The Regulation Mechanism Of Wip1 On The Development Of Hematopoietic Stem Cells In Mouse Embryonic Head

Research background and purposeHematopoietic stem cells(HSCs)are the self-renewing source of mature blood cell lineages in the adult.In clinic,HSC transplantation is used to treat various hematological diseases including leukemia.However,the limited number of HSC and the occurrence of GVHD in most patients limit the clinical application of HSCs.Therefore,the study of HSC regeneration in vitro will have a significant impact on clinical treatment,and exploring the regulation of HSC development has a very important guiding significance for HSC regeneration and clinical application.The first HSC emerges in the aorta-gonad-mesonephros(AGM)region of E10.5 mouse embryos.The head of embryo and AGM region are the hematopoietic sites at the same period.Similar to the AGM region,HSC derived from embryonic head endothelial cells can contribute to the adult hematopoietic system.The hematopoietic development of embryonic head is regulated by macrophages.Wild type p53 induced phosphatase 1(Wip1),a member of protein phosphatase type 2C(PP2C)family,is a serine and threonine phosphatase encoded by PPM1D,which is involved in the phosphorylation and dephosphorylation of many signaling proteins.It is found that Wip1 can regulate the aging of adult HSC and the development of HSC in the AGM region of mouse embryo.It is important to clarify the effect of Wip1 gene on HSC for future clinical application.However,the regulatory effect of Wip1 on hematopoietic development of mouse embryonic head is unknown.In this study,we used Wip1 knockout mouse model to investigate the regulatory mechanism of Wip1 on the development of HSC in mouse embryonic head.Research contents and methods1.To observe the effect of Wip1 on hematopoietic stem and progenitor cells in mouse embryonic head:The effect of Wip1 on the whole mouse embryonic head was observed by viable cell count,and then the effect of Wip1 on the CD31+c-kit+hematopoietic cluster of mouse embryonic head was observed by flow cytometry.Then the effect of Wip1 on the hematopoietic progenitor cells of mouse embryonic head was detected by CFU-C assay,and the effect of Wip1 on the function of hematopoietic stem cells in mouse embryonic head was detected by in vivo transplantation.Furthermore,macrophages in Wip1 deficient embryos' head was analyzed by flow cytometry,and the expression of inflammatory factors in the head of mouse embryos was detected by qPCR.The mechanism of Wip1 regulating macrophages in the head of mouse embryos was studied by co-culture.2.To study the effects of Wip1 on Erythroid and Myeloid progenitors(EMP)and macrophages in mouse embryo yolk sac:The number and proportion of EMP and macrophages in mouse embryo yolk sac were detected by flow cytometry to determine the effect of Wip1 on EMP and macrophages.Results1.The number of cells was decreased in the Wip1-/-embryos' head.2.There was no significant difference in the number of hematopoietic clusters in the Wip1-/-head,but the absolute number was decreased.3.The function of hematopoietic progenitor cells in the embryonic head of Wip1 deficient mice was decreased.4.The function of hematopoietic stem cells in the Wip1-/-head was decreased.5.Wip1 deletion resulted in increasing of TNFa and IL-1? secreted by macrophages embryos,while the expression of pro-inflammatory factor receptors in endothelial cells was unchanged.6.The number of EMP in yolk sac of mouse embryos with wip1 deletion decreased at E10.5,but the proportion increased at E11.5.7.Wip1 deficiency had no effect on the percentage and number of macrophages in the yolk sac of mouse embryos.Conclusion1.Wip1 regulates the development of hematopoietic progenitors cells and macrophages in embryos' head.2.Wip1 plays a role in the regulation of embryonic head hematopoietic cell development,which might be mediated by macrophages through secreting pro-inflammatory factors.3.The number of EMPs in yolk sac of E10.5 embryos decreased significantly after wip1 deletion,and the proportion of EMPs in yolk sac of E11.5 embryo increased significantly,but macrophages were not affected.