Asymmetrically Segregated Mitochondria Provide Cellular Memory Of Hematopoietic Stem Cell Replicative History And Drive HSC Attrition

PART ? MITOCHONDRIA PERMANENTLY REMODEL AFTER HSC REPLICATION UNDER REGENERATIVE AND HOMEOSTATIC CONDITIONSObjective:To verify the changes of function,structure and morphology of mitochondria in HSC after HSC stress,bone marrow cell transplantation as the main stress stress model to be used to detect the changes of function and morphology of mitochondria network after HSC transplantation.Methods:Healthy adult C57BL/6 mice or mitochondria-labeled transgenic mice?mito-dendra-2 mice?were used as donors and BoyJ mice as recipients.The whole bone marrow cells from the donor and the whole bone marrow cells from the BoyJ mice were mixed in proportion?10⁶:10⁶?and then transplanted to the lethal irradiated BoyJ receptor mice to construct the HSC transplantation model.HSC was used as the main cell research object for following experiment and research.Four months after transplantation,the number of active mitochondria using mitotracker green,mitochondrial membrane potential with TMRE and mitochondrial oxidative level with Mito Sox were measured by flow cytometry.And ATP production of HSC active mitochondria was detected with ATP kit.With ATPase inhibitor and electron transfer chain complex inhibitor treatment in vitro,the changes of MMP were detected by flow cytometry.Immunofluorescence was used to detect and observe the structure and morphology of HSC mitochondria after transplantation and the relationship between mitochondria and HSC.Immunofluorescence was used to detect the continuous effect of transplantation on mitochondrial remodeling.The effects of transplantation on the remodeling of mitochondrial network on HSC daughter cells were recorded by laser confocal microscopy.Results:Four months after transplantation,LSK-SLAMs?HSCs?group demonstrated the most significant difference in mitochondrial function change in transplanted group by flow cytometry(P_HSC<0.05).Compared with the control group,Mito trackergreen and MMP level significantly decreased(P_mito-G<0.01,P_MMP<0.01).?2?After the intervention of ATPase inhibitor and electron transport chain complex inhibitor on mitochondria in vitro,the response capacity of mitochondria decreased?MMP?(P_Oligomycin<0.05,P_Reotenone<0.05).?3?Immunofluorescence detection showed that mitochondrial structure in Mito-dedra2 mice was more compact and distributed to one side?P<0.0001?.?4?The area of mitochondria increased significantly?P<0.0001?and the roundness of mitochondria decreased significantly?P<0.0001?.?5?After multiple proliferation and differentiation,HSC mitochondria in the transplanted group tended to gather on one side of the cell,and the structure became more compact?P<0.0001?.?6?After HSC division,the mitochondria in the transplantation group had asymmetric divided and distributed,which showed that the surface number ratio and the volume ratio of each surface were significantly different between the paired daughter cells?P<0.0001?,and there were also statistical differences in the integrated MFI values of mitochondria?P=0.04?and the area of mitochondria?P=0.005?between the paired daughter cells.Conclusion:In this part,we found that hematopoietic stem cells are in stress state after bone marrow transplantation,which leads to some functional defects of mitochondria,and the structural morphology and distribution of mitochondria have also been permantly remodeled.PART ? THE POSSIBLE MECHANISM OF LOSS OF DRP1 DISTRIBUTION ONTO MITOCHONDRIA IN HSCS AFTER REPLICATION CAUSES HSC FUNCTIONAL DECLINE OF THE PROGENYObjective: To explore the role of Drp1 protein in the maintenance of normal structure,morphology and function of mitochondria,and the possible mechanism of permanent remodeling of mitochondria after stress,and to determine whether Drp1 protein is the key factor that causes mitochondrial structural dysfunction and changes in biological behavior of hematopoietic stem cells.Methods: Flow cytometry was used to detect the fluorescence intensity of Drp1 in hematopoietic stem cells before and after transplantation.Drp1 flox / flox: Mxcre conditional knockout mice were obtained by hybridization of Drp1 flox / flox transgenic mice with Mx Cre tool mice induced by interferon.The model of Drp1?