NUP98-HOXA10hd Fusion Protein Sustains Multi-lineage Hematopoiesis Of Lineage-committed Progenitors In Transplant Setting

Objectives:Allogeneic hematopoietic stem cells（HSCs）are widely to treat blood disorders,such as leukemia,myeloproliferative diseases,anemia.However,lack of timely available HLA matched-donor hindered the application of allogenic transplantation in clinical applications.Cord blood,as one of the cell sources for allogeneic HSCs,have advatages,relatively easy accessibility and low immunogenecity.Even HLA incompatible cord blood rarely cause graft-versus-host diseas e（GVHD）.Nonetheless,the insufficient hematopoietic stem cell number in one unit of cold blood become the main obstacle for the application of cord blood.Multipotent progenitors（MPPs）,as the immediate progeny of hematopoietic stem cells,can rapidly proliferate and regenerate all types of mature blood cells.Exploring whether extending hemtopoeisis induced by multipotent progenitor and lineage-committed progenitor could provide new insights for reconstitution of long-term multi-lineage hematopoiesis.Nup98-hoxa10hd（NA）fusion protein has been shown to expand long-term hematopoietic stem cells（HSCs）and promote engraftment competitiveness without causing obvious oncogenesis.In this study,our objectives were to explore the role of NA plays in MPPs and their downstream lineage-committed progenitors.Methods:The NA^LSL/+Vav-cre compound mice（NA mice）were identified by mouse genotyping identification experiment.Multipotent progenitors（MPPs）were isolated by staining and flow cytometric analysis.Transplantation assay and dynamic donor contribution were conducted to test whether the expression of NA in MPPs could sustain long-term multi-lineage hematopoiesis.Myeloid progenitor（MP）analysis and common lymphoid progenitor（CLP）analysis were used to study whether NA MPPs produced abundant lineage-committed progenitors to sustain long-term hematopoiesis.LSK analysis was used to test whether overexpression of NA induced MPPs to dedifferentiate into long-term hematopoietic stem cells（LT-HSCs）.Secondary transplantation experiments were conducted to futher test whether NA-MPPs could sustain long-term multi-lineage hematopoiesis.RNA sequencing was carried out with sorted MPPs and MP to explore how the NA gene altered the MPPs and MP cells at the molecular level.Results:Three hundred of NA-MPPs sucessfully reconstituted long-term multi-lineage hematopoiesis up to 44 weeks in primary mice.NA upregulated genes regulate essential pathways in MPPs,including regulation of cell cycle,epigenetic regulation and responses to stress.These molecular traits are associated with the earlier appearance of a Sca1^-c Kit⁺myeloid progenitor population,and more abundant cellularity of lineage committed progenitor as well as bone marrow nucleated cells.Further,the NA-derived primary bone marrow cells,which lack NA-LSK cells,successfully repopulated secondary multi-lineage hematopoiesis over 20 weeks.Conclusion:NA fusion protein promotes MPPs and lineage-committed progenitor engraftment via extending long-term multi-lineage hematopoiesis.Our findings pave the way for transplantation of MPPs when matched HSCs are unavailable.