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.
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.