Induction of Arterial Type of Hemogenic Endothelium from Human Pluripotent Stem Cells

De novo production of hematopoietic stem cells (HSCs) and lymphoid cells from in vitro expandable human cells, such as human pluripotent stem cells (hPSCs), represents a promising approach for modeling and treating hematologic disease and immunotherapies. While the feasibility of generating engraftable hematopoietic cells and T lymphoid cells from hPSCs has been demonstrated, further advancing these technologies to bedside requires developing clinically safe protocols that allow for scalable production of therapeutic cells in defined conditions without genetic modification. In the embryo, lymphoid progenitors and hematopoietic stem cells (HSCs) arise from hemogenic endothelium (HE) lining arteries, but not veins. The lack of venous contribution to HSCs when considered with the shared requirements for Notch, VEGF and Hedgehog signaling in both arterial fate acquisition and HSC formation also indicates that arterial specification could be critical prerequisite to HSC formation. Recent demonstration that HE represents a distinct CD73^- endothelial lineage (Choi et al., 2012; Ditadi et al., 2015) and that hematopoietic specification already occurred at the HE stage (Elcheva et al., 2014; Guibentif et al., 2017; Slukvin, 2016), raises the question whether acquisition of arterial identity per se is sufficient for HE to specify toward lymphoid cells and HSCs, or whether arterial specification and signaling creates the permissive environment that allows for definitive hematopoiesis from HE. Because prior studies demonstrated that arterial specification is controlled by DLL4 enhancer which contains ETS-binding sites, we engineered H1 human embryonic stem cells (hESC) carrying doxycycline (DOX)-inducible ETS1 and assessed the effect of ETS1 on establishing of arterial program. We demonstrated that ETS1 upregulation during the mesodermal stage of development enhances arterial specification from hPSCs differentiated in chemically defined conditions. The boost in arterial programming by ETS1 was associated with promotion of HE formation with DLL4⁺CXCR4^+/- arterial phenotype. Arterial type HE was highly enriched in cells with T and B lymphoid potential and the capacity to produce red blood cells with high expression of BCL11a and adult b-globin. As determined by limiting dilution assay the frequency of T cell precursors in DLL4⁺ HE was almost 100 times higher as compared to DLL4^- HE (1/17 vs 1/1801). In addition, we demonstrated that specifying arterial HE and enhancing definitive hematopoiesis with lymphoid potential could be achieved through modulation of MAPK/ERK pathways. Indirect ERK activation through inhibition of phosphatidylinositol 3-kinase (PI3K) of branch of VEGFR signaling using LY294002 enhanced production of DLL4⁺ arterial HE, including the DLL4⁺CXCR4⁺ fraction, while ERK inhibition with U0126 treatment almost completely abrogated formation of arterial DLL4⁺ HE. We also observed a direct correlation between the efficacy of definitive hematopoiesis and arterial specification. When ERK pathways were activated following HE specification, production of multipotent CD235a/CD41a^-CD45⁺ hematopoietic progenitors and multipotential CFCs were dramatically increased, while ERK inhibition abrogated production of these cell types. In addition, T lymphoid potential was significantly (more than six-fold) increased in cultures treated with LY294002 and entirely abrogated in cultures treated U0126. Together, these findings demonstrated that promotion of arterial specification in cultures could provide a novel strategy that aids in lymphopoiesis and eventually production of HSCs from hPSCs.