Funding opportunities

Generation of hESC lines, under defined conditions, modeling normal & diseased states from material stored at the Burnham shared embryo bank.

Funding Type: 
SEED Grant
Grant Number: 
RS1-00305
Funds requested: 
$638 000
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
The human embryonic stem cell (hESC) lines eligible for federally-funding are limited not only in number but also in their: (a) range of applications (e.g., because of their “contamination” with poorly-defined biological components); (b) genetic & cell type stability; (c) ability to represent diverse racial & genetic backgrounds, disease states, & heritable predispositions to disease; (d) ease of manipulation, expansion, differentiation; (e) safety. The field would benefit from new hESC lines that not only reflect newly emerging biological insights but that improve upon extant lines in the following 3 key areas: (1) hESC lines derived from their inception under “defined” conditions (i.e., conditions where all ingredients can be specified); (2) hESCs that can serve as models for a range of human diseases (particularly those for which poor animal models exist) which would allow the development of better therapies, including drug development; (3) hESCs that can be more facilely maintained under defined conditions in the immature state & then efficiently & reproducibly become desired cell types to the exclusion of undesired cell types. To help meet these needs, we have established a repository for normal & abnormal preimplantation embryos donated from IVF clinics nationwide for research purposes. This non-profit facility is designed to serve the research community at no cost. With numerous layers of ethical & financial oversight in place, we have already catalogued ~1000 normal & disease-bearing blastocysts. Our Stem Cell Research Center has devised improved techniques for deriving & differentiating hESCs. One of these advances was the establishment of an efficient long-term defined culture system for hESCs that is based on having determined the minimal essential components for insuring pluripotency & exploits intrinsic embryogenesis-like processes. These techniques have been used to begin deriving hESC lines from normal & diseased blastocysts (identified by pre-implantation genetic diagnosis [PGD]). The repository, because it has access to IVF services, is also staffed by an embryologist with extensive experience in nuclear transfer (NT), i.e., transfering the nuclei from skins cells into unfertilized eggs in large animals. We also maintain skin cell cultures from patients with genetic defects, the best characterized of which is called Lesch-Nyhan disease (LND). In the spirit of the Seed Grants, we propose to combine all of our skills & resources to generate well-defined disease lines not only from PGD but also by NT – as proof of concept & validation of the approach. In the course of pursuing these goals, we will also generate needed normal hESC lines. Few teams in CA are as well equipped as we to explore these critical yet admittedly high risk proof-of-concept experiments.
Statement of Benefit to California: 
The human embryonic stem cell (hESC) lines eligible for federally-funded research are limited not only in number but also in their: (a) range of applications (e.g., because of their “contamination” with poorly-defined biological components); (b) genetic & cell type stability; (c) ability to represent diverse racial & genetic backgrounds, disease states, & heritable predispositions to disease; (d) ease of manipulation, expansion, differentiation; (e) safety. The field would benefit from new hESC lines that not only reflect newly emerging biological insights but that improve upon extant lines in the following 3 key areas: (1) hESC lines derived from their inception under “defined” conditions (i.e., conditions where all ingredients can be specified); (2) hESCs that can serve as models for a range of human diseases (particularly those for which poor animal models exist) which would allow the development of better therapies, including drug-based; (3) hESCs that can be more facilely maintained under defined conditions in the immature state & then efficiently & reproducibly become desired cell types to the exclusion of undesired cell types for research & cell-based therapies. To help meet these needs, we have established a repository for normal & abnormal preimplantation embryos donated from IVF clinics nationwide for research purposes. This non-profit facility is designed to serve the research community – particularly in California-- at no cost. We have also devised improved techniques for deriving & differentiating hESCs. These techniques have been used to begin deriving hESC lines from normal & diseased blastocysts. These new lines, too, become invaluable unique reagents for drug testing, for testing the pathophysiological mechanisms underlying a wide variety of disease states, & for developmental studies. Federal funding cannot presently be used to generate such new lines. Lines generated with industrial funds would inevitably carry large licensing fees & restrictions on the use & dissemination of IP & insights derived from such studies. Our intent is that such reagents be distributed in an unencumbered manner to other investigators who would be obligated to share data with other scientists, & that the products of that research (drugs, therapies, diagnostics, technologies) be made available at minimal cost to California citizens.
Review Summary: 
SYNOPSIS: This research team, led by Dr. David Smotrich proposes to take on the challenge of deriving new human embryonic stem cell (hESC) lines from existing products of in vitro fertilization. The advantages of such lines are the potential to maintain them under defined conditions from their inception, to model disease states by deriving lines from blastocysts identified as defective by preimplantation diagnosis (PGD) and to derive specific lineages from established hESCs under defined conditions of culture. In a final aim, they propose to derive disease gene bearing hESC lines by somatic nuclear transfer (SCNT) focusing on Lesch Nyhan disease fibroblasts as a model. Extensive preliminary data are presented suggesting the feasibility of deriving new lines of hESC as proposed. INNOVATION & SIGNIFICANCE: This is a highly inovative proposal from a highly experienced group of investigators. They propose to directly explore the challenges in human stem cell biology by attempting to obtain new lines under defined conditions and specifically to derive lines affected by a variety of human disease gene mutations. Their commitment to making such lines available for research without restrictions could have a major impact on the field of stem cell research. Overall the reviewers rank this proposal very highly for both inovation and significance. This proposal is exactly the type of highly innovative and significant research the CIRM should look to fund. STRENGTHS: The researchers appear to be in a class of there own in terms of experience, access to embryonic material, and collaboration. To summarize, (1) these are experienced investigators with an established track record of working together. (2) They are experienced in preimplantation diagnosis which is necessary for identifying appropriate potential sources of hESC with human disease genes. (3) They present extensive preliminary data. WEAKNESSES: One potential weakness is the speculative nature of the research, specifically for Aim 4. However, if the real potential of hESC is to be realized, this type of research will be essential. The only major weakness of the proposal is that they would use somatic cells with Lesch-Nyhan disease (LND) as a proof-of-principle nuclear donor in their SCNT experiments. LND hESC lines are much more easily generated by targeted mutation of HPRT (previously reported) or hESC derivation from LND+ embryos that have undergone preimplantation genetic diagnosis (PGD). The incredibly valuable and rare commodity that is oocytes would demand that the researchers choose to create disease/patient specific cell lines for those diseases that can not be easily genetically engineered or tested for by PGD. DISCUSSION: This proposal is exactly the type of research that CIRM should be funding. The personnel is second to none, and it could not have been funded by NIH. Most of the time working on this research will be spent working out how to do the transfer. In summary, this proposal aims to generate new hESC lines via SCNT and has developed collaborations with a PGD clinic. A good team of researchers that is "second to none" has been assembled. One weakness is that the plan to focus on Lesch Nyhan (LN) isn't very creative. What about more complex diseases? What about issues associated with homologous recombination? Working out the mechanisms will be a challenge.
Conflicts: 

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