The role of notch modulation in spatially defined hematopoiesis

About This Grant

Abstract Hematopoietic stem cell (HSC) transplantation is a potent therapeutic strategy for the treatment of many blood disorders. Therefore, the development of methods for deriving large quantities of HSCs is a major focus in regenerative medicine. Unlike HSCs, human induced pluripotent stem cells (hiPSCs) can be grown indefinitely. Despite hiPSCs being an extremely scalable source, HSCs derived from hiPSCs using current protocols are largely devoid of long-term blood reconstitution potential. These shortcomings are suggestive of knowledge gaps surrounding HSC biology. In the Camargo lab we leveraged our in-vivo barcoding mouse model to show definitively that HSCs arise from both intraembryonic (aorta) and extraembryonic (umbilical and vitelline arteries) sites during native hematopoiesis, and that these sites play differential roles in blood production. Our findings suggest for the first time that umbilical and vitelline (UV) artery HSCs are more short-lived than aortic HSCs, and that the UV artery engages potently in embryonic lymphopoiesis. By performing single cell RNA-seq on murine blood producing endothelial cells, I observed differential NOTCH signaling strength and the presence of NOTCH inhibitors GPR183 and DLK1 in the aorta and UV arteries respectively. While it is vastly appreciated that a transient reduction in NOTCH signaling strength is required for hematopoiesis to occur, no study has detailed differential mechanisms of NOTCH inhibition at spatially distinct HSC-producing sites. Cross-referencing this site-specific data with scRNA-seq on a commonly used hiPSC hematopoietic differentiation protocol, I identified for the first time the exclusive prevalence of the UV-like hemogenic endothelium in vitro. To develop methods of producing long-lived HSCs with adult-like lymphoid potential, we plan to study the Notch pathway as a regulator of site-specific hematopoiesis and modulate NOTCH signaling strength to produce more aortic-like hemogenic endothelium from hiPSCs. From this preliminary data, we hypothesize that differential NOTCH signaling strength is crucial for producing distinct hematopoietic programs in the UV arteries and the aorta. To test this central hypothesis, we plan to pursue the following specific aims: (1) characterize the role of GPR183 in aortic hematopoiesis through murine loss of function studies, (2) describe the role of DLK1 in UV hematopoiesis by murine loss of function, and (3) determine the function of DLK1 in controlling the hiPSC spatial hematopoietic program. From these experiments we expect to elucidate the role of these NOTCH inhibitors in spatially defined hematopoiesis. By leveraging our site-specific scRNA-seq dataset, we are uniquely positioned to produce methods of deriving aortic-like blood cells. These novel blood populations have the potential to revolutionize the therapeutics landscape.