Funding opportunities

Manipulation of Hedgehog signaling in early human embryos

Funding Type: 
SEED Grant
Grant Number: 
RS1-00218
Funds requested: 
$407 718
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
We propose deriving a new generation of embryonic stem cell lines with greater potential than current conventional lines. Until recently, all human embryonic stem cell (hESC) lines have been derived from one of two clearly discernible cell types, the inner cell mass (ICM), of the blastocyst stage embryo. ICM-derived hESC are able to differentiate to a number of tissue types. Nevertheless, such conventional lines range widely in how easily they give rise to adult cell types. Investigations with ‘twin’ hESC lines, i.e. lines made from splitting a single ICM, show that cells at this stage of development give rise to lines with distinct differentiation preferences. Thus a given protocol to generate a therapeutically desired cell type has a variable chance of success when applied to any particular ICM-derived hESC line. Recently, a novel technical advance has allowed hESC to be derived from cells of significantly younger embryos (i.e. from blastomeres of 8-10 cell cleavage stage embryos). These cleavage stage hESCs are superior to conventional lines in the ease/efficiency with which they could be induced to differentiate a variety of cell types. As yet, however, only 2 such lines have been established. Through our work in mice, we have developed a protocol to keep cleavage stage embryos from advancing through the usual program leading to blastocyst formation. We hypothesize that culturing human blastomeres under such conditions will allow more efficient derivation of cleavage stage hESC. Compared to the hESC lines in circulation today, blastomere-derived hESC lines are potentially far superior in their differentiative capacities. Such properties could greatly unify and advance the field in efforts to generate cells types of therapeutic interest.
Statement of Benefit to California: 
The central hope of Proposition 71 is that human embryonic stem cell (hESC) research will alleviate devastating medical conditions such as diabetes, Parkinson's, Alzheimer's and cancer. Realization of this goal requires solving the problem of being able to reliably generate, in culture, a given cell type from any hESC line. Currently it is still not possible to differentiate (or generate) any given cell type from any given hESC line. Our proposal to create a new generation of hESC line from cells of younger embryos may move the field closer to this goal. Recent technical advances made by the private sector on the East Coast have allowed derivation of the first two hESC lines from blastomeres (cells) of 8-10 cell embryos, i.e. cells that are only about 3 days post fertilization as opposed to 6 days in conventional hESC derivations. These two novel lines have shown a greater efficiency and capacity for differentiation than conventional lines in a number of assays. We have developed a protocol in mice that suggests a modification of this new method may improve on such blastomere-derived hESC derivations. We are therefore poised to take a lead in research creating a new generation of hESC lines that may supercede those currently in circulation, including a major source of lines made by private foundations on the East Coast. Given such a leading edge, and with one of the highest concentrations of college graduates in the nation, the state of California could transform the fields of embryonic stem cell research and medical discovery.
Review Summary: 
SYNOPSIS: In this proposal the PI will attempt to develop more efficient methods to generate new hESC lines from cleavage stage embryos rather than from the traditional blastocyst stage. The rationale for cell line derivation from cleavage stage embryos is that the cell lines may be more pluripotent with superior differentiative capacities. Some previous studies suggest that it is possible to use this source of starting material and that the cell lines are pluripotent. However, these are new studies, and their generality is not yet certain. The PI hypothesizes that it may be possible to increase the efficiency of hESC line derivation from cleavage stage embryos by treating with Hedgehog inhibitors. The PI has shown that culturing murine 2-cell stage embryos in the presence of Hedgehog signaling inhibitors is permissive for cleavage, but that compaction and cavitation are inhibited. The proposed studies will attempt to apply this strategy to the human system. In the first aim, the PI proposes to take advantage of the results in the murine system that demonstrate inhibition of compaction and cavitation of early embryos by antagonizing the Hedgehog signaling pathway. The PI will start with 2-cell embryos from IVF clinics and culture these with cyclopamine or a novel inhibitor compound SD that will be provided by a collaborator. In this aim, the goal is to establish effective dose ranges. Parallel experiments with Indian Hedgehog (Ihh) wil also be performed. The results will be evaluated by analysis for caviatation and compaction, as well as continued cleavage. In addition, markers of pluripotency will be evaluated. These studies appear to be straightforward, and should set the stage for aim 2. In this aim, the PI will attempt to derive outgrowths and ultimately hESC lines. SIGNIFICANCE AND INNOVATION: The proposal is both fairly innovative and significant. The potential to derive hESC lines by starting with single blastomeres from embryos at stages earlier than the blastocyst stage is fairly innovative. The use of Hedgehog signaling inhibitors to block compaction and cavitation of the embryos is novel. The studies are also significant given the possibility of deriving hESC lines from single blastomeres without necessarily destroying the starting embryo. This could impact on the political debate regarding the establishment of novel hES cell lines. STRENGTHS: The studies in this proposal are solid and the goals - to use cleavage stage embryos to improve the quality and efficiencey of hESC line deriviation - are worthy; furthermore it is research that can not be funded by the NIH. The studies described for aim 1 are straightforward, and should provide valuable information, as well as setting the stage for aim 2. Additional strengths of this proposal include the experience and expertise of the researchers in manipulating and understanding hedgehog signaling as well as the collaborative nature of the proposal. WEAKNESSES: This proposal has a number of signficant weaknesses. The overall justification provided for these experiments is that ICM derived cell lines are heterogeneous, a claim that is substantiated by one low quality paper that is in stark contrast to a large body of work that ICM derived cell lines even from SCNT blastocysts are extremely similar in mice and humans. Moreover, there is very good evidence that blastomeres in early embryos are specificied/fated much earlier than compaction. Thus, there is no guarantee that deriving lines from cleavage stage human embryos would make them more consistent. In fact, published evidence suggest that when cell lines are derived from 8-cell stage mouse embryos, half of the cell lines become trophectodermally fated, and are not useful for creating adult cell types. Additionally, there is absolutely NO evidence that cell lines derived from early embryos are better than those derive from the ICM in terms of growth or pluripotency. Finally, the role of hedgehog signaling in early embryos is ill-defined, suggesting that manipulating this pathway may compromise the developmental potential of cell lines derived via the methods proposed here. Aside from concerns as to the underlying rationale, there was also questions as to how experiments would be analyzed to address the hypothesis of improved efficiency and quality of lines generated from cleavage stage embryos with hedgehog inhibitors. For example, it is not clear how the PI will demonstrate that any newly established hES cell lines are in fact superior to those derived by standard techniques. The PI claims that the lines will be a "fundamentally new type of stem cell line", and that the low efficiency of the Lanza studies may be due to the fact that the individual blastomeres simply go on to form blastocysts, which then go on to produce mixture of cell types. The hope is that blocking of Hedgehog signaling will prevent this. There is no discussion on how the PI will determine that any resultant cell lines are different and superior. There is also little discussion of how success or failure of the proposed studies will be defined. For example, how many 2-cell embryos will be utilized, with what success rate, and how success will be measured. These are significant weaknesses to this proposal. DISCUSSION: There was no further discussion following the reviewers comments.
Conflicts: 

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