This project will address some fundamental questions about the biology of human embryonic stem cells. Our overall, long-term goal is that the answers to these questions should help to hasten the development of novel stem-cell based strategies for treating human diseases. We will pursue the following aims. First, we will evaluate a range of antibodies directed against cytoplasmic intermediate filament (IF) proteins as markers to help identify specific classes of progenitor cells in cultures of human embryonic stem cells (hESC). We will use these antibodies to track the changing patterns of IF protein expression as cells progress to terminal differentiation. Second, we will use small interfering RNAs (siRNAs) to down regulate specific IF proteins to help determine the functional significance of the different IF protein expression profiles that emerge during development. Third, we will make a detailed study of the structure and function of nestin, a type IV IF protein widely-used as a neural progenitor cell marker, focussing on our recent finding that its C terminus interacts with the insulin degrading enzyme and may regulate its activity. The experiments will make use of an extensive collection of mono- and polyclonal antibodies raised against cytoskeletal IF proteins that we have developed and tested in our laboratory over the years. We will employ high resolution, confocal fluorescence microscopy to study fixed, immunostained cells. Time lapse studies will be made of living cells expressing various GFP-IF protein constructs and the dynamic properties of IF of varying protein composition will be studied using fluorescence recovery after photobleaching (FRAP). Public health relevance of this research: Human embryonic stem cells have the potential to give rise to all cell types of the human body, and thus have great utility for treating diseases such as Parkinsons' Disease and diabetes, as well as spinal cord injuries. The proposed research will advance our understanding of the cell biological events that take place as stem cells change into more highly differentiated cell types. This information is crucial for guiding cells down the right pathway so that they can eventually be used to treat patients.