3006 – SINGLE CELL MAP OF THE EMERGENCE OF HUMAN HEMATOPOIETIC STEM CELLS FROM HEMOGENIC ENDOTHELIUM

Abstract

Hematopoietic stem cells (HSCs) capable of sustaining life-long multilineage hematopoiesis are formed in the human embryo between 4 and 5 weeks of development. HSCs emerge from hemogenic endothelium (HE) in the aorta-gonad mesonephros (AGM) region in a process called endothelial-to-hematopoietic transition (EHT). EHT has been mainly studied in in vitro human pluripotent stem cell models, which do not fully recapitulate in vivo HSC specification. Thus, the precise molecular identity and source of the HSC-forming HE and the molecular programs governing EHT in human remain elusive. This is crucial, as the embryo and extraembryonic tissues also harbor HE that generates non-HSC hematopoietic progenitors. Here, we conducted single-cell RNA-sequencing on purified cells (CD34+/CD31+) from the AGM region of 4.5-5-week human embryos from elective pregnancy terminations. Trajectory inference analyses revealed a distinct cluster of nascent HSCs with a unique expression profile (RUNX1+HLF+HOXA+MLLT3+SPINK2+) and a closely associated putative arterial HE population with a molecular signature that distinguishes it from other arterial endothelium (ALDH1A1+COL23A1+AGTR2+DKK1+KCNK17+). We also identified a novel arterial population (IL33+SULF1+ALDH1A1+AGTR2+), herein termed pre-HE, that is largely non-proliferative and metabolically quiescent and precedes the HSC-forming HE. Pseudotime analyses showed that acquisition of hemogenic properties in endothelium is concomitant to the downregulation of Notch, TGFb and Wnt signaling, and to the induction of hematopoietic transcription factors and genes associated with autophagy, identifying key regulatory switches during EHT. Comparison of HSC-forming HE (4.5-5-week AGM) to earlier, non-HSC-forming HE (3-4-week AGM) informed that ALDH1A1, COL23A1, AGTR2 and DKK1 are HSC lineage-specific markers, while KCNK17 is a pan hemogenic marker also induced during progenitor development from HE. Altogether, these findings provide a high-resolution map of the cell types and molecular switches involved in human HSC formation from endothelium, and can ultimately enable the recapitulation of HSC development in vitro for therapeutic purposes.