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RL1-00675-1: Generation of human embryonic stem cell lines, under defined conditions, modeling normal & diseased states from material stored at the {REDACTED} embryo bank

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

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, {REDACTED} is designed to serve the research community at no cost. With numerous layers of ethical, medical, & 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). We propose to generate a library of well-defined hESC lines that authentically model the range of diseases one can diagnose in the embryo & that would compel them to be rejected for implantation. Although unethical to implant, these blastocysts could yield hESCs that might ultimately inform us of the mechanisms underlying incurable human diseases & perhaps point the way towards new therapies. Indeed, the lines could be used to screen for new drugs & diagnostics. Few teams are as well equipped as we to pursue these critical proof-of-concept experiments while providing a valuable resource for scientists. In addition, {REDACTED}, which, as an academic non-profit embryo bank providing a service to scientists & families without encumbrances, requires CIRM financial support to stay active.

Statement of Benefit to California (provided by applicant)

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 intellectual property (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. Also, California citizens should become stakeholders in the IP & benefit from any royalties & fees.

Review

The goal of this proposal is to generate and characterize a number of new human embryonic stem (hES) cell lines from normal blastocysts and blastocysts that have been found to carry disease-related alleles via preimplantation genetic diagnosis (PGD). In particular, the applicant will focus on embryos that have trisomy for chromosome 21 (Down’s Syndrome) and that carry mutations in the CFTR gene (Cystic Fibrosis, CF). The proposal attempts to generate and characterize the hES cells from normal blastocysts grown under defined conditions, and to characterize the hES cells with pathological genotypes, e.g. to validate their pathological characteristics against human diseased tissue. Finally, the applicant intends to differentiate both the normal and the disease hESCs to various lineages and to characterize their response to stressors.

The panel debated the significance of the proposal, since many felt that cell lines for these disorders already exist, and were widely available. One panelist commented that many CF lines are available throughout the world, some with a very simple material transfer agreement (MTA). Another panelist noted that perhaps the existing Down’s cell lines are less available to the broad research community.

Reviewers concurred that the proposed generation of new hES cell lines is fairly straightforward and the groups involved clearly have the expertise necessary to successfully carry out this portion of the proposal. In addition, the source tissue was already available in the applicant institution embryo bank. Reviewers also identified weaknesses with the proposal. The third aim of using Down’s Syndrome and CF hES cell lines to model these diseases was felt to be less straightforward. In particular, no reliable protocols exist to efficiently produce lung epithelial cells, which are the target cell type of CF. Moreover, as Down’s Syndrome affects multiple cell types in very different ways, the proposed experiments for this disease lack focus and clear, measurable cellular pathologies. Another reviewer’s largest concern with this proposal was the amount of work proposed and the number of key personnel that will be committed to it. A large amount of tissue culture time is required just for manipulation and maintenance and expansion of cell lines, and additional time is required for their characterization. This reviewer also noted that no one is assigned to the project with 100% effort, and worried about commitment to the project.

This proposal was felt to be responsive to the RFA, and should culminate in the development of novel pluripotent stem cell lines. The proposed work is likely to shed some light on early stages of diseases and result in some valuable research tools. Panelists, however, would have been more enthusiastic about this proposal if it addressed genetic defects for which cell lines were not currently available.

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