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RL1-00635-1: Derivation of human embryonic stem cell lines from human blastocysts: analysis of genetic stability and ethnic diversity

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

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 (provided by applicant)

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

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.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None