Exosomes From Human Umbilical Cord Mesenchymal Stromal Cells Promote Proliferation And Inhibit Apoptosis Of Acute Leukemia Cells In Vitro

Background:The pathogenesis of acute leukemia(AL)is still unclear.Mesenchymal stem cells(MSCs),as important regulatory factors in the bone marrow microenvironment,participate in the transformation from normal hematopoietic stem cell niche to malignant leukemia microenvironment.Some studies have shown that mesenchymal stem cells can inhibit apoptosis by secreting exosomes(Exos),thus achieving the purpose of tumor protection.The effect of exosomes derived from mesenchymal stem cells on the biological characteristics of leukemia cells is still controversial.Objective:In this study we aim to investigate whether human Umbilical Cord Mesenchymal Stem Cells have any effects on the proliferation and apoptosis of AL cells,and try to find out its possible mechanism.Methods:1.Human umbilical cord mesenchymal stem cells were extracted by tissue adherent method,and their morphology,specific surface markers,osteogenic and adipogenic differentiation abilities were identified.2.Exosomes derived from human umbilical cord mesenchymal stem cells were extracted by ultracentrifugation.The morphology of exosomes was identified by transmission electron microscope and nano-particle tracking analysis.Specific surface markers of exosomes were identified by Western Blot.3.The exosomes were co-cultured with acute leukemia cells,and the effects of hUC-MSC-Exos on the proliferation of Jurakt cell line and HL-60 cell line of acute lymphoblastic leukemia with different concentrations and different treatment time were detected by CCK8 coloriometry.4.Annexin V-FITC/PI detection hUC-MSC-Exos biology effects on leukemia cell apoptosis;5.Western blot was used to detect the expression levels of apoptosis-related proteins(Bcl-2,Caspase3)in leukemia cells before and after treatment with exosome.Results:1.Under a light-inverted microscope,the hUC-MSCs were spindle-like or polygonal,arranged in a whirlpool-like shape.Six-color flow cytometry analysis exhibited that almost all the hUC-MSCs were positive for classical MSC surface markers CD73,CD90,CD105 and negative for hematopoietic markers CD34,CD45,HLA-DR.The results of Alizarin Red S and Oil Red O staining indicated that hUC-MSCs had the potential for adipogenic and osteogenic differentiation.2.Transmission electron microscopy identified a cup-shaped structure of vesicles ultracentrifuged from culture media of hUC-MSCs.Nanoparticle tracking analysis revealed that most vesicles had a diameter of approximately 100 to 160?nm,both of which accord with typical morphologic characteristics of exosomes.What’s more,Western blotting demonstrated that vesicles expressed common exosome protein markers,including CD9 and CD63;however,Calnexin was not detected.All of these results confirmed that we had successfully isolated hUC-MSC-Exos from the cultured medium.3.We labelled hUC-MSC-Exos with green fluorescent cell linker PKH67 and then cocultured PHK67-labelled hUC-MSC-Exos with HL-60 cells for 24 hours.PKH67-labelled exosomes exhibited green fluorescence in the cytoplasm of HL-60 cells.The results showed that exosomes were taken up and transported into the cytoplasm of HL-60 cells.4.Compared with the control group,different concentrations of hUC-MSC-Exos showed different degrees of proliferation promoting effect on h L-60 cell line and Jurkat cell line,and the proliferation promoting effect on HL-60 cell line was more obvious.5.After treatment with Jurkat and hl-60 cells for 72 h,hUC-MSC-Exos at the concentration of 400ug/ml and 200ug/ml showed inhibitory effect on the apoptosis of leukemia cells,and the inhibitory effect on hl-60 cells was more obvious.6.Western blot analysis of apoptosis-related proteins in Jurkat cells before and after exosome treatment showed that caspase3 protein expression was down-regulated and Bc12 expression was up-regulated in HL-60 cultured for exosome group compared with HL-60 alone groupConclusion:These results showed that hUC-MSC-Exos had the potential of promoting proliferation and inhibiting apoptosis of acute leukemia cells in vitro,suggested that interference with exosome production or internalization may be a new entry point for stem cell therapy in acute leukemia.