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RL1-00665-1: Derivation of scalable clinical grade human embryonic stem cells under current Good Manufacturing Practice conditions using conventional and single blastomere biopsy protocols

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

New human embryonic stem cell lines for the clinical repair of damaged or missing tissues. Human embryonic stem cells have great therapeutic potential because of their ability to grow indefinitely and their potential to produce almost any cell type and tissue in the body. This makes it theoretically possibly to produce an inexhaustible supply of cells, tissues, and organs to repair damaged or diseased tissues in patients. However, several problems exist that must be resolved before human embryonic stem cells can be used for human therapies. Almost all the currently available human embryonic stem cell lines have been grown under conditions using animal products. This is problematic because the human embryonic stem cells may have been contaminated with animal viruses, prions or other animal pathogens making them unsuitable or, at least, suboptimal for therapeutic use in humans. We propose to develop a cell culture system to grow human embryonic stem cells that does not use animal products. Furthermore, in conventional methods used to develop new embryonic stem cell lines, the embryo is typically sacrificed, which raises an ethical concern about the destruction of the embryo. We propose to develop new human embryonic stem cell lines by removing a single cell from the embryo to develop a new embryonic stem cell line. Since human embryonic stem cell can divide indefinitely, we can expand that single cell into a new embryonic stem cell line. By removing only a single cell from the embryo, the embryo will remain alive, obviating the ethical concerns about sacrificing embryos to produce new human embryonic stem cell lines. Finally, many cell types in the body do not divide (duplicate themselves) well when grown in tissue culture plates, thus, making it difficult to produce enough cells for use in patients. We propose to develop new tissue culture systems to grow human embryonic stem cell in mass cultures so that they can be expanded in sufficient quantities and then converted into the desired cell type for use in patients.

Statement of Benefit to California (provided by applicant)

California, like much of the United States, is facing a staggering challenge to its health care system. The large investments made in recent decades by the National Institutes of Health (NIH) have largely ignored the problems of age-related degenerative disease. As a result, increasingly physicians are treating the chronic, debilitating, and therefore expensive diseases associated with aging. This is made all the worse by the demographic wave caused by the entry of the Baby Boomers into retirement. It is estimated that by the year 2010, the Baby Boomers will be 25 percent of the population of California. By 2020 they will be approaching 64 years of age. As a result, the percentage of the elderly in California is expected to grow from 14 percent in 1990 to 22 percent in 2030. (Source: California Department of Finance, Population Projections 1993). Many of the chronic devastating diseases of an aging population are the degenerative diseases. Generally speaking, degenerative diseases are those diseases caused by the loss or dysfunction of cells. Examples include osteoarthritis (loss of cartilage cells that protect the ends of the bones), Parkinson’s disease (the loss of dopaminergic neurons), osteoporosis (dysfunction of osteoblasts), macular degeneration (dysfunction of retinal pigment cells) and so on. More significantly, the loss or dysfunction of cells in the heart (or the vessels that supply the heart with blood) results in heart disease, the most frequent cause of death in California. In 2001 (the most recent year data is available) heart disease caused 68,234 deaths (29% of all the deaths in the state). Stroke is also a vascular disease and the third leading cause of death in California. In 2001, stroke caused 18,088 deaths (8% of all of the deaths in the state). Regenerative medicine represents the effort of cell biologists to invent a new approach to the problem of degenerative disease. Human embryonic stem (hES) cells have the potential to become all of the cells in the human body, and their unique properties give researchers the hope that from these primitive cells new therapies can result that may be available in time for the looming health care crisis. It is estimated that are over 200 cell-types in the adult human and hES cells are capable of making all of these. However, to turn this new technology into actual therapies that can alleviate human suffering, researchers need new tools to generate large numbers of purified cell types. This proposal describes a project to derive new embryonic stem cells lines that are clinical grade and suitable for therapeutic use in regenerative medicine.

Review

The primary goal of this proposal is to produce clinical grade human embryonic stem cell (hESC) lines under current good manufacturing practices (cGMP) conditions using conventional and single blastomere biopsy protocols, the latter having been developed at the applicant’s institution.

In aim one, the applicant proposes to develop animal product free (APF) hESC culture media and cell substrates under cGMP conditions. The rationale for this goal is based on the fact that most existing hESC lines have been in contact with animal products during their derivation or propagation, thereby making them suboptimal for therapeutic applications. In the second aim, the applicant proposes to derive new cell lines from single blastomeres using the media and culture conditions derived in aim one. This technology has the advantage of leaving the embryos unharmed. In the third aim, the applicant plans to develop GMP procedures to expand cells en masse for differentiation of the cell lines to useful cell types, using a process developed by the applicant’s institution.

The significance of this proposal lies in the development and optimization of an APF hESC derivation protocol, and the derivation of new hESC under cGMP conditions. However, reviewers questioned whether the proposed approach was truly APF, since some of the proposed components are known to contain animal products, others were not described in sufficient detail for the reviewers to assess their APF status, and the applicant makes no mention of the fact that the embryos themselves are not collected in a cGMP-compliant manner. So it was unclear to what extent cGMP-compliant hESC lines will be produced. Furthermore, reviewers felt that preliminary data were insufficient to assess the potential for a successful large scale expansion of the cell lines.

Although one reviewer felt that the proposed derivation of hESC from single blastomeres is a useful goal, others criticized the use of this technology in the present proposal. While the single blastomere technique has the potential to yield hESC lines without destroying the embryo, thereby avoiding a major ethical concern, this holds little significance for the present proposal. The embryos used in this proposal are surplus embryos supplied by an in vitro fertilization (IVF) bank. It appears that these embryos will be consented for donation for research purposes and are not expected to be returned to the fertility clinics for implantation. There does not seem to be a shortage of surplus IVF embryos available for this project and others like it, as indicated by the letter of support submitted with the proposal. The researchers will most likely have better success rates using more traditional hESC derivation techniques, i.e. isolation and culture of the entire inner cell mass. Moreover, although some of the co-investigators have strong, relevant backgrounds in hESC research, it was unclear from the proposal whether the applicant actually has sufficient expertise to accomplish the proposed work. The most relevant experience with stem cell research in the applicant’s organization resides with a collaborator at a location outside of California, and the extent to which the collaborator will directly interact with the research team in California is not clear. Additional concern was raised with regard to the proposed time line, which was viewed as unrealistic. Overall, the enthusiasm for the proposal was not very high.

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