Dormancy-dependent determination of hematopoietic stem cell fate from hemogenic endothelium

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Title: Dormancy-dependent determination of hematopoietic stem cell fate from hemogenic endothelium Project Summary/Abstract: Hematopoietic stem cells (HSCs), uniquely defined by their simultaneous capacity for multilineage blood cell formation and life-long self-renewal, represent a valuable resource for both the treatment and study of blood and immune disorders. Methods to generate HSCs de novo, such as from pluripotent stem cells (PSCs), are of great interest, as they could significantly enhance availability of HSCs for research and therapeutic purposes. However, reproducible protocols to produce functional HSCs from PSCs have been largely elusive. This likely reflects our incomplete understanding of the molecular programs required for HSC specification during embryonic development. The primary objective of this project is to address this critical barrier by identifying the unique factors necessary to impart HSC fate from the embryonic precursors to hematopoiesis, hemogenic endothelium (HE). Toward this goal, we will leverage an innovative ex vivo vascular niche platform that supports the development of murine embryonic HSCs, combined with integrated single cell techniques, to uncover the distinctive molecular properties of HE that can acquire functional HSC fate in vitro. In initial studies using this approach, we determined that HSC-competent HE possess a transcriptional signature uniquely characterized by relative metabolic and mitotic dormancy associated with decreased MYC target gene activity. We further determined that HSC emergence in vitro is dependent on niche-derived chemokine, CXCL12, and that its receptor, CXCR4, is expressed on HSC-competent HE. Based on these studies, we hypothesize that the dormant state of HSC-competent HE functions to delay hematopoietic differentiation and establish self- renewal programs essential for HSC specification, which will be tested in Aim 1. We further hypothesize that CXCL12-CXCR4 signaling reinforces dormancy and self-renewal programs in HE, supporting their transition to functional HSCs in the vascular niche, which will be tested in Aim 2. Our broader objective is to determine whether modulation of signal pathways promoting dormancy during HE specification from murine and human PSCs can enhance functional HSC development in vitro, which will be pursued in Aim 3. Success in these studies would provide new insight into the molecular mechanisms orchestrating HSC genesis and facilitate progress toward the long-term goal of de novo generation of HSCs from PSCs for therapeutic applications.