The Inhibitory Effect And Mechanism Of Multiple Myeloma Cells Derived Microvesicles On Mscs Osteogenic Differentiation

Objective:Our aim was to observe the shape and immunological phenotype of MM-MV; To explore a better method of isolation human bone marrow stem cells in vitro; To confirm MM-MV messengers of extracellular communication with MSCs.Methods:Transmission electron microscope was used to observe the shape and size of MV; the immunological phenotype was determined by flow cytometry. MSCs were isolated and cultured by using whole bone marrow adherence method. The cell morphology was observed under the inverted phase contrast microscope.The cells cycle and immunological phenotype were examined by flow cytometry. Laser confocal microscopy and flow cytometry were used to confirm the interaction between MM-MV and MSCs.Results:Flow cytometric analyses demonstrated that MVwere positive for CD138which was the surface marker of parent cells.They were heterogeneous, membrane-encapsulated small vesicles ranging in size from0.3to1.0μn as revealed under electron microscopy. The passage MSCs were homogeneous in shape, to be similar to fibroblasts.The passage3cells were postive for CD90, CD13, CD105. CD44, but negtive for CD34, CD45, CD14, and the overwhelming majority of cells were in G0/G1phase. MV were taken up and internalized by MSCs with two hours, we detected CD138surface marker on MSCs after incubation with MV, suggesting transfer of CD138from MV into MSCs because MSCs do not express CD138.Conclusion:.Multiple myeloma cells release CD138-bearing MV. Whole bone marrow adherence method with advantages of simple, cheaper and high performance is a better culture method of MSCs. MV are new mediators of extracellular communication between myeloma cells and MSCs Objective:Our aim of this study was to explore the effect of MM-MV on osteogenic differentiaion of MSCs.Methods:Alkaline phosphatase and alizarin red staining were used to evaluate CFU-Fs and CFU-OB respectively. Cell viability of MSCs in24-72hours cocultures was assessed by MTT method. The number of deaths and apoptotic cells in long-term(2weeks) cocultures has been evaluated by flow cytometry with annexin V-FITC(AV) and propidium iodide (PI) double staining method. The expression of osteoblast-specific genes RUNX2, ALP, OC and collagen I were evaluated with quantitative realtime RT-PCR method at d3, d6after induction. Osteocalcin levels in cocultures were detected using an enzyme-linked immunosorbent assay (ELISA). Alkaline phosphatase activity leves in the cell culture supernatant were detected by colorimetric assay kit.Results:①The culture supernatant and conditioned supernatant of RPMI8226and U266inhibited CFU-F formation after14days, but we found that the effect of conditioned supernatant is weaker than supernatant (P<0.05); The inhibitory effect of KM3culture supernatant,but not conditioned supernatant, on CFU-F formation was also observed(vs control:P<0.05, P>0.05respectively);We found that KM3supernatant, but not conditioned supernatant, significantly inhibited CFU-OB formation after21days of culture(vs control:P<0.01, P>0.05respectively).②50～100ng/mlMV are lack of toxic and apoptotic effect on MSCs.③PMI8226U266, KM3MV inhibited CFU-F formation (vs control:P<0.01, P<0.05, P<0.05respectively), and the inhibitory effect of MV on CFU-OB formation was more pronounced (vs control:P<0.01); The inhibitory effect of RPMI8226MV was much stronger than U266MV. but K562MV had no inhibitory effect on CFU-F, CFU-OB formation.④Expression levels of osteocalcin mRNA were significantly down-regulated at day3and remained down-regulated up to day6of osteogenic differentiation (P<0.01); MM-MV, but not K562MV, inhibited RUNX2mRNA expression at day6after induction (P<0.05); Expression level of ALP mRNA were down-regulated in U266, KM3MV group at day6(P<0.05);Collagen I mRNA was up-regulated in UJ266, KM3MV group at day3((P<0.05),but it was down-regulated at day6and U266MV inhibited its expression(P<0.05).⑤Osteocalcin levels were measured at different time points during osteogenic differentiation. Osteocalcin level was reduced after6days in cocultures with U266MV(P<0.05);At day9.12, MM-MV significantly inhibited osteocalcin levels(P<0.01).⑥We found a significant reduction in the alkaline phosphatase activity after6,12days in cocultures with RPM18226MV(P<0.01);KM3MV reduced alkaline phosphatase activity at day6and remained down-regulated up to day12of osteogenic differentiation(P<0.05);The reduction was also observed at day12in cocultures with U266MV(P<0.05).Conclusion:.MM-MV inhibit MSCs osteogenic differentiation Objective:The aim of this part was to investigate the possible mechanisms of MM-MV inhibitory effect on MSCs osteogenic differentiaion.Methods:Western blot method was used to detect the expression of DKK-1, sFRP2, IL-7in myeloma cells and MV. Alkaline phosphatase staining was used to evaluate whether blocking anti-IL-7Ab can reduce the the inhibitory effect of U266, KM3MV on CFU-F formation.The protein expression of Runx2/Cbfal, β-catenin, dephospho and phospho-catenin were analyzed by western blot. The activity of Runx2/Cbfal was evaluated by an ELISA-based method.Results:①M-MV selectly packed osteoblast inhibitors sFRP2, IL-7. RPMI8226, KM3cells, but not MV, expressed DKK1protein; RPMI8226, KM3MV, but not myeloma cells, expressed sFRP2protein; U266MV"condensely"expressed IL-7protein which is higher than parent cells; But the expression of IL-7in RPMI8226, KM3MV is lower than parent cells,especially in KM3MV;②locking anti-IL-7Ab blunted the inhibitory effect of U266MV on osteoblast differentiation(P<0.05), but it had no effect on KM3MV(P>0.05);③RPMI8226, U266MV inhibited Runx2/Cbfal protein expression and binding activity;④We found that MM-MV at low concentration significantly increased inactive phosphorylated β-catenin cytosolic levels in MSCs; Consistantly with sFRP2expression levels in MV, the effect of RPMI8226, KM3MV was much stronger compared with U266MV.Conclusion: MV inhibitory effect on MSCs osteogenic differentiaion depending on their contents. RPMI8226MV pack sFRP2and IL-7,which block Wnt/β-catenin, RUNX2/Cbfal dual pathways;U266MV mainly express IL-7inhibiting RUNX2/Cbfal activity; KM3MV inhibit osteoblast differentiation through sFRP2involved in Wnt pathway; Consistantly, the inhibitory effect of RPMI8226MV is much stronger compared with U266, KM3MV.