3003 – TARGETED CRISPR-MEDIATED GENE ACTIVATION DURING IN VITRO IPSCS DIFFERENTIATION SUCCESSFULLY EXPANDS ARTERIAL-FATED CELLS IN ASSOCIATION WITH HIGHER PROGENITOR ACTIVITY.

Abstract

Hematopoietic stem and progenitor cells develop from hemogenic endothelium located in various sites during development. Particularly, Hematopoietic Stem Cells (HSCs) develop from specialised endothelial cells within the wall of the dorsal aorta. This process has proven especially challenging to recapitulate in vitro from pluripotent stem cells. Here, we compared iPSC-derived (PSD) endothelial cells and in vivo (IV) hemogenic endothelium and identified 9 transcription factors expressed at significantly lower levels in vitro. We then developed a novel DOX-inducible CRISPR activation system to induce the expression of those genes during in vitro differentiation. We then employed single-cell RNA sequencing to study the phenotypical changes induced by the target genes. Furthermore, by engineering the gRNA we were able to detect them within the sequencing pipeline. Our data showed a significant expansion of arterial-fated endothelial cells with associated higher in vitro progenitor activity. Interestingly, the expanded arterial cluster is distinct from that expressing hemogenic markers, suggesting that the arterial cells might play a supportive role in the emergence of progenitor cells from the hemogenic endothelium. Hematopoietic stem and progenitor cells develop from hemogenic endothelium located in various sites during development. Particularly, Hematopoietic Stem Cells (HSCs) develop from specialised endothelial cells within the wall of the dorsal aorta. This process has proven especially challenging to recapitulate in vitro from pluripotent stem cells. Here, we compared iPSC-derived (PSD) endothelial cells and in vivo (IV) hemogenic endothelium and identified 9 transcription factors expressed at significantly lower levels in vitro. We then developed a novel DOX-inducible CRISPR activation system to induce the expression of those genes during in vitro differentiation. We then employed single-cell RNA sequencing to study the phenotypical changes induced by the target genes. Furthermore, by engineering the gRNA we were able to detect them within the sequencing pipeline. Our data showed a significant expansion of arterial-fated endothelial cells with associated higher in vitro progenitor activity. Interestingly, the expanded arterial cluster is distinct from that expressing hemogenic markers, suggesting that the arterial cells might play a supportive role in the emergence of progenitor cells from the hemogenic endothelium.