Project Summary Uterine fibroids (also known as leiomyomas) are the most common tumors in the female reproductive tract. Some estimates indicate that up to 75% of American women have uterine fibroids, depending on race and ethnicity. There is a significant disparity in the incidence of fibroids since African-American women are 2-3 times more likely to develop fibroids, often with earlier onset and greater severity of symptoms. These tumors can be very painful and are the leading cause of hysterectomies in the US. Despite the healthcare burden caused by uterine fibroids, their etiology and pathophysiology are essentially unknown. The vast majority of fibroids either have mutations in Mediator Complex protein 12 (MED12) gene or over-express high mobility group AT hooks (HMGA)1/2 transcription factors, but the molecular mechanism that are disrupted and lead to tumor development or growth are unknown. Another well known characteristic of fibroids is that they are clonal, indicating a single cell of origin for each tumor. Our overarching hypothesis is that fibroids develop from myometrial stem cells that have become dysregulated by either MED12 mutation or HMGA1/2 over- expression. We have developed complementary models to study myometrial stems cells and determine the mechanisms disrupted that lead to fibroid development or growth. We have identified and characterized label- retaining cells (LRCs) in the mouse myometrium. These cells express putative stem cell markers, and divide in response to postpartum repair, as would be expected for myometrial stem cells. We have also used side population analysis and cell surface markers to identify human myometrial cells with stem cell properties. However, none of these methods leads to a highly enriched, homogeneous population necessary for some of the next generation sequencing approaches to study molecular mechanisms driving disease etiology and progression. We propose to use establish RNA-seq profiles for these putative myometrial stem cells and map them to single-cell sequencing clusters of myometrial cells to identify other cell surface markers to further enrich for highly purified, human myometrial stem cells. Once these putative myometrial stem cells are validated, we can then use them to study and better understand possible disrupted chromatin and epigenetic mechanisms that lead to fibroid development and growth, with the ultimate goal of identifying targets for therapeutic intervention.