Human embryonic stem (hES) cells are pluripotent stem cells that can theoretically give rise to every cell type in the human body. Consequently, hES cells have enormous promise for the treatment of human disease. Differentiated cell types derived from hES cells could be used to treat a wide variety of diseases and disorders or used to develop drugs that would ameliorate disease. Finally, the analysis of hES cell differentiation into specialized cell types could reveal important information about the embryonic and fetal development of our own species. But significant hurdles must be overcome if hES cell-derived cells are to be used in these ways. The goals of this project are to gain a fundamentally better understanding of the mechanisms controlling hES cell growth and differentiation. We discovered a unique role for neurotrophins (NTs), acting through their receptors, the Tropomyosin-related kinases (TRKs), in mediating hES cell survival. NTs improve both hES survival and the ability to genetically manipulate the cells. But both the role of the NTs and the other factors regulating hES cell growth remain largely obscure. The goals of the grant take advantage of our improved ability to genetically manipulate hES cells and are designed to fill gaps in our knowledge about factors regulating their growth. In the second year of studies we have carried out studies that have defined the role of key stem cell regulatory genes, including, SOX2, OCT4 and NANOG, in regulating the growth of hES cells via regulation of the TRKs. We have found that SOX2, OCT4 and NANOG likely control the expression of the TRKs in hES cells. We have also carried out further studies on the pathways downstream of the TRKs that are activated in hES cells and have identified a key role for the Forkhead transcription factors in this process. In addition, we have developed tools for identifying new genes that regulate stem cell growth, especially kinases and phosphatases that are known regulators of growth of other cell types. These studies should aid in growth and expansion of hES cells as normal healthy cells for cell-based transplantation and other uses of hES cells in the development of new treatments for human disease.