$3 139 600
Embryonic stem (ES) cells not only hold considerable promise for the treatment of a number of devastating diseases, they also provide an ideal tractable culture system of studying early embryonic development of all cell types. Harnessing these potentials of ES cells will require an improved ability to manipulate their self-renewal and differentiation, and a better understanding of the signaling pathways that control their fate. Among many technical challenges to overcome, a robust self-renewal and clonal expansion condition for hESCs would be highly desirable, which would facilitate practical applications of hESCs toward therapies (e.g. large scale manufacture of desired cell types); provide a reliable platform for hESC engineering (e.g. genetic manipulation to create reporter or disease-specific model cell lines); and promote faster advance in basic understanding of hESC/developmental biology. Because the hESC field is still at its infancy, unbiased and functional discovery approach would be highly suited, e.g. providing tools and laying ground for scientific investigations. This proposal will integrate technology-driven approaches (for discovery of small molecules and genes that regulate hESC self-renewal and differentiation processes) and hypotheses that are derived from these discoveries for a better understanding and controlling of hESCs. Collectively, these proposed studies will provide new chemical tools, functional genomic technologies and substantial knowledge for better understanding and controlling hESC self-renewal and differentiation, and may ultimately allow development of therapeutics employing hESCs for treating diseases.
Statement of Benefit to California:
The proposed studies will provide new chemical tools, functional genomic technologies and substantial knowledge for better understanding and controlling hESC self-renewal and differentiation (e.g. development of robust self-renewal and clonal expansion conditions for hESCs) , and may ultimately allow development of therapeutics employing hESCs for treating diseases.
SYNOPSIS: This proposal represents a complete list of high priority topics in stem cell research, covering everything from large scale chemical screens for characterization of compounds that maintain self renewal, characterization of compounds that guide the differentiation of human embryonic stem cells toward ectodermal, mesodermal, endodermal lineages; neural formation to cardiomycte specification to pancreatic differentiation, and all of this in specific aim one. The proposal is primarily technology-driven, using small molecule screens and combinatorial chemistry to identify novel chemical mediators (and possible molecular pathways) of ESC processes. This approach has led to the development of simple chemically defined media conditions that apparently support efficient long term self-renewal of hESCs and to other small molecules that appear to promote differentiation to various stem or progenitor cell fates. The screen has recently also revealed two classes of small molecules that support clonal expansion which will be studied in Aim 1. These molecules will be further characterized and optimized, particularly focusing on the chemical SHI-2-134, which unpublished data shows to interact with AMPK. Phosphoproteomic analysis of human and mouse ESCs have identified two candidate proteins that may have a role in the epigenetic chromatin state of pluripotent ESCs. These are MBD3 and EZH2, which will be further studied for their role in self-renewal and in relation to nanog, Oct 4 and Sox 2 in Aim 2. A genome-wide RNAi functional analysis of hESCs will be performed in Aim 3. Here they will further characterize RAS GAP-RAS in the regulation of hESC self-renewal and differentiation and extend the arrayed RNai functional screen to the full SI RNA library. IMPACT AND SIGNIFICANCE: The goals of the proposal are extremely relevant to the hESC community. While substantial progress has been made on development of defined culture conditions for hESCs, important issues remain unsolved including robustness, stability, and cost. In addition identification of molecular mechanisms of fate choice decisions is a critical challenge facing the community. The tools developed in this study (RNAi, high throughput screening methods) would have value to a variety of hESC labs. The project may provide new insights into small molecule control of self-renewal and clonal expansion of hESCs. These studies may also facilitate the development of better techniques for maintaining hESCs and controlling their differentiation. QUALITY OF THE RESEARCH PLAN: The “chemical biology” approach to understanding and influencing cell function has substantial promise. The discovery-driven experiments proposed in aim 1 are likely to identify interesting compounds. The global effects of these compounds on cell behavior may be difficult to ascertain, however. The PI proposes to identify lead compound targets and investigate signaling pathways affected by each compound. Any compounds identified would be useful tools in understanding mechanisms of hESC self-renewal and differentiation, although the proposal is a little vague on how this will be accomplished. The main problem with this proposal is that it is unrealistically ambitious: each aim is in itself an ambitious research project. Aim 1 focuses on chemical biology approaches to hESC differentiation, aim 2 on phosphoprotein regulation of differentiation, and aim 3 on RNAi screening and knockdown of signaling pathways. Completion of these activities in the time frame of this grant is not reasonable. STRENGTHS: This is an innovative proposal that uses chemical biology and molecular screening of compounds and genes that regulate hESC self-renewal, differentiation, and cloning potential. The applicant has extensive experience in two of the key technologies that will be used: ESC characterization and high throughput screening. The prelimary studies illustrating development of defined media for hESC culture and directed differentiation and those identifying lead compounds that may affect hESC self-renewal and cloning are very strong. WEAKNESSES: The primary weakness of this study is that the aims are extremely overambitious. While each of the three aims addresses an important problem, taken together they appear unrelated and disjointed. Aim 1 will identify compounds, characterize their effects on hESC phenotype, optimize compound activity, identify molecular targets of these compounds, and determine they genetic and protein pathways affected by these compounds in eliciting the observed phenotypes. This aim alone would be an ambitious, though more manageable project. Adding aims 2 and 3 calls into question the ability to achieve the stated goals within the project period. Each of the proteomic and RNAi aims would also potentially be an independent proposal. Given the PI’s preliminary data and experience in the chemical biology field, the experiments proposed in aim 1 seem the strongest. The screening methodology and compound characterization are described very well. The use of the compounds in media development is clear, but how they will be used to dissect signaling pathways is less obvious and could be described better. For example, Affymetrix genechip readouts do not necessarily provide direct evidence of pathway activation. When combined with proteomic and RNAi data, a large number of testable hypotheses could be generated. It is not clear how these hypotheses would be prioritized. A substantial amount of preliminary data are presented. However, without captions describing the experiments, interpretation of the results is difficult. The proposal indicates the PI will use human oocytes, in vitro embryos, and derive a covered stem cell line. It is not clear how or why (or if) these procedures will be performed. DISCUSSION: The main weakness of the application was viewed as its unrealistic breadth. The independence of the applicant was also questioned as the applicant is not the senior author in most of the publications listed.