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RL1-00648-1: Optimization of Human Embryonic Stem Cell Derivation Techniques and Production/Distribution of GMP-Grade Lines

Recommendation: Recommended for funding
Scientific Score: 82

First Year Funds Requested: $462,372
Total Funds Requested: $1,383,419

Public Abstract (provided by applicant)

The government has strict rules for producing cells that will be transplanted into patients. For example, these regulations discourage the use of animal products that could transmit diseases to humans. In this context, the high-quality and tightly regulated procedures that govern other cell-based therapies, e.g., bone marrow transplants, will be applied to regenerative-type clinical applications that employ human embryonic stem cells (hESCs). We need to produce these cells now so that they will be ready to use when research findings are translated into patient therapies. Our goal is to supply researchers in California and outside the state with the highest-quality hESCs. To achieve this goal, we will build on our previous work, published in the scientific literature, which includes deriving hESCs from intact embryos and their single-cell components. We also study the basic properties of embryos and hESCs so that we can formulate theories about how to improve the derivation process, which we then test in our laboratory. For example, adult humans need very precise levels of oxygen. Our work shows that the same is true for embryos and hESCs. We have also developed novel culture conditions that use defined components such as those that are required by the governmental agencies that set the standards for the production of cells used in therapeutic applications. Accordingly, we propose a two-phase approach. During the first two years, guided by advances made by our group and others, we will derive hESCs from embryos in a biologically relevant oxygen environment using defined, high-quality materials. We will also derive hESCs from single cells removed by biopsy from embryos at specific stages of development and/or from particular regions. We think that these lines might have more predictable properties than hESCs that arise randomly, the current practice. Thus, at the end of the first phase we will have produced and banked the next generation of lines, which will be derived under defined conditions that more closely comply with government regulations regarding the production of clinical-grade cells. In the second phase, year 3 of the project, we will use the conditions that best support hESC derivation/propagation to produce lines that can be transplanted into patients. Our efforts will benefit greatly from the infrastructure of our institution, which includes a government-approved facility for doing this work, and from colleagues with the requisite specialized expertise. With the resources provided, we think that we can generate and bank 12 to 20 cell lines; one-third will be produced in a manner that complies with government regulations pertaining to cell-based therapies. All will be widely distributed, as we believe that the pace of translational and clinical research depends on the availability of the highest-quality hESCs and on the important information about their fundamental properties that will emerge from this work.

Statement of Benefit to California (provided by applicant)

California citizens were overwhelmingly in favor of proposition 71, due in large part to the public's belief that laboratory scientists working together with their clinical colleagues could develop therapeutic approaches that utilize human embryonic stem cells and their derivatives in regenerative medicine applications. The research teams are equally excited about cell replacement strategies for treating a variety of medical conditions, as in many cases a cure might be feasible. However, we know from the collective experience of the biotechnology industry that filling the pipeline that leads from basic research to clinical applications inevitably takes time. How do we start this process and shorten the timelines for delivering cell-based therapies to patients? Much of the work in the individual pipelines that focus on specific diseases/conditions can happen in a nonlinear fashion. For example, we need not wait until safe and robust strategies are devised for differentiating human embryonic stem cells into specific cell types to develop the lines that meet government regulations regarding the production of cells for transplantation into patients. There are many reasons that deriving these human embryonic stem cell lines now is crucial. For example, it is likely that production of the cells that will eventually be used in clinical applications will be an iterative process. That is, we will continue to make key discoveries about the fundamental properties of human embryonic stem cells, about which basic information is still needed to improve conditions for growing and deriving lines. This is particularly relevant to the production of clinical-grade cells, as it will be much easier to meet Food and Drug Administration requirements if cells are produced using defined materials that contain only human and recombinant components. It will also be important to know if clinical-grade cells, which for regulatory reasons must be derived under streamlined conditions, have the same properties as other human embryonic cell lines that are used for research purposes. Thus, extensive preclinical testing will be required before these cells are approved by the Food and Drug Administration for use in humans. Thus, accomplishing the major goals set forth in this application will be of enormous benefit to California's citizens. We envision that production of the next generation of human embryonic stem cells that can be used in clinical applications will speed the delivery of therapeutic applications to patients. Along the way these cells will have many other valuable applications. For example, they can be used to screen pharmacologically active compounds for both beneficial and detrimental effects. They will also be valuable tools for understanding the molecular etiology of disease processes. Accordingly, accomplishing the goals of this project will greatly benefit the people’s health and California’s economy.

Review

The overall objective of this proposal is to generate new human embryonic stem (hES) cell lines either from intact human embryos, or from individual blastomeres from cleavage stage embryos. The principal investigator (PI) proposes to optimize techniques and culture conditions to improve derivation of hES cell lines. The PI and his/her group have considerable expertise with hES cell derivation. In addition they have spent considerable effort in developing new culture technology, including the use of defined substrates and serum-free, chemically defined, culture medium. Previous data from this lab suggest that already existent cell lines can be cultured using these methods with pluripotency retained for at least 20 passages. In this application, the applicants would like to extend this work by attempting to generate new cell lines using these techniques. The PI also proposes to generate cell lines from single blastomeres. Previous work from the PI’s laboratory shows morphological heterogeneity among blastomeres which may contribute to functional heterogeneity observed in stem cell line derivatives obtained from these blastomeres. If successful, the proposed work may be useful for the derivation of more homogeneous hES cell lines. In the later part of the project the PI plans to use the optimized culture conditions to generate current Good Manufacturing Practices (cGMP)-grade hES cell lines destined for clinical use. The PI proposes to generate and bank a total of 12 to 20 new cell lines; one third will be produced under cGMP conditions.

There was general agreement among the reviewers on the validity of the scientific rationale, and reviewers believed that the approach is sound and well developed. The PI’s institution has established an embryo bank with hundreds of embryos available for the derivation of new cell lines. However, it was pointed out during the discussion that these embryos were not collected under cGMP conditions, and therefore, cGMP production of cell lines proposed in this application will require further release testing required by the FDA.

Reviewers concurred that the PI is qualified to lead this program. The PI is an established investigator in the stem cell field and collaborators listed in the application all have excellent credentials and scientific backgrounds to facilitate the derivation of cell lines under cGMP conditions.

Overall, reviewers thought this to be an excellent application from a very competent group. Research proposed here will likely enhance our understanding of ES cell biology and differentiation. Research grade and cGMP grade new cell lines produced under this grant will be available to other investigators.

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:

• Heimfeld, Shelly