Summary/Abstract

Human pluripotent stem cells are a tremendous tool to model early human development and disease including their use in the in vitro generation of blood cell fates. Hematopoietic progenitors and stem cells are the primary source of blood and the immune system from early development to adulthood and arise through successive waves of hemogenic mesoderm either in the yolk sac or embryo proper. Researchers have long sought a tractable human model for observing and distinguishing these waves of hematopoiesis in the dish for human developmental and disease modeling. Here we report a high-efficiency method for differentiating human pluripotent stem cells into an aorta-gonad-mesonephros-like definitive hemogenic mesoderm capable of giving rise to definitive hematopoietic progenitor and stem cells. The hematopoietic progenitor and stem cells exhibit robust multilineage in vitro colony forming potential. Gene expression analysis and single cell sequencing strongly support the developmental timing and notion that the pluripotent stem cell derived hematopoietic stem and progenitors are strikingly like bone fide hematopoietic stem cells. The hematopoietic progenitors can be subsequently differentiated into polarized macrophage and T-cells in vitro . Minimal silencing was observed upon differentiation of the pluripotent stem cells to hematopoietic lineages when conducting gene editing. Finally, upon engraftment into immunodeficient animals the hematopoietic progenitors and stem cells differentiate into multiple lineages including B-cells, T-cells, NK-cells, and monocytes.