ABSTRACT Despite substantial experimental evidence for the important functional role of myeloid-biased HSC (myHSC) in stress, inflammation and aging, the mechanisms which control myHSC quiescence and self-renewal are not known. The long-term goal of this project is to understand how myHSC self-renewal can be regulated by the bone marrow microenvironment in order to identify novel signaling pathways that can be activated to enhance hematopoietic regeneration, and blocked if they become dysregulated in myeloid malignancy. The overall objective in this application is to understand at the mechanistic level how Sema4a, a newly-defined niche factor, selectively promotes quiescence and self-renewal of myHSC, and to test potential therapeutic utility of its pro-regenerative effect in clinically relevant models of proliferative stress and HSC exhaustion. The central hypothesis is that Sema4a is released from osteoprogentiors and endothelial cells in the bone marrow microenvironment and binds to Plexin D1 on myHSC, which triggers a cascade of inhibitory signals that shield myHSC from exhaustion, premature differentiation and DNA damage. The rationale for this project is that identification of Sema4a-induced myHSC-specific pathways and testing their protective effect on myHSC in pre-clinical models of proliferative stress is likely to offer a strong scientific framework for future therapeutic strategies to improve myHSC self-renewal and hematopoietic regeneration. The central hypothesis will be tested under two specific aims: 1) Determine molecular and cellular mechanisms of Sema4a effects on myHSC; and 2) Define therapeutic opportunities for Sema4a as a myHSC “protector” from proliferative stress. Under the first aim, genetic mouse models, single-cell RNA Sequencing and intravital microscopy will be used to evaluate the effect of microenvironmental deletion of Sema4a or its putative receptor, Plexin D1, on myHSC and to uncover the role of Sema4a as myHSC-specific hematopoietic regulator. Under the second aim, my- HSC protective properties of Sema4a will be explored in clinically relevant models of functional HSC loss, such as exposure to cytotoxic chemotherapy and ex-vivo HSC culture during gene modification procedure. The research proposed in this application is innovative, in candidate’s opinion, because it builds on a novel discovery of Sema4a as a potent and indispensable regulator of myHSC in vivo, and provides substantive new experimental evidence for the concept that functionally specialized HSC are controlled by distinct regulatory circuits. The proposed research is significant because it is expected to serve as a scientific justification for future therapeutic strategies to improve hematopoietic regeneration following chemotherapy and stem cell transplantation, enhance engraftment of gene-modified HSC and prevent myeloid bias, age-related HSC dysfunction and evolution to myeloid malignancy in the elderly.