| Background and objectivesHematopoietic stem cells(HSCs)were among the first discovered adult stem cells and have been widely administered in clinical practice with growing clinical potentials.As the source of the entire blood cell lines in adult mammals,HSC exhibits both self-renewal and multiple-lineage potentials which subsequently leads to the development of all mature and functional cells.In the bone marrow(BM),HSC maintains delicate balance between self-renewal and downstream differentiations with the assistance of coordinated transcription factor(TF)and cytokine regulations.HSC dysfunction may cause severe hematological disorders and therefore unveiling the molecular mechanism regulating HSC function holds great significance in that sufficient theoretical evidence may ultimately allow for potential treatment strategies to hematological diseases.Myosin heavy chain 9(MYH9)gene encodes the heavy chain of non-muscle myosin Ⅱ A,which interacts with actin to convert energy produced during ATP hydrolysis into mechanical force and functions in various biological processes:such as cell migration,cell adhesion,cytokinesis.In humans,mutations in MYH9 can cause a series of autosomal dominant disorders characterized by macrothrombocytopenia and other symptoms,known as MYH9-related diseases(MYH9-RD).Although the first symptom of MYH9-related diseases is hematological abnormality,little is known regarding its role in hematopoiesis due to the lack of in vivo models.In this study,Mx1-Cre;Myh9flox/flox conditional knockout mice was constructed for the first time to address this issue and elucidate the role of Myh9 in adult normal hematopoiesis and its mechanism.Method1.The expressions of Myh9 in multiple blood lineages of mice were mapped GEO database digging.2.Mx1-Cre transgenic mice were mated with Myh9flox/flox conditional knockout mice to obtain Mx1-Cre;Myh9flox/flox mice.3.The survival rate,PB complete blood count,hematopoiesis of BM were analyzed after Myh9 knockout by intraperitoneal injection of Poly I:C.4.Hematopoiesis of mice thymus was analyzed by flow cytometry.5.Hematopoiesis of mice spleen was analyzed by flow cytometry.6.Noncompetitive transplantation assay was performed to determine whether the loss of HSPC of Myh9 deficient mice was cell intrinsic.7.Competitive transplantation assay was performed to further evaluate the capability of Myh9 deficient HSCs in hematopoietic reconstitution.8.The apoptosis of HSPCs from mice BM was evaluated by Annexin V and 7-AAD staining.9.Myh9 deficient LSK cells were collected for RNA sequencing(RNA-seq),and the mechanism of Myh9 on HSPCs function was analyzed by GO enrichment,KEGG enrichment and GSEA analysis.10.The role of MYH9 during erythroid differentiation of UCB-derived CD34+HSPCs was evaluated by deceased MYH9 expression with shRNA interference.Results1.The induced Mx1-Cre;Myh9flox/flox mice suffered fatal death in 10-12 days post the initial Poly I:C injection.2.Myh9 deficient mice exhibited PB pancytopenia and markedly decreased bone marrow cellularity.Flow cytometric analysis revealed a significant decrease in the total numbers of myeloid(CD11b+Gr-1+),B lymphocyte(CD19+)and erythroid(CD71+Ter119+,CD71-Ter119+)precursors in bone marrow of Myh9 deficient mice compared with controls.3.Myh9 deficient mice exhibited significantly decreased thymus cellularity.The total numbers of CD4+CD8-,CD4-CD8+,CD4+CD8+cells in the thymus were significantly decreased in Myh9 deficient mice compared to the controls.But Myh9 deficient mice did not exhibit any significant change in the frequencies of CD4+CD8-,CD4-CD8+,CD4+CD8+,CD4-CD8-and total numbers of CD4-CD8-cells in the thymus compared with controls.4.The frequencies and total numbers of LK,LSK,LT-HSC,ST-HSC,MPP,CMP,GMP,MEP in the BM were significantly decreased after loss of Myh9.5.Myh9 deficient mice exhibited significantly decreased spleen cellularity.The total numbers of LK,LSK,LT-HSC,ST-HSC,MPP in the spleen were markedly decreased in Myh9 deficient mice compared with controls.6.The noncompetitive transplantation assay revealed that the deletion of Myh9 in the recipient mice resulted in obvious decrease in bone marrow cellularity and the total number of HSPCs.Myh9 deficient recipient mice exhibited peripheral blood pancytopenia,bone marrow failure and became moribund or died between 12-14 days after first Poly I:C injection just like primary Myh9 deficient mice.7.In the competitive transplantation assay,chimerism analysis in PB and BM of transplanted mice revealed that the Myh9 deficient HSCs failed to compete with wild type HSCs.And the contribution of Myh9 deficient HSCs to the hematopoietic system reconstitution of recipient mice hematopoietic system was minor.8.The apoptosis of LK,LSK,Gr-1+,CD 19+and whole BM cells were significantly increased after loss of Myh9.9.The results of RNA-seq revealed that Myh9 deletion resulted in dysregulation of numerous genes and signaling pathways responsible for HSC self-renewal,maintenance,differentiation and survival.1O.In erythroid differentiation of UCB-derived CD34+HSPCs ex vivo,we found that the downregulation of MYH9 did not affect differentiation kinetics but reduced erythroblasts enucleation.ConclusionOur present study provided the first evidence that Myh9 played an essential role in the maintenance and survival of adult HSCs.The inducible Myh9 knockout mouse model offered a solid tool to further explore the role of Myh9 in different hematopoietic lineages and tissue development.A better understanding of the MYH9 in normal hematopoiesis would specify its contribution in hematologic abnormalities and facilitate the development of accurate therapeutic approaches for MYH9-related diseases. |
