PROJECT SUMMARY The central process of female reproduction is the formation of oocytes within the developing ovary, known as oogenesis. This process is crucial for the formation of healthy oocytes and proper transmission of genetic and epigenetic information to begin embryonic development. Abnormalities in ovarian development and oogenesis are a leading cause of female infertility and disorders of sexual development, and furthermore are the cause of many developmental disorders in the subsequent generation, such as Down syndrome and Angelman syndrome. However, relatively little is known about the genetic regulation of human ovarian development. This is in contrast to other organisms such as the mouse, where transgenic and knockout lines, and a short reproductive cycle, have allowed much research in this area. An in vitro organoid model of human ovarian development would help fill this gap, and enable an improved understanding of human ovarian development that could lead to treatments for infertility and prevention of developmental disorders. Ovarian development involves interactions between primordial germ cells (PGCs) and somatic cells (granulosa cells). The granulosa cells enclose the PGCs within ovarian follicles, and support their differentiation into oogonia, progression through meiosis, and development as oocytes. Therefore, both lineages will be required to model this process in vitro. Existing methods allow differentiation of induced pluripotent stem cells (iPSCs) into PGC-like cells, but these cells are in an immature state, retaining epigenetic characteristics of iPSCs. For an in vitro model of oogenesis to be successful, improved methods must be developed to generate mature germline cells and granulosa cells from iPSCs. Reprogramming of cellular identity by expression of transcription factors (TFs) is a powerful technique that can allow both reprogramming of somatic cells into iPSCs, and directed differentiation of iPSCs to specific cell types. The Church lab has recently developed computational tools to predict TFs that specify cell identity, as well as screening methods for combinatorial TF expression to find sets that can differentiate iPSCs to a cell type of interest. The currently proposed research will identify TFs that can promote maturation of PGC- like cells and produce granulosa cells from iPSCs. Results will be evaluated by single-cell transcriptomic and epigenetic profiling, and by functional validation of key phenotypes. This research will provide an improved understanding of the genetic regulation of ovarian development, leading to an in vitro model of human oogenesis.