Engineering a Niche Supporting Hematopoietic Stem Cell Development Using Integrated Single Cell Transcriptomics

Hematopoietic stem cells (HSC) develop from hemogenic endothelium (HE) within embryonic arterial vessels such as the aorta of the aorta-gonad-mesonephros region (AGM). To identify the signals responsible for HSC formation, we used single cell RNA-sequencing to simultaneously analyze the transcriptional profiles of AGM-derived cells transitioning from HE to HSC, and AGM-derived endothelial cells which provide signals sufficient to support HSC maturation and self-renewal. Pseudotemporal ordering revealed dynamics of gene expression during the HE to HSC transition, identifying surface receptors specifically expressed on developing HSC. Transcriptional profiles of niche endothelial cells enabled identification of corresponding ligands, including those signaling to Notch receptors, VLA-4 integrin, and CXCR4, which, when integrated in an engineered platform, were sufficient to support the generation of engrafting HSC. These studies provide a transcriptional map of the signaling interactions necessary for the development of HSC and advance the goal of engineering HSC for therapeutic applications single cell RNA-sequencing (10X Genomics platform) of murine E10-E11 AGM-derived cultured endothelial cells expressing mAkt (AGM-EC) used as stroma to support HSC development ex vivo; and murine E10-E11 AGM-derived FACS-isolated VE-Cadherin+CD61+EPCR+ cells enriched for HSC precursors (primary cells and progeny following co-culture with AGM-EC)