Control of Human Embryonic Stem Cell Self-Renewal, Pluripotency and Differentiation
All of the diverse cells in a human body contain the same genetic information, and originally arose from a single cell, a fertilized egg. Embryogenesis is a result of cell division followed by differential gene expression, to selectively activate only the genes needed for development of each specialized cell type. By understanding the multiple gene activities required to either maintain stem cell pluripotency or effect cell specific differentiation, it should be possible to define conditions under which undifferentiated stem cells may be grown in large volume in culture, or induced to become mature cell types of therapeutic interest.
The experiments described in this proposal are directed at understanding the regulation of gene expression in stem cells as they self-renew or exit the pluripotent state and begin to differentiate. In preliminary studies, our laboratory has identified two protein complexes, SCC-A and SCC-B, required for the activity of genes needed for stem cell self-renewal. Here we propose to characterize the component proteins of these complexes. SCCs are potential targets for drugs aimed at increasing or decreasing the ability of stem cells to divide. We have also identified another protein, TAF3, that may play a central role in influencing whether stem cells self-renew or differentiate into various cell types. Understanding TAF3 function may provide a strategy to better control stem cell differentiation, a necessary step toward tissue replacement therapy.
A third focus of the proposal is to understand the impact of eliminating specific stem cell gene regulatory proteins via intracellular protein degradation pathways. Our preliminary data suggest that degradation of proteins crucial to controlling gene expression in pluripotent stem cells is an important regulated step in initiating differentiation. We propose biochemical and molecular biological studies to identify protein degradation pathways involved in maintaining these gene regulatory proteins, or marking them for destruction. Similar to the SCC complexes, these proteins could provide excellent targets for drug design, to increase or decrease the ability of stem cells to divide or differentiate.
The ultimate goal of these studies is the development of therapies for diseases that are fundamentally the result of inappropriate levels of gene expression, cell division and differentiation. The proposed experiments will demonstrate how gene activity is controlled, either to maintain a renewing population of stem cells, or to direct differentiation of specific mature cell types. Because all biological activities; cell division, differentiation and function, are the result of differential gene expression, an understanding of the gene regulatory networks controlling these processes will be crucial for drug development and testing.
The proposed experiments will benefit the people of the State of California immediately by supporting the state economy. This project will directly employ three people, supporting two graduate students, and one highly trained Research Specialist. Additionally, it will support the purchase of reagents and services necessary to conduct the proposed research, indirectly contributing to the employment of many other individuals in the biotechnology, education and service industries. In the longer term, this research will benefit the people of California by helping our state to maintain its status as a major force in biomedical research. A thorough understanding of basic stem cell biology will be necessary to support the ultimate development of safe and effective therapeutic applications, and the research efforts toward this end will contribute to the continued success of the outstanding universities and robust biomedical research community that have made California a leader in the fields of biotechnology and medicine.
The work described in this proposal will likely reveal gene activities that are essential for the establishment, survival and maintenance of stem cells and differentiated cell populations. This knowledge may potentially identify previously unknown drug targets that will allow the screening of novel classes of pharmeceuticals, or to allow stem cells to be grown in culture as large volumes of homogeneous cells, a necessary prelude to their use in stem cell therapy. Understanding the role of coordinated turnover of transcriptional regulators during stem cell differentiation may also represent a promising new strategy to predictably influence the pluripotent state and differentiation program in stem cells.