Funding opportunities

Derivation of human embryonic stem cell lines from human blastocysts: analysis of genetic stability and ethnic diversity

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00635
Funds requested: 
$1 708 542
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Human embryonic stem cells have enormous potential for expanding our knowledge of human disease, for developing safer, more effective drugs, and for therapeutic solutions to incurable disease. However, forward progress has been severely slowed by the lack of available hESC lines. Since President Bush's Executive Order on August 9, 2001 banned NIH funding for any hESC lines derived after that time, there has been little funding available for derivation of new lines, with the consequence that the most widely studied hESCs were developed seven years ago . A great deal has been learned about hESCs in the last seven years; most importantly, we have learned that the available hESC lines develop genetic abnormalities during culture, and that the methods used for derivation of the NIH-approved lines are likely to have caused genetic abnormalities. There is also a great need for hESC lines that pharmaceutical companies can use to speed the pace of drug development. The goals of this research program are to increase the number and diversity of hESC lines available to researchers and for clinical applications, and to study the genetic stability of the cells as they expand in culture. Ultimately, we plan to determine whether the genetic changes affect the ability of hESC cells to become certain types of adult cells, and whether genetic abnormalities increase the probability that the cells will form tumors after transplantation. We will thoroughly analyze the new cell lines and provide both the cells and this information to the scientific and clinical communities.
Statement of Benefit to California: 
Californians are a large and diverse population that poses unique challenges for the future of medical care. Fortunately, California has a tradition of taking the lead in technology and medical breakthroughs and following through from the first idea to the final product. A major goal for California's supporters of stem cell research is development of stem cell-based products that have medical use, and the mandate for the research community is to provide the best possible fundamental information to help guide clinical applications. We have already laid the groundwork for research that encompasses both federally approved and non-approved human embryonic stem cells (hESC) by establishing a privately funded resource to collect excess embryos that have been donated for research. This embryo bank, which has multiple layers of ethical oversight, currently has more than 1000 donated embryos. We propose to use this resource to generate new hESC lines that reflect the genetic diversity of California. Our long term goals are 1. to speed the development of drugs that are safe and effective for all people, regardless of their ethnicity, and 2. to make human embryonic stem cells as safe as is possible for cell therapy, by ensuring that they retain normal, noncancerous qualities.
Review Summary: 
Executive Summary The applicant intends to derive new human embryonic stem cell (hESC) lines from blastocysts using defined culture conditions, and then to analyze the genetic stability of these cells over time in culture. The applicant proposes to generate a number of new cell lines over the next 3 years. Once established, these cells will be followed over time in culture and analyzed for genetic stability using standard karyotyping and genotyping methods. The genotyping analysis will then be used in comparison with a public database to determine ethnicity of the cell lines and to provide a genetic fingerprint of the cells, with the intent to encourage the use of hESCs in pharmacogenetic drug development. A secondary objective is to correlate observed genetic changes with differences in phenotype, differentiation capacity, and/or tumorigenicity. The PI plans to distribute these well characterized cell lines to the research community. Reviewers felt this proposal addressed an important question of quality assurance in the derivation of new cell lines. The plan would fulfill the critical function to develop an analytical fingerprint to ensure correct identity of cell lines. The proposal is focused on establishing metrics for characterizing new hESC lines. These metrics will be useful in establishing quality control standards as use of hESC lines moves towards the clinic. The PI was noted to have expertise in brain development, but less in hESC biology than is ideal for the project. The PI has documented previous success with the derivation of new hESC lines; however, the PI acknowledges that only minimal characterization was previously completed due to a lack of funding. In this proposal, the design for the characterization was felt to be sound, and that it will provide the detailed information to characterize the ancestry and stability of the derived hESC lines. The collaborator associated with the project has great expertise in karyotyping and genotyping methods, and this collaborator’s skills should be transferable to the proposed platform. These strengths were offset by a number of weaknesses with the proposal. Reviewers were intrigued with the idea to correlate ethnic diversity with drug sensitivity, but could not assess the feasibility of this concept, since no data were presented about how this will be done. Since the plan did not include donor screening, ethnic diversity of the resultant lines can only be determined relatively late in the derivation process. Ultimately, reviewers felt that the project will be dependent on random chance to achieve ethnic diversity. Reviewers also cited issues with manpower in the proposal. Overall, inadequate personnel resources were identified for the proposed scope of work. One reviewer expressed concern that the PI on this application may be overly-committed in terms of percent effort. Finally, the proposal describes the possible use for clinical applications, but reviewers noted demonstration of only minimal understanding of FDA regulations or requirements for GMP clinical qualification. The application was felt to be responsive to the RFA. One of the key aims of this project is to distribute cells which have authenticated identity, ethnic composition, genetic stability information, and documented pluripotency. The analysis of all cell lines relies on well established techniques to assess pluripotency, and the cell lines will be distributed to researchers under a simple letter of agreement, although the applicants will charge a fee to recover some of the cost of scaling up the cells for distribution. In summary, the panel felt that the goal to correlate observed genetic changes with differences in phenotype, differentiation capacity, and tumorigenicity was feasible, but not significant to the field at the low number of cell lines proposed in the application. Furthermore, the intriguing part of the concept, to characterize a relationship between ethnic diversity and drug sensitivity, could not be accomplished by studying only the relatively small number of cell lines proposed in the application. Reviewer One Comments Significance: Dr Chun and his team plan to: • develop 10 new hESC lines under the CIRM program that will provide a limited sample of hESC with diverse ancestry. • use defined conditions, applying new methods as they are validated by other