Expansion And Identification Of Functional Megakaryocyte-biased Hematopoietic Stem Cells Based On Three-dimensional Biomimetic Microniche

Objective:Hematopoietic stem cells are a highly heterogeneous population in biological function.molecular level and cell fate.With the development of single-cell technology.more and more evidences have confirmed the existence of lineage-biased differentiation and lineage-restricted hematopoietic stem cell subsets.The classic balanced hematopoietic hierarchy is constantly revised and supplemented.Researches have clarified that mouse megakaryocyte-biased long-term hematopoietic stem cells reside at the apex of the hematopoietic hierarchy.Under pathological conditions,there is a Subpopulation of human megakaryocyte-biased stem cells.Under normal physiological conditions,human megakaryocytes can be directly derived from pluripotent stem and progenitor cells and it is reasonable to speculate that at least some stem cells can directly differentiate into the megakaryotic lineage without going through the pluripotent stage.Based on the biomimetic microniche technology,this study aimed at the unsolved scientific problem of the differentiation path from stem cells to megakaryotic lineage cells,looking for and identifying human megakatyocyte-biased stem cells under physiological conditions,and establishing effective expansion in vitro.This study aims to find and identify such a subpopulation of human megakaryocyte-biased hematopoietic stem cells in physiological conditions,to expand this rare and specific subset that is easily lost in vitro by the three-dimensional microniche-based culture system,to explore its immunophenotype,biological function and regulatory mechanism,and to optimize the existing effectiveness evaluation system of hematopoietic stem cell expansion.Methods:In this study,flow cytometry and colony-forming unit assay of megakaryocyte progenitros were used to evaluate the in vitro expansion of microniche-cultured c-Kit+cells on mouse megakaryocvte-biased hematopoietic stem cells.Then,the mononuclear cells or CD34+ cells derived from human bone marrow,peripheral blood,and umbilical cord blood were cultured in vitro by the three-dimensional biomimetic microniche system with no serum and low factors.The spatial state of cells and microniche was characterized by scanning eletron microscope and confocal laser scanning microscope and other imaing techniques.Cell morphology was observed by Wright-Giemsa staining.Stem cell immunophenotype was detected by flow cytometry.Colony-forming cell assay and colony-forming unit assay of megakaryocyte progenitros were used for in vitro functional evaluation.Limiting dilution transplantation and specific subpopulation sorting for transplantation were used to evaluate the function of stem cells in vivo,especially the reconstruction of the megakaryotic system.The effects of micronichebased culture on stem cell-related signaling pathways were explored through bulk RNA sequencing,real-time fluorescent quantitative polymerase chain reaction,and proteomic analysis.Single-cell RNA sequencing explored the specific transcriptional characteristics of heterogeneous populations of stem cells during microniche-based culture,and found the megakaryocyte-biased hematopoietic stem cell subpopulations at the single-cell level,and verified them through phenotypic and functional experiments.Results:Microniche-based culture significantly expanded mouse long-term stem cells and megakaryocyte-biased hematopoietic stem cells,and the colony-forming ability of large megakaryotic progenitor cells was improved.Besides,it had a significant expansion capacity for human bone marrow/peripheral blood/umbilical cord blood hematopoietic stem cells,and the stem cells with stronger stemness,the more pronounced the effect.On this basis,it was found that human long-term hematopoietic stem cells and megakaryotic progenitor cell subsets maintained their viability in longterm microniche-based culture.Single-cell transcriptome sequencing revealed specific markers SELL(CD62L)and PROM1(CD 133)for human megakaryocyte-biased stem cell subsets.Immunodeficient mouse transplantation confirmed that human functional megakaryocyte-biased long-term stem cells with megakaryotic reconstitution ability were enriched in CD34+CD38-CD45RA-CD90+CD49flow CD62L-CD133+subpopulation,while microniche-based culture could maintain and expand this subpopulation that originally existed in physiological conditions and easily lost under culture conditions.Imaging observations revealed that there were a small number of stem cell subpopulations inside the microniche.Transcriptomics,proteomics,qRT-PCR,and cytokine rescue and blocking experiments jointly revealed that the microniche-based culture system played a role in expanding megakaryocyte-biased stem cells by reprogramming the cytokine milieu and providing appropriate physical support.Conclusion:Human functional megakaryocyte-biased hematopoietic stem cells could be retained and significantly expanded by microniche-based culture in vitro,and this specific subpopulation was enriched in the CD34+CD38"CD45RA"CD90+CD49flow CD62L-CD133+immunophenotype.The three-dimensional structure of biomimetic microniche could provide suitable physical support and cytokine milieu similar to physiological stem cell niches for primitive megakaryocyte-biased hematopoietic stem cells to grow and exert specific physiological functions.