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RL1-00656-1: Generation of human ESC lines using SCNT

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Transplantation of somatic stem cells (SC) such as hematopoietic stem cells is currently used in the treatment of blood disorders and immunodeficiencies due to chemotherapy. There is growing evidence that transplantation of other somatic SCs, including mesenchymal and neural SCs, can be beneficial for patients with a variety of degenerative conditions. However, somatic SC use has several disadvantages: 1) incompatibility of donor and host cells; 2) restricted differentiation potential; and 3) only limited quantities can be obtained from adult donors or neonatal tissues or by in vitro expansion. In contrast, ESC lines are capable of developing into all tissues in the body and can be expanded ad infinitum. Thus, ESC may serve as an excellent, alternative source of transplantable cells for regenerative medicine. However, similar to SCs, the use of ESCs produced conventionally using in vitro fertilization will be limited by host-graft rejection. Experimental approaches designed to avoid this outcome involve the use of retroviral vectors and the introduction of exogenous DNA or the creation of stem cell banks. A third option is the use of somatic cell nuclear transfer (SCNT) to generate patient specific, histocompatible ESCs in a process referred to as therapeutic cloning (TC). While TC has not yet been accomplished in humans, recent success has been reported in the rhesus monkey using a novel protocol for SCNT. Here, we propose to adapt this novel patented technology to the generation of human, patient-specific, SCNT-ESCs. Because of similarities between primates, monkeys and humans, it is likely that this novel research will result in the generation of the first human SCNT-ESC lines thereby providing a basis for further development of cell-based therapeutic approaches in the treatment of degenerative disorders.

Statement of Benefit to California (provided by applicant)

The concept of stem cell based therapy implies that damaged tissues can be repaired by tissue-specific stem cells that generate mature, functionally active progeny. One long standing example of such an approach is the use of hematopoietic stem cells. However despite obvious progress in the somatic stem cell transplantation approach, there are obstacles in effective stem cell-based therapy, namely a shortage of HLA-matched donors and technical limits on the in vitro expansion of somatic stem cells. Only one in three needy patients receive hematopoietic stem cell transplants. Thus, alternative sources for transplantable cells are needed, and pluripotent human histocompatible, embryonic stem cells (ESCs) generated by somatic cell nuclear transfer (SCNT) theoretically represent an excellent source. Recently, a novel protocol for SCNT in primates was established and the first non-human primate SCNT-ESC lines were generated. An important aspect of this technology is that it has been patented and licensed by our California based company, thus providing a basis for further commercial development. Using this licensed technology, we will generate and characterize the first human, histocompatible, patient specific, SCNT-ESC lines setting the stage for preclinical trials. Eventually benefits generated by the use of such cells in regenerative medicine should accrue to all peoples but Californians will also benefit from the generation of novel products (i.e. SCNT-ESC lines and SCNT technology media kits) commercially available to clinical, academic and for-profit research laboratories and from the regional experience and expertise in regenerative medicine.

Review

This proposal is designed to create human embryonic stem cell (hESC) lines using somatic cell nuclear transfer (SCNT) and donor cells obtained from a fibroblast cell line. This project is based on a collaboration between the applicant and the only group known to have successfully cloned primate cells.

The ability to generate individualized hESC lines using SCNT from either healthy individuals or patients with specific disease states is an exciting and yet technically demanding prospect. To date, this has not been achieved, although recently a group has successfully derived primate ESC with an efficiency of approximately 0.3%. The strength of this proposal comes from the collaboration between the principal applicant, who has stem cell biology expertise, and the group mentioned above.

This is a highly focused project, proposing a step by step evaluation of the enucleation process, fusion of the donor somatic cell and the oocyte, nuclear remodeling, cytoplast activation and progression to blastocyst stage by in vitro embryo culture. However, the proposal lacks a detailed description of how the resultant cell lines will be assessed and characterized for pluripotency. No data is presented suggesting that these assays are routinely available in the applicant’s laboratory.

One interesting component of this proposal is that the collaborator with the primate cloning expertise proposes to spend 25% of his/her time in the applicant’s laboratory in California. However, reviewers expressed serious concern that it will be logistically difficult for the out-of-state collaborator to be available at the time when the oocytes are donated.

Regarding the source of the oocytes, which will be obtained from an in vitro fertilization (IVF) clinic, one reviewer felt that the procurement of 100 oocytes per year seems reasonably achievable. However, others felt that not enough information is presented in the application to firmly support that the applicant can obtain the high quality eggs. Furthermore, even if they have access to 100 eggs, some reviewers argued that that would not be enough to make a cell line in 1 year, given the primate success rates.

Finally, reviewers mentioned that there is no consideration of how the resultant cell lines, if any, will be distributed to other research groups and under what conditions as requested 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