The goal of the project is to define the role of key regulators of pluripotency and differentiation in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Work completed in the first reporting period of this project focused on a regulator named TCF-3. The role of this factor is known in mouse embryonic stem cell cultures to be a key regulator that helps define the pluripotent state in a very important way: loss of TCF-3 protein results in a refractory state of pluripotency and inability to differentiate. The molecular and cellular characteristics of TCF-3 and its function in human ESCs and iPSCs is not known. Therefore, it is not known whether human TCF-3 performs functions in hESC and hiPSC that are similar to its functions in mouse ESC. The work performed in the first funding period focused on defining the basic properties of human TCF-3 mRNA and protein expression under different culture conditions – preparatory experiments that will enable us to define its functions. Multiple conditions that enable maintenance of an embryonic stem cell state, or conditions that promote differentiation into different cell fate lineages were established. Biological tools were developed to study TCF-3 mRNA and protein under those conditions, including tools to re-introduce mutant and protein-modified TCF-3 and reagents to remove TCF-3 mRNA and protein from hESC cells. The following results were obtained: i) TCF 3 is expressed and active as a negative regulator, ii) TCF-3 protein is modified by protein cleavage and covalent modifications, iii) TCF-3 is an important regulator of differentiation genes. These data suggest that TCF-3 is an important modulator of stem cells and differentiation. While this might imply that TCF-3 performs similar functions in mouse and human ESC, we observe differences in the genes regulated and in hESC colony phenotypes when TCF-3 levels are modified. These observations suggest there are important differences in TCF-3 function that distinguish it from its counterpart in mouse ESC. Experiments have been designed to test this hypothesis and to define how the apparent differences of human TCF-3 are established at the molecular and cell signaling level.