The Role Of WAP4 Disulfide Core Domain 2(Wfdc2) During The Migration Of Hematopoietic Stem Cells From Fetal Liver To Bone Marrow

Objective:During the development of mouse hematopoietic stem cells(HSCs),HSCs significantly expand in fetal liver and migrate to bone marrow,which will maintain the stability of hematopoietic system for whole life.However,little is known about which factor mediate HSCs translocation from fetal liver to bone marrow.The purpose of the current study is to investigate the role of WAP 4-disulfide bond core domain 2(Wfdc2)in the migration of HSCs from fetal liver to bone marrow.Methods:The expression of Wfdc2 was detected by q RT-PCR.Heterozygous Wfdc2^+/-mice(C57BL/6 genetic background)were produced by CRSIPR/CAS9 technique.The percentages and numbers of HSCs in E14.5 Wfdc2^-/-fetal liver cells and E18.5Wfdc2^-/-bone marrow cells were analyzed by flow cytometry.E14.5 Wfdc2^+/+and wfdc2^-/-fetal liver cells(CD45.2)were transplanted into lethal dose irradiated mice(CD45.1)by simple and competitive bone marrow transplantation.The percentages of CD11b,Gr-1,CD3 and B220 positive cells in peripheral blood of recipient mice were analyzed at 1,2 and 3 months after transplantation.The numbers of HSCs in bone marrow of recipient mice were analyzed at 3 months after transplantation.5-fluorouracil(5-FU)was injected into the recipient mice at 3 months after competitive bone marrow transplantation.The percentages of peripheral blood cells in each group were detected at 9 days after 5-FU treatment.The numbers of HSCs in the bone marrow of the recipient mice were detected at 10 days after 5-FU exposure.CD45.1 bone marrow cells were incubated with purified Wfdc2 protein or PBS,and transplanted into irradiated CD45.1/2 with the same numbers of CD45.2 bone marrow cells.The percentages of CD11b,Gr-1,CD3 and B220 positive cells in peripheral blood of recipient mice were examined at 1,2 and 3 months after transplantation.The effects of Wfdc2 protein on the in vivo reconstruction ability of HSCs were evaluated.Results:1.Wfdc2 was specifically overexpressed in mouse HSCs,and the expression of Wfdc2 in CD45⁺Lin^-c-kit⁺cells in E14.5 fetal liver was significantly higher than that in CD45⁺Lin^-c-kit⁺cells in adult bone marrow(p(27)0.01).2.Wfdc2 gene knockout did not affect the survival of E14.5 embryos while none of Wfdc2^-/-mice survived at 3 weeks after birth.3.Compared with the numbers of immature cells(Lin^-),LSK(Lin^-c-kit⁺Sca1⁺)cells and HSCs(Lin^-c-kit⁺Sca1⁺CD48^-CD150⁺)in E14.5 Wfdc2^+/+fetal liver cells,the numbers of immature cells in Wfdc2^-/-fetal liver cells were slightly increased without statistical significance.There was no significant difference in the numbers of LSK and HSCs between E14.5 Wfdc2^+/+and Wfdc2^-/-fetal liver cells.4.Compared with the numbers of immature cells,LSK cells and HSCs in E18.5Wfdc2^+/+fetal bone marrow cells,the percentages of LSK cells and HSCs in Wfdc2^-/-fetal bone marrow cells were significantly decreased(p(27)0.01).5.The results of direct bone marrow transplantation and competitive bone marrow transplantation showed that knockout Wfdc2 did not affect the ability of the reconstitution of bone marrow cells and multiple differentiation of mouse HSCs in vivo.6.After competitive bone marrow transplantation recipients were treated with5-FU,the percentages of Wfdc2^-/-CD11b,Gr-1and B220 positive cells in peripheral blood were significantly decreased(p(27)0.01)when compared to Wfdc2^+/+counterparts.Compared with Wfdc2^+/+-derived HSCs in bone marrow,the numbers of Wfdc2^-/-LSK cells and HSCs were significantly decreased(p(27)0.01).7.Purified Wfdc2 protein enhanced the ability of HSCs engraftment in vivo.Compared with PBS group,the percentages of hematopoietic cells derived from HSCs incubated with Wfdc2 protein were significantly increased in peripheral blood(p(27)0.01).The percentages of CD11b?Gr-1?CD3 and B220 positive cells were significantly increased in peripheral blood(p(27)0.05).Conclusion:1.Wfdc2 is highly expressed in mouse HSCs.After knocking out Wfdc2 gene,the numbers of hematopoietic stem cells in E14.5 fetal liver were not affected while the numbers of hematopoietic stem cells in E18.5 fetal bone marrow cells were significantly decreased.These data indicate that the expression of Wfdc2 regulates the migration of HSCs from fetal liver to bone marrow.2.Genetic knockout of Wfdc2 does not affect the engraftment ability of hematopoietic stem cells in vivo.3.Genetic knockout of Wfdc2 reduces the regeneration ability of hematopoietic of HSCs under 5-FU stress condition.4.Purified Wfdc2 protein enhances the engraftment ability of hematopoietic stem cells in vivo under transplantation setting.