Project Summary Hematopoietic stem cells (HSCs), specialized cells responsible for generating billions of new red and white blood cells each day, reside within a complex bone marrow microenvironment. How specific cells within the niche regulate HSC activity and function is incompletely understood. We’ve previously demonstrated a pivotal role for bone marrow CD169+ macrophages (MΦ) in retaining HSCs within the bone marrow niche, as depleting MΦ causes HSC mobilization into blood. This was originally attributed to an indirect mechanism where MΦ act upon stromal cells to promote HSC retention. However, we have since obtained preliminary evidence suggesting that MΦ regulate HSCs by a direct cell contact mediated mechanism. First, we have discovered a population of HSCs within the bone marrow that express the putative macrophage marker F4/80, which is transferred from MΦ to HSCs through direct cell contact in a process similar to trogocytosis. Importantly, HSCs that have interacted directly with MΦ cannot be mobilized from the bone marrow, while those that have not interacted with MΦ can be mobilized. These data suggest a direct MΦ interaction with HSCs that is critical for determining which HSCs can be mobilized and which are retained in the bone marrow niche. Therefore, the overall goal of the current proposal is to determine the molecular mechanisms which mediate MΦ-HSC interactions, and whether this interaction facilitates HSC engraftment. I hypothesize that direct cell-cell contact between MΦ and HSCs, mediated by gap junctions and ATP, is an essential mechanism specifying HSC residence within the bone marrow and resilience to enforced mobilization. Experiments in Aim 1 will use CRISPR/Cas9 gene editing techniques to investigate the role of connexin gap junction proteins in mediating the MΦ-HSC transfer. Next, Aim 2 will quantify ATP during MΦ-HSC transfer to determine if MΦ regulate ATP in HSCs, and deplete ATP to investigate the role of ATP-mediated trogocytosis in facilitating MΦ-HSC transfer. Finally, Aim 3 will determine whether MΦ-HSC interactions improve acute HSC homing and long-term engraftment in bone marrow in cell transplantation assays. Discoveries from the proposal have the capacity to advance the current understanding of MΦ-HSC interactions within the bone marrow niche and provide potential therapeutic avenues to alter HSC mobilization from, and homing to, the bone marrow in the clinical context of HSC transplantation.