[unreadable] Human Embryonic Stem (HES) cells have great potential for the study of basic human development, as tools for the testing and discovery of human pharmaceuticals, and as replacement cells for transplantation therapies Progress in HES cell research will lead to better therapies for multiple diseases, such as birth defects, diabetes, Parkinson's, leukemia, heart diseases, and many other degenerative diseases. The WiCell Research Institute (WiCell) was established in 1999 to support HES cell research and to distribute HES cells to external researchers. During the past two years, WiCell has undertaken large-scale production of its five established HES cell lines for distribution to academic and commercial researchers. Current culture conditions for HES cells are sub-optimal and optimization of HES cell culture conditions will enhance our distribution efforts and the efforts of all researchers who work with HES cells. The specific aims of this proposal are to (1) Expand the five WiCell HES cell lines to quantities sufficient to meet the demand, perform quality control tests on the five lines, and make the cell lines and technical support available to researchers, and (2) Optimize the cell culture media used to culture HES cell lines to maximize their expansion and distribution and their utilization by researchers. These aims will be accomplished with (1) Tested HES cell expansion protocols; (2) Standard assays for the analysis of human pathogenic viruses, mycoplasma, genetic identity, and karyotype; and (3) A newly developed HES self-renewal differentiation assay using a genetically modified HES cell line that expresses green fluorescent protein (GFP) in undifferentiated HES cells, but not in their differentiated derivatives. The cell lines to be used in these studies are listed on the NIH registry as WA01, WA07, WA09, WA13, WA14 [unreadable] [unreadable]