Pluripotent stem cell research is just on the verge of beginning to fulfill its promise to revolutionize medicine. Whether they are derived from embryos, or from adult cells that have been reprogrammed, human pluripotent stem cells can be propagated indefinitely in the laboratory and can turn into a wide range of mature cell types, providing an renewable source of a wide range of types of human tissue for research or therapy. We are still learning about the best ways to grow and manipulate pluripotent stem cells, and how best to reprogram adult cells to the pluripotent state. In fact, our concept of what a pluripotent stem cell is and what it looks like is still emerging. Recent work has shown that pluripotent stem cells, in the embryo or in the laboratory, are not simply homogeneous monocultures. Rather, stem cell cultures are complex and highly dynamic ecosystems. They contain a spectrum of cell types, from the most primitive cells, to cells that are already well on the way to becoming particular specialized types of cell. Different subpopulations of cells within these ecosystems communicate with one another, and these interactions dictate cell behavior. Cells even produce a type of scaffold on which they grow, called the extracellular matrix, that helps guide its fate. Thus, the microenvironment of the stem cell-its neighbors, and the signaling molecules they produce, is a critical component to guiding the cells fate. Our understanding of this microenvironment is however still rudimentary. This project will study two key processes-stem cell renewal, or the means by which stem cells divide to produce more stem cells, and specification, or how stem cells chose to begin to specialize into more mature cell types. We will look at the regulation of these processes at very high resolution, to see how individual cells within the various subpopulations respond to signals around them, and to identify the critical signals that maintain stem cells in the primitive unspecialized state or help adult cells to become reprogrammed. By carefully dissecting the stem cell population, and identifying its various subcompartments, we will provide critical information for other scientists that will enable them to study stem cell regulation at a much more refined level. And, an enhanced knowledge of the signaling systems cells use to talk to one another will help us to propagate stem cells and to enhance the reprogramming of adult cells. All of these fundamental discoveries will facilitate work towards the application of pluripotent stem cells in medicine.