Bulk RNA-sequencing of CD49fHigh and CD49fLow populations from hESCs-derived HSPCs and Hemogenic Endothelium

CD49f has been used to define human hematopoietic stem cells (HSCs). Tracing the origin of CD49f+ HSCs shall enhance our understanding of the regulatory mechanisms and improve in vitro generation of HSCs. Here, we employed the hematopoietic differentiation system from human pluripotent stem cells and performed a computational trajectory analysis of the cells throughout this process based on integrated calculating the levels of 10 HSC markers. We observed two clades of cell populations in the trajectory tree, one of which represents the phenotypic CD49fHigh HSCs, and the other diverges only by low CD49f level. Live cell imaging revealed that CD49f expression is persistent during endothelial-to-hematopoietic transition, suggesting that the phenotypic CD49fHigh HSCs are pre-defined in hemogenic endothelial cells (HECs), while the concurrently emerged CD49fLow cells represent HSC-independent progenitors. Colony forming assays showed that the CD49fHigh cells have multilineage potential, whereas the CD49fLow cells lack lymphoid potential but show a strong erythroid preference. The aim of the experiment was to compare newly defined CD49fHigh and CD49fLow populations from hESCs-derived HSPCs and Hemogenic Endothelium. Overall design: CD49fHigh and CD49fLow cells were sorted from hESCs-derived HSPCs (CD43+CD34+CD38-CD45RA-CD90+) at day 8, and also CD49fHigh and CD49fLow cells were sorted from hESCs-derived hemogenic endothelium (CD34+CD31+CD43-) at day 6