Project Summary/Abstract All mature blood cells are derived from rare hematopoietic stem and progenitor cells (HSPCs) that reside in the adult bone marrow. Evidence shows that proper gene expression in HSPCs is essential for maintenance of homeostasis. Moreover, recent work suggests that progenitor cells may be as important as hematopoietic stem cells (HSC) in maintaining steady-state blood cell production and establishes a new level of complexity in blood cell production. Several transcription factors are critical for the formation and function of HSPCs, and for the differentiation to specific blood lineages. However, following hematologic injuries that reduce blood cell numbers, such as chemotherapy or toxic insult, a slow HSC response can result in life threatening complications from infection, bleeding, and anemia. Furthermore, malfunctioning HSPCs through dysregulated transcription factor expression can result in bone marrow failure or malignancies. Cell isolation through flow cytometry using specific surface markers has significantly refined the HSPC population. However, since tissues and populations are rarely homogeneous and consist of numerous distinct subsets, it has become increasingly clear that studying bulk populations of cells provides only limited knowledge, while interrogation of individual cells would provide insight into biological processes masked at the population level. Therefore, it is important to identify the molecular events regulating HSPC self-renewal, proliferation, and differentiation during both steady-state and stress hematopoiesis at the single cell level. Additionally, the hereditable properties of individual clones suggested that some HSPCs are imbalanced with respect to myeloid versus lymphoid cell potential as well as cell cycle status that can alters their reconstitution potential. Our proposal will use droplet- based Barcoding and Analysis of Single Cells (dBASC) to discover distinct transcriptional and epigenetic clonality of the earliest events in adult hematopoiesis. Functional heterogeneity of HSPCs based on analysis of self-renewal and differentiation at a clonal level has been appreciated for some time through cell transplantation assays but our collaborators, Camargo, Scadden, and Zon and have taken this a step further to uncover steady-state clonal hematopoiesis. The transcriptional network within HSPCs to promote self-renew is maintained through several well-characterized pathways. Together with Dr. Orkin?s single cell RT-PCR approach, we will validate the importance of transcriptional and epigenetic regulation on HSPC clonality. By studying the transcriptional network that regulate self-renewal, stress and clonal hematopoiesis, our unique single cell approach will provide an important step toward our fundamental understanding of biology of HSPCs and progenitors.