Development of a Technology to Increase the Availability of Human Oocytes for Use in Regenerative Medicine
Tools and Technologies I
Human embryonic stem (hESC) cells are one of the most promising tools for treating numerous human diseases. To use hESC in regenerative medicine, it will be necessary to derive lines of hESC that are immunologically compatible with patients. The most promising way to achieve this would be use of somatic cell nuclear transfer (SCNT), in which the nucleus from a patientís cell is transferred into an enucleated oocyte. The oocyte can then be activated to develop into a blastocyst from which a line of patient specific hESC would be derived. The hESC could then be differentiated into the appropriate cell type for treatment. However, a major critical barrier prevents the implementation of the above technology. This barrier is the lack of sufficient human oocytes to development methods for SCNT. Our goal is to develop and evaluate a technology that will increase the availability of human oocytes and thus enable us to cross this critical barrier and move forward toward development of patient specific therapies using hESC. We propose to grow and mature follicles to produce mature human oocytes that can be used for SCNT. This technology will have two major benefits. First, it will overcome the shortage of human oocytes needed for SCNT. Secondly, it will establish a method for culturing human ovarian follicles to maturity. It probably will be possible in the future to derive follicles with oocytes from hESC. When this becomes possible, the method for follicle culture that we develop will be in place. Our strategy is to compare and evaluate two methods of culture that enable maintenance of the three dimensional architecture of the follicle. These methods are alginate encapsulation of follicles that are then cultured in dishes vs. culture of follicles in low shear bioreactors. A progression of experiments is planned that will establish the most efficient method for producing high quality oocytes in culture. We will first determine if alginate encapsulation facilitates growth in bioreactors. The results of this experiment will determine if follicles grown in bioreactors are encapsulated in the following experiments. We will then compare growth of alginate encapsulated follicles to follicles grown in bioreactors. Finally we will determine if collagen I, a protein that facilitates follicle growth in mice, can also improve follicle growth in humans. In each experiment, we will monitor three types of endpoints: (1) morphological (e.g. follicle diameter over time, number of mature oocytes), (2) physiological (level of steroid and peptide hormones in culture media), (3) functional (formation of blastocysts following activation of oocytes or SCNT). The increased availability of human oocytes that our technology will provide is crucial to the development of future technologies using SCNT and subsequent therapeutic treatment of patents with stem cells.
Statement of Benefit to California:
In November 2004, the voters of California passed Proposition 71 by a large margin indicating the importance of stem cell technology to citizens of our state. Stem cell technology is one of the most promising approaches for treatment of numerous degenerative diseases, such as diabetes and Parkinson's disease, in humans. Most, perhaps all, citizens of California would find direct or indirect benefit from biomedical advances and eventual treatment of disease by stem cell technology. Even young citizens, who currently do not have major medical problems, could benefit in the event of future illness or accidents. Development of hESC technology and its application to disease will also contribute to the overall economy of the state by creating opportunities for the formation of new biotechnology companies and by stimulating growth of existing companies. If hESC are to be applied to treatment of human disease, it will be necessary to derive lines of hESC that are patient specific to prevent immunological rejection. Somatic cell nuclear transfer (SCNT) would be the most probable method to achieve derivation of patient specific stem cells. However, the lack of enough human oocytes to develop and use SCNT presents a major critical barrier to development of hESC technology for therapeutic applications. The goal of our proposal is to overcome this shortage of human oocytes and remove this critical barrier so that hESC therapy can be developed to benefit the citizens of California.
Derivation of human embryonic stem cells (hESC) from blastocysts following somatic cell nuclear transfer (SCNT) would result in immunologically compatible hESC with potential therapeutic value. This proposal is an attempt to address the problem of shortage of human oocytes to be used in SCNT. The principal investigator (PI) proposes to isolate human follicles, which contain immature oocytes, and culture them in vitro until maturation. The culture systems that they will test are alginate encapsulation for aim 1 and bioreactors for aim 2. In aim 3, they will determine if an added extracellular matrix component improves growth of the follicles. Endpoints for these experiments include morphological criteria, whether steroids and peptide hormones are produced and whether oocytes will develop to the blastocyst stage when activated or following SCNT. Reviewers differed in their assessment of the significance of this proposal. Some felt that there is a need for an alternative source of human oocytes. Even with the development of induced pluripotent stem (iPS) cell technology; development of human SCNT is still needed since oocytes are capable of reprogramming somatic nuclei via a mechanism not yet well understood. Technologies for in vitro maturation of immature human oocytes would potentially enable development of human SCNT and thus the generation of patient-specific hESC lines. The impact of successful results would be high, since a major hurdle in the field, access to human oocytes, would be addressed. However, other reviewers argued that the usefulness of the proposed technique for stem cell therapies may be limited given recent successes with iPS cells and the dependence of this project on human ovarian material. Thus, they regarded the potential impact on regenerative medicine to be low. The likelihood of success in obtaining fully mature oocytes, a prerequisite for nuclear reprogramming, depends on the quality of the follicle culture system. Some reviewers considered the use of bioreactors for this type of tissue culture to be novel and capable of providing an improved environment for the growth of oocytes. They regarded the proposed approach to be feasible since it is based on related mouse work published by another group. However, others pointed out that the in vitro maturation of equivalent starting material has not been accomplished in any other model system to date, calling the feasibility of this project into question. This concern was substantiated by the scarcity of preliminary data and the fact that the applicant does not elaborate significantly on alternative approaches, only two culture methods are proposed and may not be translatable to human immature follicles. The task of growing these follicles is probably not as easy as described, and inclusion of innovative culture method development would have raised reviewers’ enthusiasm. Some of the proposed endpoints for assessing the maturity of oocytes were judged to be reasonable, whereas attempts to activate blastocysts are not trivial and will be an important milestone if reached. Generating SCNT blastocysts is probably too much to ask within the scope of this application but the PI recognizes this. The PI has expertise in reproductive biology and has assembled a group of experts to accomplish all the objectives. In general, reviewers felt that the proposed budget is appropriate, although one reviewer pointed out that only additional culture technology development would justify the budget as proposed. In conclusion, reviewers differed in their assessment of the significance of the proposed work. Feasibility of the project was called into question, based on lack of success in other model systems, scarcity of preliminary data, and limited scope of proposed culture methods. Although the expertise of the team balances some of these criticisms, it was not enough to convince reviewers to recommend this application for funding.