The Preliminary Study Of Hematopoietic Stem Cell Expansion Regulation In The Mouse Fetal Liver

During mouse embryogenesis,hematopoiesis consists of two successive waves:primitive hematopoiesis and definitive hematopoiesis.The primitive hematopoiesis occurs in the blood islands of extraembryonic yolk sac.Then,the transient primitive hematopoiesis is replaced by the definitive hematopoiesis.Hematopoietic stem cells?HSCs?,which are a population of multipotent cells that possess the abilities of self-renew and multi-lineage differentiation,are generated in the aorta-gonad-mesonephros?AGM?region.Moreover,HSC activity can be detected in the head,vitelline artery,umbilical artery and placenta.Definitive HSCs will migrate into the fetal liver?FL?through blood circulation,for rapid expansion and differentiation at embryonic day?E?12.Finally,hematopoietic stem and progenitor cells will colonize into thymus for lymphopoiesis and home to the bone marrow to maintain adult hematopoiesis.During the embryonic development,FL is an indispensable hematopoietic organ for HSC expansion;however,the underlying regulatory mechanism of HSC expansion in FL remains elusive.Firstly,we analyzed the spatial distribution of HSCs in FL from E12.5 to E15.5 by immunofluorescence assay to analyze the position relationship of arteries?marked by Ephrin-B2?,veins?marked by EphB4?and HSCs?marked by Runx1 and c-Kit?,and the results suggest that FL HSCs are close to the arteries during embryogenesis.To determine the effects of different niche cells on the HSC expansion,we sorted the stromal cells?CD45^-Ter119^-CD31^-?and endothelial cells?CD45^-Ter119^-CD31⁺?,and then co-cultured them with HSCs?Lin^-Sca-1⁺c-Kit⁺CD150⁺CD48^-?,flow cytometry analysis and colony-forming unit culture?CFU-C?assay show that stromal cells and endothelial cells can support HSC expansion.HSCs present rapid expansion in FL,and most HSCs are in the cell cycle.It's uncertain whether HSCs at distinct phases of cell cycle have distinct biological function.By using Hoechst and PyroninY staining,we resolved FL HSCs into 4 subpopulations according to their cell cycle states.We performed the CFU-C assay,CFU-spleen assay and transplantation assay to analyze the difference of biological function,the results suggest that HSCs at G0phase possess higher CFU-C ability,CFU-S ability and self-renewal ability than HSCs at other phases.BLOS2,encoded by Bloc1s2,is a component of BLOC-1 complex,which is involved in the endolysosomal trafficking.Previous studies demonstrated that BLOS2 regulates HSC generation in the AGM region through mediating Notch signaling pathway.To determine the function of BLOS2 in FL HSCs,we performed in vitro culture assay,rescue assay and systematic phenotype analysis.The results show that the Bloc1s2^-/-embryos present dysplasia,smaller FL size,excessive expansion of HSCs.Mechanistically,BLOS2 deficiency leads to over-activation of Notch signaling pathway,and further leads to abnormal expansion of HSCs.These results suggest that balanced Notch signaling pathway is essential for HSC maintenance.During embryogenesis,HSCs have a close physical location to the arteries in FL,and niche cells?stromal cells and endothelial cells?can contribute to HSC expansion.Compared with HSCs in cell cycling,quiescent HSCs possess higher self-renewal and reconstitution abilities.At the molecular level,BLOS2 can regulate the FL HSC expansion through balanced regulation of Notch signaling pathway.