laboratories. • characterize these new lines, along with existing lines, using a genome-wide panel of single nucleotide polymorphisms. • Use the SNP profile to determine the population ancestry of the hESC lines • examine the genetic stability of these lines as they are continuously cultured in vitro. • employ a number of other tests to determine pluripotency. The team addresses the important question of quality assurance - how are cell lines to be authenticated and what are the standards that should be in place to ensure that hESCs are characterized sufficiently to be used eventually as a therapeutic? The addition of Dr. Kelly Frazer to the team is a good move in this direction. Feasibility: My comments are directed to the genomic characterization of the stem cell lines. A combination of karyotyping and SNP genotyping will be employed. Dr. Frazer has great expertise in the application of genome-wide SNP panels to the study of population diversity and also the fine-mapping of DNA structural elements that may change during culture. Her experience with the Affymetrix platform should transfer to the Illumina platform, as will her analysis tools. I think the design is sound and will provide the detailed information to characterize the ancestry and the stability of the derived hESC cell lines. Responsiveness to RFA: The proposal is responsive in that 10 new hESC cell lines will be developed using defined conditions and their ancestry characterized. Dr. Chun explicitly states that that “we don’t have plans to use more challenging methods of derivation” so the value in this work is in establishing metrics for characterizing new hESC lines. These measures may allow: • Comparison of the robustness of different method for the derivation of hESC • Genetic stability of hESC lines in culture • Differences that relate to the ancestry of the hESC line These metrics will be useful in establishing quality control standards as use of hESC lines moves towards the clinic. Reviewer Two Comments Significance: The applicant intends to derive new human ES cell lines from blastocysts using defined culture conditions, and then to analyze the genetic stability of these cells over time in culture. Finally, the applicants propose to determine the ethnic diversity of each of these cell lines and to create the genetic fingerprint using SNP genotyping and the HapMap database. Feasibility: The goal of this project is to develop new human embryonic stem cell lines which have been derived under defined culture conditions including the use of serum free medium and human feeder layers. The applicants propose to generate at least 10 new cell lines over the next 3 years. Once established, these cells will be followed over time in culture and analyzed for genetic stability using both karyotyping and SNP genotyping methods. The SNP analysis will then be used in comparison with the HapMap database to determine ethnicity of the cell lines and to provide a genetic fingerprint of the cells. The PI on this application may be overly-committed in terms of percent effort; there is poor justification of costs associated with the project and the travel costs to CIRM meetings seem overly inflated. Responsiveness to RFA: One of the key aims of this project is to distribute cells which have authenticated identity, ethnic composition, genetic stability information, and documented pluripotency. The analysis of all cell lines relies on well established techniques to assess pluripotency, and the cell lines will be distributed to researchers under a simple letter of agreement, although the applicants will charge a fee to recover some of the cost of scaling up the cells for distribution. Reviewer Three Comments Significance: Creation of many more hESC lines, of high quality and with greater ethnic diversity, would greatly benefit the field. Feasibility: The PI plans to derive new hESC under more “defined” conditions. The goal is to derive “at least” 10 lines during project, which is a relatively minimal goal. They have some documented success in the past with new derivation of hESC lines, however they acknowledge that due to lack of previous funding only minimal characterization was completed. They also acknowledge that the use of feeder cells complicates their analyses. With the newly derived lines they plan to characterize by relatively standard functional, molecular, and cellular methods. They will assess genetic stability and pluripotency using classical karyotyping, SKY, and SNP genotyping. They plan on analysis of 20 metaphase spreads instead of usual 10. They state that pluripotency and teratoma assays are challenging. They will perform gene expression profiling (300 genes identified as highly expressed in pluripotent cells). Teratoma formation will be assessed in immunodeficient mice There is a somewhat novel use of SNP analyses, by which they plan to fulfill the critical function to serve as a fingerprint to ensure correct identity of cell line, there have been well-documented problems in the past with other cell line mix-ups. They plan to use this SNP data, along with the HapMap database, to assess ethnic diversity. This is an interesting approach for characterizing existing hESC and the newly derived lines in the absence of specific donor information. However, there was only limited preliminary data (text only) which supports success with this approach. They hypothesize that ethnic diversity may correlate with drug sensitivity, but again there was no direct data presented on this point. The PI is well-funded, although mostly focused on brain development rather than hESC derivation, including the following projects: 1. Development of cerebral cortex, extracellular signaling by neurons 2. Schwann cell biology – lysophospholipid signaling 3. Embryo implantation – lysophosphatidic acid and prostaglandins 4. Receptor signaling in embryonic brain 5. Aneuploid neural cells in brain disease Concerns: Inadequate personnel resources are identified for the proposed scope of work: PI is only listed for 10% effort. Co-Investigator Loring only listed for 10% effort, Co-investigator Smotrich only listed for 5%. One tech with 2 years experience on hESC culture at 20%, and a senior Post-doc trainee will perform the bulk of the karyotyping, SKY, and FISH genotyping, while another TBD Post-doc will be responsible for ethnic diversity studies. Because there is no donor screening, ethnic diversity can only be determined relatively late in the derivation process. If there is no way to pre-select or pre-screen the starting material, in the end the project is dependent on random chance to achieve ethnic diversity. Finally, the proposal describes the possible use for clinical applications, but there was only minimal understanding of FDA regulations or requirements for GMP clinical qualification. For example, the proposal states no use of animal products, but does state use of media (StemPro) and human feeder layers (foreskin fibroblasts from a commercial supplier), neither of which are qualified for clinical manufacturing. Responsiveness to RFA: This proposal only meets one of the four overall goals of the RFA (increasing ethnic diversity). The methods and description did not seem generally applicable for generation of clinical-grade hESC or alternative patient matched or disease-specific cell lines. In addition, with only 10 new lines generated, and no method to preselect for greater diversity, the overall increase in ethnic diversity of hESC would likely be trivial.
Conflicts: 

© 2013 California Institute for Regenerative Medicine