Study Of Small Molecule Compounds On Ex Vivo Expansion Of Hematopoietic Stem And Progenitor Cells

BackgroundHematopoietic stem cells?HSCs?are adult stem cells that have self-renewal activity and the potential ability to differentiate into cells of all mature blood lineages.Hematopoietic stem cell transplantation?HSCT?could restore a new functional hematopoietic system in the recipients,which is used in the clinic to treat hematological malignancies and other malignant tumors,as well as in gene therapy settings.For HSCT,the main sources of HSCs include bone marrow?BM?,mobilized peripheral blood?m PB?,and umbilical cord blood?UCB?.The number of HSCs transplanted correlates with successful engraftment and patient survival.For successful HSCT,the minimum recommended doses are 2×10⁶ CD34⁺cells/kg in allogeneic HSCT?allo-HSCT?and 5×10⁶ CD34⁺cells/kg in autologous HSCT?AHSCT?,which would lead to rapid and continuous engraftment.HSCT is widely used as an important clinical treatment method.However,the number of HSCs from any source is very limited.The low number of HSCs in UCB units has largely restricted its widespread application,which is mainly used in pediatric patients.Adult patients often have delayed neutrophil and platelet engraftment after umbilical cord blood transplantation?UCBT?,which easily increases the risk of infection.It is convenient to mobilize and collect m PB-derived HSCs,which avoids multiple aspirations and anesthesia.Nevertheless,reported failure rates with the traditional mobilization are still high.Plerixafor,the novel stem cell mobilizing agent,could save some patients with poor or failed mobilization but cannot improve the mobilization of patients with severe damage to HSCs and BM niche.In summary,the insufficient number of HSCs is one of the key problems restricting the application of HSCs.Therefore,the effective ex vivo expansion of HSCs has become an important method to solve this problem.High-throughput screening of small molecule compounds libraries identified pyrimidoindole derivative UM171 and purine derivative SR1 that could stimulate ex vivo expansion of UCB-derived HSCs.The ongoing clinical trials have obtained safe and effective results.However,the expansion and molecular mechanism of HSCs from different sources are still unclear.ObjectiveThe purpose of this study is to investigate the effects of the small molecule compounds UM171 and SR1 on ex vivo expansion,differentiation and the expression of CXCR4 on hematopoietic stem and progenitor cells?HSPCs?from human UCB,donor mobilized peripheral blood?Donor-m PB?,lymphoma patients autologous mobilized peripheral blood?Auto-m PB?;explore the conditions for effective ex vivo expansion of Auto-m PB-derived HSPCs in lymphoma patients,providing a rescue measure for lymphoma patients with poor or failed mobilization,and further gain insights into the molecular mechanism of small molecule compounds acting on HSPCs.Methods1.CD34⁺cells from UCB,Donor-m PB and Auto-m PB were enriched by Ficoll and CD34 Micro Bead Kit Ultra Pure and used as uncultured groups.HSCs expansion medium composed of Stem Span^TM SFEM?supplemented with 100 ng/m L stem cell factor?SCF?,100 ng/m L FMS-like tyrosine kinase 3 ligand?Flt-3L?,100 ng/m L thrombopoietin?TPO?,100 ng/m L interleukin 6?IL-6?was used for ex vivo expansion.CD34⁺cells from three sources were divided into control group,UM171 group,SR1 group and UM171+SR1 group and expanded ex vivo for 10d.The proportion and absolute number detected by flow cytometry?FCM?and cell counting,and the expansion fold was calculated by the number of cells in each ex vivo culture group divided by the number of input cells.2.In order to explore the multi-lineage differentiation ability of HSPCs after ex vivo expansion for 10d,expanded cells were harvested to perform colony-forming unit assay?CFU?and FCM.3.CD34⁺cells from three sources were cultured in control group,UM171 group,SR1 group and UM171+SR1 group for 48h,the expression of CXCR4 on CD34⁺cells,CD34⁺CD38^-cells and CD34⁺CD38^-CD90⁺cells was detected by FCM.4.According to different mobilization results,lymphoma patients were divided into poor mobilization group?CD34⁺cells collection<2×10⁶/kg?and satisfactory mobilization group?CD34⁺cells collection?2×10⁶/kg?.CD34⁺cells from two groups were cultured with UM171 for 10d.The proportion and absolute number of CD34⁺cells,CD34⁺CD38^-cells and CD34⁺CD38^-CD45RA^-CD90⁺cells were detected by FCM and cell counting,and the expansion fold was calculated.5.CD34⁺cells from three lymphoma patients were ex vivo cultured in control group and UM171 group for 10d.RNA sequencing?RNA-seq?was performed to find differentially expressed genes?DGEs?and do Gene Ontology?GO?enrichment analysis,thus to explore the molecular mechanism of UM171 acting on ex vivo expansion of Auto-m PB-derived HPSCs in lymphoma patients.Results1.UCB-derived CD34⁺cells were expanded ex vivo for 10d,compared with control group,the proportion of CD34⁺cells and CD34⁺CD38^-cells in UM171 group,SR1 group and UM171+SR1 group was significantly increased?P<0.05?.The expansion folds of CD34⁺cells and CD34⁺CD38^-CD45RA^-CD90⁺cells in UM171+SR1 group was significantly increased?P<0.05?.There was no significant difference in the expansion folds of total nucleated cells?TNCs?among four groups?P>0.05?.Donor-m PB-derived CD34⁺cells were expanded ex vivo