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Maturation of Human Oocytes for SCNT and Embryonic Stem Cell Derivation

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01553
Principle Investigator: 
Institution: 
Funds requested: 
$1 432 197
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
A major issue for tissue and cell therapy in regenerative medicine is the immune rejection of grafts originated from a non-compatible individual. Mature eggs contain factors essential for the re-programming of cell nuclei from patients to allow the establishment of patient-compatible pluripotent stem cells for the treatment of diverse degenerative diseases. Although up to half a million dormant small follicles are present in young women reaching puberty, only 400 of them developed to the large follicle stage and release mature eggs during a women’s reproductive life. The present application proposes to overcome the major obstacle dealing with the shortage of human mature oocytes for the generation of patient-compatible pluripotent stem cells. The {REDACTED} preserved ovarian tissues from cancer patients before chemo- and radiation therapy to avoid damages. We have obtained patients' consent and propose to promote the growth of arrested small follicles from ovaries of cancer patients with specific activators to allow the development of hundreds of large follicles containing mature eggs. The surplus eggs can be used to re-program cell nuclei from patients, thus allowing the generation of patient-compatible pluripotent stem cells for the treatment of diverse degenerative diseases. After identifying key embryonic hormonal factors important for the optimal growth of human early embryos in the test tube, we will promote the development of re-programmed cells with hormone-enriched culture media for the generation of patient-compatible stem cell lines. In addition to providing pluripotent stem cells for future generation of diverse cells in the body (neurons, muscles, pancreatic insulin-producing cells, etc), the present generation of mature oocytes will benefit cancer patients and diverse infertile women who still have arrested small follicles but are unable to respond to the present gonadotropin therapy.
Statement of Benefit to California: 
Because human ovaries contain thousands of arrested small follicles and only a few of them grow into large follicles containing mature eggs, we devised a new approach to promote the growth of these dormant follicles to generate a large surplus of mature oocytes. Future adaptation of this procedure for the re-programming of the nuclei of cells from patients with degenerative diseases will allow the generation of patient-compatible pluripotent cell lines. Because these cells can be induced into diverse cell types of the body, establishment of optimal culture conditions for the derivation of these cells would allow new approaches for assisted reproductive technologies and provide new treatment modalities for diverse patients with degenerative diseases. We will submit patent applications on our findings to protect intellectual property rights according to {REDACTED} guidelines. In addition to applications in regenerative medicine, the present findings are also expected to substantially broaden the patient population for and improve the success rate of in vitro fertilization procedures that are presently used for an estimated one million treatment cycles per year worldwide. We anticipate the successful completion of the present proposal could benefit patients with degenerative diseases and infertility in California and throughout the world. The P.I. already has a pending patent application on the use of brain-derived neurotrophic factor in the promotion of early embryo development. We expect future findings will lead to additional patent applications and licensing agreements that would benefit the State of California.
Review Summary: 
EXECUTIVE SUMMARY The goal of the proposed research is to achieve human somatic cell nuclear transfer (SCNT). Acknowledging that the paucity of human oocytes is the single limiting factor for working out human SCNT approaches, the Principal Investigator (PI) proposes to address this limitation by utilizing dormant primordial follicles which, unlike mature oocytes, cannot support in vitro fertilization for reproductive purposes and are thus available for research uses following appropriate consent. In Aim 1, the PI will generate oocytes from human ovarian tissue using a technique for maturing primordial follicles that has proven successful with mouse tissue. In Aim 2, the matured oocytes will be used for SCNT, followed by development to the blastocyst stage and derivation of human embryonic stem cell (hESC) lines. Since the protocol pursued in Aim 1 involves in vivo maturation in immune-compromised mice, work in Aim 3 then seeks to develop a procedure to mature primordial follicles both from mouse and human into mature oocytes entirely in vitro. Reviewers were unanimous in their enthusiasm for this highly significant proposal as it promises to enable the development of important technology for the derivation of patient-specific pluripotent stem cell lines. The relative ease of induced pluripotent stem (iPS) cell derivation when compared to SCNT has caused a bias in research efforts. While it has been suggested that the emergence of iPS cell technology has rendered SCNT less urgent, defining the differences and similarities between iPS cells, SCNT-ESC and fertilization-derived hESCs remains an important priority in regenerative biology; different reprogramming technologies are needed to investigate reprogramming mechanisms. This application approaches the pursuit of human SCNT in a novel way and, if successful, will have a major impact both on fundamental research and reproductive medicine. The in vitro culturing technology to be developed in this application will allow for the generation of large numbers of human oocytes without the requirement for ovarian stimulation and oocyte retrieval from patients, thus increasing the availability of oocytes for SCNT and, potentially, for infertility treatment. Reviewers felt that the rationale for the intended studies was clearly presented. Feasibility of the proposed work was supported by very strong preliminary results based on studies of both mouse and human cells. The proposed investigation follows a sound approach with logical and achievable aims. Reviewers appreciated that this project builds upon work accomplished using previous CIRM grant support. Reviewers expressed some minor concerns that the applicant had not adequately considered potential problems with the SCNT protocols or adequately developed alternative methodologies should difficulties arise. Furthermore, some reviewers suggested that a direct comparison of the genomic and epigenetic status of SCNT-hESC and human iPS cells derived from the same somatic cell donor could be worthwhile. However, reviewers were enthusiastic about the project’s overall feasibility and its prospects for success. Reviewers noted the applicant’s long and productive history in reproductive biology, many important contributions to the field, and strong publication record. The PI commits adequate time to this project, and the quality and expertise of the assembled team further increases the likelihood of success. This is a productive team in a strong environment with appropriate access to required tissues. In summary, the overall enthusiasm for this project’s potential impact on the stem cell field and the quality of the preliminary data resulted in strong support by the reviewers.
Conflicts: 

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