| Objective: Hematopoietic stem cells(HSCs)are adult stem cells with self-renewal potential and capable of multi-directional differentiation to form multi-lineage blood cells.Clinically,hematopoietic stem cell transplantation(HSCT)is mainly used to treat various hematological diseases such as hematopoietic malignancies,genetic defects,autoimmune diseases and certain solid tumors.However,the difficulty in matching human leukocyte antigen(HLA)is a serious challenge to limit bone marrow or mobilize peripheral blood stem cell transplantation.Cord blood hematopoietic stem cells have low immunogenicity,can reduce the incidence of graft-host disease(GVHD),and have strong cell proliferation potential and easy collection,so they have broader clinical application prospects.Unfortunately,due to the lack of a single umbilical cord blood cell,which limits its clinical application,compounds that can expand cord blood hematopoietic stem cells in vitro have become new research hotspots.UM171 is the best small molecule compound for the expansion of cord blood hematopoietic stem cells at this stage.In this study,37 UM171 analogs were synthesized.We evaluated the expansion effects of these analogs,tried to find new molecules similar to UM171,and provided reference for the structure activity of UM171.Method:First,we used immunomagnetic beads to enrich CB-CD34+ cells,construct an ex vivo culture system containing a combination of cytokines,and a drug screening system using flow cytometry as the main detection method.On this basis,the primary screening and secondary screening of 37 small molecule compounds were performed.According to their expansion effect and biological activity(EC50 value)of cord blood CD34+CD38-cells,the optimal compounds were selected as well as its optimal amplification concentration.Thereafter,the expansion effect was verified by flow cytometry,and the hematopoietic function of the cultured cells was evaluated using Metho CultTM H4434 semi-solid medium.Next,in order to explore the mechanism of the expansion effect of compound 11,we examined the apoptosis of each subtype of cells(Annexin-V apoptosis detection),the cell cycle(Ki67/7-AAD intracellular staining cycle detection)and cell proliferation division(CFSE detection),and the expression of self-renewal related genes was analyzed by real-time quantitative PCR(RT-q PCR).Conclusion: Six potential compounds were found out in the primary screening,two of them were selected for secondary screening.According to their expansion effects and EC50 values,compound 11 was obtained as the optimal compound,and its optimal expansion concentration was 165 n M.The expansion effect was significantly better than that of various HSCs expansion drugs(unfortunately,the effect of compound 11 failed to meet UM171),and the expanded cells maintained intact hematopoietic colony forming ability.The mechanism study found that compound 11 had no significant effect on apoptosis,and there was no significant change in cell cycle progression of long term-Hematopoietic stem cells(LT-HSCs)and Hematopoietic stem/progenitor cells(HSPCs).Cell division assays showed that compound 11 slowed the division of HSPCs,and that compound 11 maintained a higher BMI1 gene expression,which is important for the self-renewal of HSCs.In general,this new molecule has high biological activity and good amplification effect,the cultured cells maintain a complete multi-lineage differentiation potential as well.Next,mechanistic studies found that compound 11 may effectively avoid cell exhaust by slowing the division of HSPCs and promoting self-renewal division.Significance: In this study,37 new molecules were synthesized,and their effects on the expansion of cord blood HSPCs were screened and tested.A novel small molecule compound with potential for HSCs ex vivo expansion was discovered.It is expected that the structure activity relationship of UM171 will be explored from this new compound structure,and more importantly,it can be used as a lead compound to guide further research to find better HSCs amplification compounds. |
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