Single Cell Transcriptomics Maps the Embryonic Emergence of HSC and Identifies Intercellular Interactions Regulating HSC Genesis

Hematopoietic stem cells (HSC) are generated during a transient window of embryonic development from endothelial-like hemogenic precursors within specific arterial vessels such as the aorta of the AGM (aorta-gonad-mesonephros region). During HSC emergence, hemogenic precursors must acquire and maintain HSC-defining properties such as the ability to self-renew, home, and provide multilineage hematopoiesis, properties which distinguish rare HSC from a multitude of other hematopoietic progenitors arising simultaneously in the developing embryo. However, the precise niche-derived signals necessary and sufficient to support the acquisition and maintenance of these properties remains poorly defined. Toward identification of these signals, we generated a platform consisting of endothelial cells from the embryonic AGM (AGM-EC) which supports the specification and self-renewal of engrafting HSC from clonal embryo-derived hemogenic precursors in vitro. Using this platform to assay functional HSC potential at the single cell level, we determined a phenotype (VE-Caderin⁺CD61⁺EPCR^high) that encompasses the population of hemogenic precursors during their asynchronous transition to HSC between E9.5 and E11.5 in murine embryonic development. To elucidate the transcriptional changes associated with the emergence of HSC from hemogenic precursors, we analyzed the global transcriptional profiles of FACS-purified VE-Caderin⁺CD61⁺EPCR^high cells at various stages of embryonic development by single cell RNA-sequencing and reconstructed their developmental trajectory in "pseudotime" based on incremental changes in their transcriptional profiles. Complementary analysis of AGM-EC by bulk and single cell RNA-sequencing revealed a unique transcriptional profile of niche endothelial cells supporting HSC development enriched for immune/inflammatory signals. Combining the transcriptional profiles of emerging HSC with niche AGM-EC, we have identified candidate ligand-receptor pairs regulating intercellular interactions during HSC specification and self-renewal and have begun to validate the functional importance of these interactions in supporting HSC generation from hemogenic precursors in vitro. We expect these studies will enhance our understanding of the unique signal pathways necessary for the development of functional HSC, a critical step toward engineering HSC in vitro for clinical applications in disease modeling, drug discovery, and gene modification to identify novel therapies for hematologic and immunologic disorders.