/? conditional knockout mice were constructed and Drp1 flox / flox mice as the control group.Imaris imaging software was used to reconstruct the three-dimensional surface images of mitochondria and Drp1 protein spots to understand the expression level of Drp1 and its relationship with mitochondrial structure.Immunofluorescence was used to detect the difference of Drp1 expression between the two groups,and to observe the difference of mitochondrial content and morphological structure between the two groups.The effect of Drp1?/? on the remodeling of mitochondrial network and the change of activity on HSC progeny was observed and recorded by laser confocal microscopy.Flow cytometry was used to detect how Drp1 protein deficiency affected on the differentiation of bone marrow stem cells,progenitors and the formation of peripheral blood cells.Results: After successfully constructing Drp1?/? conditional knockout mouse model,compared with the control group of Drp1 flox / flox,?1?In the Drp1?/? group,Drp1 protein level decreased significantly?P = 0.0057?,and the amount of Drp1 spots attached to the top of mitochondria and 0.3?m close to mitochondria also decreased significantly?Pattached = 0.0006,Pclose = 0.006?.At the same time,the number of mitochondria separated by Drp1 decreased significantly after transplantation?P < 0.0001?.?2?HSCs containing condensed and compact mitochondria in Drp1?/? group was significantly higher than that in the control group?P < 0.0001?.?3?It was found that the dynamic activity of mitochondria in Drp 1?/? group was significantly lower than that in control group?P< 0.0002?after HSC activation entering the interphase.During the process of HSC self-replication cell division,the mitochondria of Drp1?/? group also distributed asymmetrically between the paired daughter cells,and the fluorescence intensity of mitochondria between paired daughter cells was significantly different?P= 0.0084?,but the difference of the area of mitochondria among the paired daughter cells analyzed in this study was not statistically significant?P= 0.19?.?4?Flow cytometry was used to detect the cell numbers of LSK-SLAM,MPP and CP in the two groups of mice.The number of cells in Drp1?/? group was higher than that in the control group?PHSC < 0.05;PMPP < 0.05?,but there was no significant statistical difference between the two groups.?5?After the first bone marrow transplantation,the proportion of donor derived cells in the peripheral blood of the patients in the Drp1?/? group was significantly lower than that in the control group?8K: P = 0.1;16K: P < 0.001?;after the second consecutive transplantation,the ability of HSCs recombination and regeneration in the Drp1?/? group was still significantly lower than that in the control group?P < 0.05?;after the transplantation,the mitochondrial membrane potential water in the Drp1?/? group was detected by flow cytometry.The decreases were statically significant.?6?In the mdivi1 group,mitochondria became more aggregated and fused,and their structures became more compact.At the same time,mitochondria also redistributed,showing stronger polarity.Through imaris image analysis software,it was found that the number of mitochondria surface of each cell in the mdivi-1 treatment group was less than that in the control group,but the volume of each surface in each cell in the mdivi-1 treatment group increased significantly?P < 0.05?.?7?The percentage of donor cells in the bone marrow of the mice treated with 5-FU and DMSO was significantly lower than that of the control group?* P < 0.05?11 days after the treatment.The percentage of peripheral blood cells in the mdivi group was significantly lower than that of the control group?50K: * P < 0.05;140k: * P < 0.05?.Conclusion: Those results show that the deletion of mitochondrial dynamic protein Drp1 will lead to the consistent changes of the morphological structure of mitochondria after transplantation,which will lead to more compact and fused mitochondria.More asymmetrical segregation of mitochondria happened in HSCs due to loss of Drp1 gene,thus affecting the function of mitochondria.Moreover,the malfunctional mitochondria will be carried by HSCs to the progeny cells after consecutive cell replications and divisions,and ultimately change the fate and future outcome of HSCs.