Chemical Cocktail Induces Expansion Of Human Hematopoietic Stem Cells Ex Vivo

Background:Due to the limited amount of cells in a single umbilical cord blood,the clinical application of umbilical cord blood is limited.A suitable and efficient culture system for in vitro expansion of hematopoietic stem cells(HSCs)has become a research focus.Our recent research found that chemical cocktail can reprogram mouse fibroblasts and differentiated blood cells into hematopoietic stem and progenitor cells,and we hypothesized that it could maintain the stemness of HSCs.It has not been reported whether this chemical cocktail can induce the expansion of human HSCs ex vivo.Methods:In this study,CD34+cells were sorted by magnetic beads from umbilical cord blood or bone marrow.We cultured CD34+cells in serum-free medium containing cytokines.The cells were divided into three groups based on treatment,which were DMSO(control group),VPA alone and the combination of VPA and Repsox.Then the phenotype and function of the expanded cells were detected by Wright-Giemsa staining,flow cytometry,and CFU assay.Additionally,CFSE assay was used to observe the pattern of cell division.In order to verify the effectiveness and safety of the chemical cocktail,withdrawal experiments were performed.The chemical cocktail was withdrew on the sixth day,and cells were detected every two days.Finally,to explore the mechanism of the induction of the compounds,we use the treatment of combination of different subtypes of HDAC inhibitors and TGF?pathway inhibitors and RNA sequencing.Results:After culturing umbilical cord blood-derived hematopoietic stem cells with the chemical cocktail for 7 days,there was no significant difference in the number of total nucleated cells,but the chemical cocktail treatment can improve the percentage of phenotypically-defined HSCs by 5.80 fold in comparison to the VPA single compound treatment group and 69.41 fold to the control group.In the CFU assay,the chemical cocktail group formed more GEMM clones.In the first four days of cell culture,cell divisions in the chemical cocktail group was less than that of the control group,and the cells rapidly differentiated after withdrawal.Among the six HDAC inhibitors,pan-HDAC inhibitors and selective HDAC 1/3 inhibitors can sustain a higher percentage of CD34~+CD90~+cells,which can be increased by adding diverse TGF?pathway inhibitors.Among them,the synergistic effect of Repsox is the most obvious.Conclusion:The chemical cocktail of VPA and Repsox can induce the ex vivo expansion of human HSCs,and the TGF?pathway inhibitors can greatly enhance the effect of HDAC inhibitors on promoting the proliferation of human HSCs.