Statement of Benefit to California:
In this proposal a broad array of experimental approaches will be applied to analyze the roles of mitochondria in cell fate decisions and physiology of hES cells. Virtually nothing is known regarding mitochondrial function in hES cells. ES cells are generated from hypoxial inner cell mass blastocyst cells, and mitochondria not only maintain energy supplies, but also generate a wide range of metabolites that are crucial for normal cell growth and function. A large number of mitochondrial disorders are implicated in human diseases. These disorders can arise from mutations in mtDNA or in nuclear DNA. The hypothesis that underlies the proposed studies is that mitochndrial function will play important roles in the regulation of hES cell self-renewal and differentiation decisions. The studies encompass a broad-based approach to evaluate the role of mitochondria in hES cells. SIGNIFICANCE AND INNOVATION: These studies are very likely to provide fundamentally important insights. This is a well organized proposal which seeks to use established methodology to characterize mitochondrial morphology and function in several hESC lines under normoxic and hypoxic conditions. Although exploratory in nature the integration of methodology and the breath of colloboration suggest that important results will be obtained. Almost nothing is known about mitochondrial function in regulating aspects of hES cell biology such as the control of self-renewal versus differentiation. Therefore, the application of mitochondrial analyses to hES cells is highly innovative and significant. STRENGTHS: There are numerous strengths to this proposal. The basic idea is to first characterize the basic properties of mitochondria in hES cells. This will be accomplished in the first Aim. In the second Aim there are extensive proposed experiments that are very well-developed, and that will clearly shed light on mitochondrial function in hES cells. A number of inhibitors including RNAi will be utilized. In the third Aim, studies to address mitochondrial functions during lineage-specific hES cell differentiation will be pursued. All of the proposed studies are elegant, and well-developed. This is an outstanding proposal. WEAKNESSES: There are no major weaknesses to the proposed studies. Possibly, they are a bit over-ambitious; however, the importance of the proposed experiments far outweighs any concerns. DISCUSSION: This is a very well-written proposal from a premier mitochondrial lab. Note that prior to preimplantation, embryos are hypoxic; applicants propose to test mitochondria in hESC grown under normal and hypoxic conditions. This is a very important, timely and well-developed proposal.