Ricolinostat Promotes The Differentiation Of Hematopoietic Stem/Progenitor Cells Into Megakaryocytes

Platelet transfusion has important clinical application value,but growing demand for platelets is severely limited by blood donor supply.The preparation of megakaryocytes and platelets from stem cells in vitro is expected to solve the problem of platelet shortage.Umbilical cord blood represents a large and readily available source of hematopoietic stem cells and can be used to prepare megakaryocytes and platelets in vitro.At present,the yield and efficiency of differentiation of umbilical cord blood hematopoietic stem cells into megakaryocytes need to be improved.Based on the expression of the megakaryocyte specific gene PF4 reportor,we screened the small molecules to promote megakaryocyte differentiation from the small molecule compound library of epigenetic regulation,so as to improve the differentiation efficiency and yield of hematopoietic stem/progenitor cells to megakaryocyte and platelet.Methods:1.The human PF4 gene promoter DNA sequence was amplified,ligated to the GFP sequence and inserted into the PLVX-tight-puro vector to construct a recombinant plasmid PLVX-PF4-promoter-GFP.The recombinant plasmids were packaged into lentivirus and transduced into MEG01 and K562 cells.GFP positive rate of MEG01and K562 cells was determined by flow cytometry to verify the transcriptional activity of the reporter gene.2.A library of 120 epigenetic regulating small molecules was screened in K562cells with PF4 reporter by high-content cell imaging technology,and the candidate small molecules were verified in other cell lines.3.A stepwise differentiation protocol was used to induce CD34~+hematopoietic stem/progenitor cells into megakaryoctes.Ricolinostat was added in the second stage.After 7 days of Ricolinostat treatment,the differentiation efficiency of megakaryocyte progenitor cells were detected by flow cytometry and colony formation assays.In the third stage,Ricolinostat was withdrawed and the cells were further induced to mature megakaryocytes,which were evaluated by examining the surface markers of mature megakaryocytes,the expression of related genes,and the ability to produce plates.Results:The human PF4 gene promoter reporter vector was successfully constructed and transferred into MEG01 and K562 cells.Based on the PF4 reporter system,small molecules which enhanced PF4 expression were screened out from 120 small molecule compounds.We found that Ricolinostat can effectively increase the PF4expression in K562 and MEG01 cells.Hematopoietic stem/progenitor cells were induced into megakaryocytes by a stepwise differentiation protocol,and Ricolinostat was added to the second stage.It was found that Ricolinostat had no significant effect on the overall cell proliferation,but it could increase the percentage and the number of CD34~+CD41a~+cells and CD41a~+CD61~+cells.Furthermore,Ricolinostat significantly increased the percentage and the number of CD34~+CD38~+CD45RA~-CD123~-CD41a~+megakaryocyte progenitor cells.The colony forming ability of megakaryocyte progenitor cells was significantly improved after Ricolinostat treatment.And the expression of megakaryocyte progenitor-related transcription factors was significantly increased in the Ricolinostat-treated group.In the third stage,the cells from the second stage were further induced into mature megakaryocytes after Ricolinostat withdrawal.And we showed that the megakaryocytes induced in the Ricolinostat treatment group and the control group both had typical megakaryocyte morphology,expressed the megakaryocyte-specific cell surface markers,and could produced functionalplatelets.Conclusions:In this study,a small molecule compound Ricolinostat which enhanced the expression of PF4 was screened out through high-throughput screening system.And we found that Ricolinostat can promote the differentiation of megakaryocyte progenitors from hematopoietic stem/progenitor cells.Our study provides new insight for the optimization of of megakaryocyte differentiation system,future large-scale in vitro culture of megakaryocyte progenitor cells,and the preparation of artificial platelets.