for 10d,compared with control group,the proportion of CD34⁺cells and CD34⁺CD38^-cells in UM171 group,SR1 group and UM171+SR1group as well as the proportion of CD34⁺CD38^-CD45RA^-CD90⁺cells in UM171 group and UM171+SR1 group was significantly increased?P<0.05?.The expansion folds of CD34⁺CD38^-cells and CD34⁺CD38^-CD45RA^-CD90⁺cells in UM171 group as well as the expansion folds of CD34⁺CD38^-CD45RA^-CD90⁺cells in UM171+SR1 group was significantly increased?P<0.05?.There was no significant difference in the expansion folds of TNCs among four groups?P>0.05?.Auto-m PB-derived CD34⁺cells were expanded ex vivo for 10d,compared with control group,the proportion of CD34⁺CD38^-cells and CD34⁺CD38^-CD45RA^-CD90⁺cells in UM171 group as well as the proportion of CD34⁺cells and CD34⁺CD38^-CD45RA^-CD90⁺cells in UM171+SR1 group was significantly increased?P<0.05?.The expansion folds of CD34⁺cells,CD34⁺CD38^-cells and CD34⁺CD38^-CD45RA^-CD90⁺cells in UM171 group as well as the expansion fold of CD34⁺cells in UM171+SR1 group was significantly increased?P<0.05?.There was no significant difference in the expansion folds of TNCs among four groups?P>0.05?.2.CFU assay showed that the number of CFUs,including CFU-G,CFU-M,CFU-GM,CFU-E and BFU-E,CFU-GEMM,from the same source,in UM171 group,SR1group and UM171+SR1 group did not change significantly compared with control group?P>0.05?.FCM showed that compared with control group,UM171 treatment led an increase in the proportion of CD33⁺?myeloid?cells as well as a decrease in the proportion of CD41⁺?megakaryocyte?cells from UCB-derived HSPCs,SR1 treatment led an increase in the proportion of CD3^-CD56⁺?natural killer?cells from HSPCs from three sources?P<0.05?.3.CD34⁺cells from three sources were cultured for 48h,compared with uncultured group,the expression of CXCR4 on CD34⁺cells,CD34⁺CD38^-cells and CD34⁺CD38^-CD90⁺cells in control group,UM171 group,SR1 group and UM171+SR1 group was significantly increased?P<0.05?.While compared with control group,the expression of CXCR4 on CD34⁺cells,CD34⁺CD38^-cells and CD34⁺CD38^-CD90⁺cells in UM171group,SR1 group and UM171+SR1 group did not change significantly?P>0.05?.4.CD34⁺cells from the patients with poor mobilization and those with satisfactory mobilization were expanded ex vivo for 10d with UM171.There was no significant difference between poor mobilization group and satisfactory mobilization group in the proportion of CD34⁺cells,CD34⁺CD38^-cells and CD34⁺CD38^-CD45RA^-CD90⁺cells as well as the expansion folds of TNCs,CD34⁺cells,CD34⁺CD38^-cells and CD34⁺CD38^-CD45RA^-CD90⁺cells?P>0.05?.5.RNA-seq data showed that compared with control group,1,287 DEGs,including840 up-regulated genes and 447 down-regulated genes,were identified in UM171 group.These up-regulated genes mainly included HSC-,mast cell-,endothelial cell-specific markers and the genes regulating tumor cell proliferation and differentiation,etc.Down-regulated genes were mainly related to megakaryocytes,erythrocytes,chemokine,cell adhesion and transcription factors,etc.GO enrichment analysis revealed that these DEGs mainly regulated cell adhesion molecules activity and chemokines activity,multiple cell surface receptors binding,channels and transporters activity;and regulated non-canonical Wnt signaling pathway,cellular response to reactive oxygen species?ROS?,stem cell proliferation,cell differentiation,and extrinsic apoptotic signaling pathway,as well as tumor necrosis factor?TNF?superfamily cytokine production,and response to transforming growth factor beta?TGF-??.Conclusions1.Both UM171 and SR1 could increase the proportion of HSPCs from UCB,Donor-m PB,Auto-m PB,and UM171 could effectively expand the number of HSPCs from three sources.2.Both UM171 and SR1 could maintain the potential of multi-lineage differentiation of HSPCs from three sources.UM171 could promote the differentiation of UCB-derived HSPCs into myeloid cells and inhibit their differentiation into megakaryocytes.SR1could promote the differentiation of HSPCs from three sources into natural killer cells,suggesting SR1 is expected to be used as an effective small molecule compound to induce natural killer cells production.3.UM171 and SR1 did not affect the expression of CXCR4 on HSPCs,but the expression of CXCR4 on HSPCs from three sources was increased significantly after ex vivo expansion,which could contribute to HSPCs homing and long-term engraftment.4.UM171 could increase the proportion and number of Auto-m PB-derived HSPCs in lymphoma patients with poor mobilization,and the expansion folds of Auto-m PB-derived HSPCs were no significant difference between the lymphoma patients with poor mobilization and those with satisfactory mobilization.For patients who cannot undergo transplantation because of poor or failed mobilization,the source of UM171-expanded HSPCs provides them with the opportunity to receive AHSCT.5.Mechanistically,UM171 could promote Auto-m PB-derived HSPCs in lymphoma patients to expand by suppressing the erythroid and megakaryocytic differentiation,activating non-canonical Wnt signaling,regulation of intracellular ROS levels and the balance between inflammation and anti-inflammatory/detoxification.In addition,the cytokines TGF-?and TNF also play an important role.Moreover,mast cell-and endothelial cell-specific genes identified in our study are well worthy of further study.