Dynamic roles of hematopoietic notch signaling in extramedullary hematopoiesis

Abstract During embryonic development, hematopoietic stem cells (HSC) in the fetal liver undergo expansion and self-renewal. Fetal HSCs migrate to the bone marrow (BM) niche towards birth and remain there during adulthood. Extramedullary hematopoiesis (EMH), or hematopoiesis outside of the BM, physiologically occurs in the fetal liver or when the adult BM niche ceases to be a site for functional hematopoiesis. While fetal liver HSCs are extremely robust in transplantation and reconstitution assays, the functional potential of adult liver EMH HSCs is not fully explored and the mechanism by which the adult liver becomes reactivated as a site of EMH for itinerant HSCs is unknown. We investigated EMH in the adult liver by inducing acute hemolytic anemia with phenylhydrazine (PHZ) injections in WT and Notch pathway transgenic mice. PHZ treatment induces egress and homing of hematopoietic progenitors to the liver and spleen. Our work demonstrates that long-term HSCs (SLAM-LSK-EPCR+) isolated from the portal vein branch point in the adult liver can serially reconstitute lethally irradiated mice. Thus, the adult liver serves as a potential reservoir for long-term, functional HSCs. In pursuit of a mechanism, we focused on Notch-signaling, an evolutionarily conserved pathway that has been shown to play a critical role in HSC emergence and fetal liver development. When comparing WT liver donors to Notch1 hypomorph (N1 ΔTAD) or Notch1 knockout (Notch1f/fVavCre+) liver donors, hematopoietic transplants reveal that Notch mutant progenitors fail to reconstitute irradiated recipients efficiently. Using an HSC-specific lineage tracing alpha-catulin-GFPmodel, we can track the expression of GFP+ liver-originating HSCs after hematopoietic reconstitution. We show that mice reconstituted by EMH-activated, a-catulin-GFP+ liver HSCs can provide long-term reconstitution in lethally irradiated recipients and resume residence in the BM niche of recipients. Given the transient nature of both fetal and adult EMH, long-term characterization and biochemical analysis of the niche interactions and signaling pathways has not been possible. So we have developed a fetal liver hepatoblast spheroid platform utilizing E14.5 mouse embryos, fetal endothelial and mouse embryonic fibroblasts to generate a stable fetal EMH niche that can sustain both fetal and adult BM HSC functionality over the course of 2 weeks in culture. Finally, we have utilized a micropatterned co-culture (MPCC) human hepatocyte and portal fibroblast platform to establish a platform for adult liver EMH for continuous study and differentiation potential of adult EMH ex vivo. In both platforms, we show that hematopoietic Notch signaling activation is required for functional potential of EMH HSCs and that the source of the ligand is spatially constricted with Jag1 being of hematopoietic origin, while Dll1 and Dll4 of stromal and endothelial, respectively. Our findings reveal a developmentally conserved Notch signaling mechanism that is reactivated in the adult liver to retain the functional potential and maintenance of HSCs as they adapt to EMH due to the inhospitable conditions in the bone marrow niche.