Generating Hematopoietic Stem Cells from AGM-derived Hemogenic Precursors in a Stroma-free Engineered Niche

Abstract Our previous studies demonstrated the capacity of a stroma layer consisting of AGM-derived myrAKT-transduced endothelial cells (AGM-EC) to support the in vitro conversion of hemogenic endothelium (HE) to hematopoietic stem cells (HSCs) that functionally reconstitute long-term multilineage hematopoiesis in adult recipients following transplantation. Here we present our protocol for an engineered stromal-free niche developed using complementary single cell transcriptomic analyses of HE/HSC and the AGM-EC niche to define ligand-receptor interactions sufficient to support HSC formation from embryonic hemogenic precursors in vitro. This protocol will be useful to identify and characterize additional niche-derived signaling ligands/pathways that may further enhance HSC generation from HE in vitro in a well-defined platform, and provides a basis for further translational efforts toward HSC production in vitro such as from embryonic or pluripotent stem cells.