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RL1-00636-1: Generation of Pluripotent Cell Lines from Human Embryos

Recommendation: Recommended for funding
Scientific Score: 82

First Year Funds Requested: $390,048
Total Funds Requested: $1,177,648

Public Abstract (provided by applicant)

Human embryonic stem cells (hESCs) hold significant promise for regenerative medicine. In this application our goal is to derive hESC lines from pre-implantation embryos to generate a source of low passage lines that can be used in research and to develop the procedures required to generate a clinic grade cell-based product. In this application we will develop the procedures for deriving, expanding and banking using current good manufacturing practices (cGMP), which are necessary for producing a clinic grade line. We aim to do this using chemically defined media and surfaces in consultation with the Director of Regulatory Compliance at our Institution. Secondly, we aim to derive hESC lines from embryos that have genetic disorders. These genetic aberrations are identified following pre implantation genetic diagnosis (PGD) or pre implantation genetic screening (PGS) of consented embryos from couples undergoing in vitro fertilization (IVF). Deriving hESC lines from these embryos will be essential for understanding the underlying causes of spontaneous miscarriage, or as a tool to improve the quality of life of individuals born with these chromosomal abnormalities including Trisomy 21 (Down Syndrome), or Monosomy X (Turner Syndrome).

Statement of Benefit to California (provided by applicant)

Human embryonic stem cells could revolutionize modern medicine if used in cell-based therapies. However, the translational use of hESCs will not be realized unless an appropriate repository of low passage cells is derived and made available. This resource will solidify California as a recognized world leader in ESC research through the generation of a centralized and comprehensive resource of hESC lines and the creation of jobs to support this effort, and validate the decision of California voters who ensured the passage of Proposition 71. Our proposal works toward generating lines that make treatment strategies from hESCs one-step closer.

Review

This proposal is a request for funds to generate new human embryonic stem cell (hESC) lines from both normal and genetically abnormal early embryos. One main goal is to develop consistent and reproducible standard operating procedures (SOPs) for the derivation, expansion and banking of clinical grade hESC lines using current Good Manufacturing Practices (cGMP). The second goal is to generate and bank lines from embryos rejected after pre-implantation genetic diagnosis (PGD). The applicant expects to have access to approximately 100 such embryos per year. The chromosomal abnormalities to be targeted for hESC derivation are trisomy 21, monosomy X, and additional aneuploidies.

This is a fairly straight-forward proposal. Although the level of innovation is not great, the experiments have the potential to yield highly significant results. The availability of a high quality, efficient protocol for the derivation of hESC lines under GMP conditions is essential for developing hESC for clinical trials and eventually cellular therapies, and would represent a step forward for the field. The project represents a collaboration between the applicant, who has access to a derivation lab within the cGMP suite at the applicant’s institution, and a prominent stem cell researcher from a nearby institution with extensive expertise in hESC derivation. The GMP facility is established and functioning, and has been used for FDA-approved clinical studies. The authors clearly intend to share the new lines and have considered mechanisms to facilitate that. The derivation of hESC without immunosurgery and without feeder cell layers is a laudable goal, but the proposed solutions carry their own significant difficulties with FDA approval. Furthermore, the principal investigator (PI) proposes to systematically remove the xeno and undefined components of the system, but makes no mention of the actual in vitro fertilization (IVF) procedures, which include animal and undefined components for culturing and freezing embryos. Also, commercially available media, whose use is proposed in this application, may be chemically defined, but this is not the same as GMP grade. Thus, a reviewer was unsure whether the PI understands all GMP issues, and whether the new lines will be truly GMP-compliant. Furthermore, derivation of hESC in feeder cell-free conditions has proven to be difficult in the past, and a reviewer pointed out that preliminary data showing that the investigators would be able to accomplish this goal was lacking. Overall, though, the reviewers felt that based on past expertise within the applicant’s group, it seems likely that new cell lines can be derived.

The derivation of hESC lines from genetically diseased embryos is also an important goal, since it can lead to insights and potential treatments of genetic diseases. Members of the research team have performed several successful hESC derivations from normal and PGD embryos, including hESC lines from aneuploid embryos. There is little doubt that the group will succeed with this project. Appropriate characterization of all the cell lines for pluripotency and other attributes was planned, although one reviewer felt that not enough detail was provided. Furthermore, a genetic identity assay for each hESC line, although not proposed, will need to be developed, since the likelihood of cross-contamination is high when deriving and banking multiple cell lines. Additionally, one potential benefit of the availability of many newly derived hESC lines is the breadth of the population they may represent. Therefore, each line should be subjected to genetic profiling in order to record the unique “DNA fingerprint” of that line.

The PI is a recently appointed, promising young Assistant Professor. S/he does not yet have significant outside funding nor has s/he published a senior or first author paper since 2005. Therefore, a reviewer expressed some concern regarding how much effort s/he can apply at this stage of his/her career for this project, which is primarily an important banking project, rather than a defined scientific investigation. Furthermore, one reviewer pointed out that despite the ambitious nature of the proposal, the PI is only devoting a 10% effort to it, and co-investigators are also listed with low effort, so it was somewhat unclear who would be doing all of the proposed work. Concern was raised about the commitment of a prominent collaborator, who was judged to be critical to the success of this proposal, but did not request salary on the application, and has only a part time appointment at a nearby institution. On the other hand, another reviewer felt that key personnel with significant experience in developmental and stem cell biology and regulatory affairs were identified and strengthen the application.

This application constitutes a well written proposal from a strong group of stem cell researchers. The topic is important and the cell lines to be created during this award period will likely be quite useful for regenerative medicine and basic research.

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