first time the extensive characterization of these rare human cells and the development of strategies of NC cell-based protocols in clinics.
Statement of Benefit to California: 
Abnormalities in neural crest (NC) cells involved in numerous human pathologies including various skeletal syndromes (e.g. Apert syndrome), diseases of nervous system (e.g. Hirschsprung’s disease) and pigment disorders (e.g. Waardenburg syndrome).The lack of fundamental knowledge about human NC impedes technological advancements. Human NC cells have never been isolated and characterized on cellular and molecular levels. The goal of this proposal is to fill this gap in our knowledge. The PI laboratory has recently developed an efficient procedure for the rapid differentiation of human ES cells into uniform neural precursors (hES-NPCs), which was hitherto unachievable. Intriguingly, our preliminary data show that many markers associated with NC cells are upregulated in hES-NPCs or during their derivation process. Our hypothesis is that human NC cells are present in hES-NPCs, established in our laboratory. Our analysis will unequivocally determine the in vitro and in vivo fates of hES cell-derived cells with the neural crest cell markers present in our cultures. We will specifically assay the subsets of cells using specific NC-associated markers to enrich for candidate NC lineages. If our hypothesis is correct, hES cell-derived neural precursors will be a rich source for NC cells, and subsequently for an array of human NC derivatives such as bones, cartilage, muscles, peripheral and enteric neurons and melanocytes. Fundamentally, this will allow for the first time the extensive characterization of rare human NC cells and in the future will help the development of strategies of NC cell-based protocols in drug discoveries and clinics. An effective, straightforward, and understandable way to describe the benefits to the citizens of the State of California that will flow from the stem cell research we propose to conduct is to couch it in the familiar business concept of “Return on Investment”. The novel therapies and reconstructions that will be developed and accomplished as a result of our research program and the many related programs that will follow will provide direct benefits to the health of California citizens. In addition, this program and its many complementary programs will generate potentially very large, tangible monetary benefits to the citizens of California. These financial benefits will derive directly from two sources. The first source will be the sale and licensing of the intellectual property rights that will accrue to the state and its citizens from this and the many other stem cell research programs that will be financed by the CIRM. The second source will be the many different kinds of tax revenues that will be generated from the increased bio-science and bio-manufacturing businesses that will be attracted to California by the success of the CIRM.
Review Summary: 
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.
Conflicts: 

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