To fulfill the promise of pluripotent stem cells, both embryonic and induced pluripotent stem cells, it is essential to fully understand their properties and how those properties can be manipulated to make any cell in the human body. The best way to reach that goal is to understand the relationships between these cells that grow in a culture dish in the laboratory and the equivalent cells in the developing embryo. As working with human embryos comes with many ethical concerns, an important alternative is the mouse model. Indeed, much of what we have learned in the mouse model has later been confirmed in human. Therefore, we use a combination of the mouse model and human cells to dissect the molecular basis of stem cell function and differentiation toward adult tissues. In particular, we have been focusing on a class of molecules called small RNAs that were only discovered in the 1990s and became widely appreciated in the past decade. There are several classes of these small RNAs, two of which our lab focuses on, microRNAs and endogenous siRNAs. We have found these small RNAs are essential for normal mammalian development and growth and differentiation of stem cells. In the past year of this grant, we have been expanding on these findings dissecting the relative roles of the two classes of small RNAs, and the individual small RNAs within those classes that are responsible for specific functions. We recently discovered that endogenous siRNAs are absolutely essential for maturation of oocytes and, therefore, are very important in human fertility. However, we do not know how they exactly function and what other molecules they interact with, a new focus we are pursuing. We also discovered that microRNA function is transiently silenced during oocyte maturation and early embryonic development. This finding was surprising and suggests that miRNA suppression almost certainly has an essential role in early development. We have made progress toward determining the mechanism of suppression, which will allow us to understand its role. We are also testing a potential role for microRNA suppression in the efficient production of high quality pluripotent stem cells. Additionally, we have found that microRNAs play important functions in the earliest differentiation events of embryonic stem cells and their embryonic counterpart, the transition of the inner cell mass to the epiblast. We are making progress on which microRNAs are responsible and how they interact with the other molecules in the cell. Finally, we are dissecting for the first time the role of endogenous siRNAs in embryonic stem cells by removing them and determining the cellular consequence. This research is expected to enable to us to more easily manipulate cell fates to produce high quality cells that could be used to study diseases of many types as well as reintroduce healthy tissue into patients with degenerative diseases.