The Biological Character And Clinical Significance Of Bone Marrow Mesenchymal Stem Cells In Multiple Myeloma

Background and ObjectivesMultiple myeloma(MM)is a plasma cell neoplasm characterized by the bone lesions,anemia,renal failure and immunosuppression caused by the clonal expansion of plasma cells within the bone marrow.MM is the second most frequent hematologic malignancy,with a median age of 68 years at diangosis.With the increase in an aging population,the incidence of MM will further increase in China.Although application of novel anti-myeloma drugs targeting on the bone marrow microenvironment components such as bortezomib and lenalidomide have significantly improved the the overall survival of myeloma,MM remains incurable.Therefore,it is extremely necessary to further study the mechanisms of MM pathogenesis,especially its relationship with the bone marrow microenvironment in order to find novel therapeutic targets for the disease.Previous research findings showed that the pathogenesis of MM was not only correlated with the genomic alterations of plasma cells,but also with the bone marrow microenvironment which contributes to the development and drug resistance of the disease.The bone marrow derived mesenchymal stem cells(BMMSCs)are one of the important cell types localized in the MM nich,which can differentiate to adipocytes,osteoblasts and chondrocytes.The role of BMMSCs in the pathophysiology of MM has appeared to be an area of great interest.The BMMSCs not only play an important role in the drug resistance,growth and survival of MM,but also in the bone destructions.Previous studies showed that the BMMSCs derived from multiple myeloma patients(MM-BMMSCs)were significantly distinct from the normal controls.For example,MM-BMMSCs' activity in supporting myeloma growth is increased,but their capacity to differentiate into osteoblasts is impaired.Moreover,the MM-BMMSCs contained alterations in the gene expression profiling,which might participate in the pathogenesis of MM.Telomeres consist of repetitive DNA sequence and associated proteins in the ends of chromosomes and protect chromosomes from injuries.The loss of telomeres is associated with aging and tumor development in cells.It has reported that telomere damage affects the function and gene expression of mesenchymal stem cells(MSCs).But the telomere length of MM-BMMSCs and whether the telomere length is associated with the gene expression of MM-BMMSCs has never been reported in the literature.Moreover,there are rare reports on the relationship between the biologic characteristics of MM-BMMSCs and clinical features of the patients.What is more,there are still divergences in the biologic characteristics of MM-BMMSCs containing osteogenic differentiation,senescence,immune regulation and gene expression,due to the difference in the study population and the extraction and culture system of BM MSCs between groups.Therefore,the biologic characteristics of MM-BMMSCs need further studies in order to provide the theory evidence for the pathogenesis of MM and the new strategy for the management of MM.The BMMSCs from control group and MM were obtained by adherent culture method in this study,and they were confirmed by test of immunophenotype and differential ability.The difference in the immunophenotype,gene expression,cell cycle and telomere length was evaluated between the two groups.The regulation effect of the myeloma associated condition medium(MCCM)on the biological character of MM-BMMSCs was also studied.Moreover,the relationship between the gene expression or telomere length of MM-BMMSCs and the patient's clinical features was also investigated.Materials and MethodsThe culture and identification of the BMMSCsBone morrow samples from patients and control group were obtained by ilium puncture.The adherent culture method was used to obtain BMMSCs.Then the BMMSCs was confirmed by flow cytometry analysis,osteogenic differentiation and morhpology.Morphological character and proliferative capacity of BMMSCs from the two groups were compared.Cell cycleThe cell cycle of BMMSCs from the two groups was valued by flow cytometry.Gene expressionThe expression of interleukin-6(IL-6),macrophage inflammatory protein-1 alpha(MIP-la),indoleamine 2,3-dioxygenase(IDO),Dickkopf-1(DKK1),receptor activator of nuclear factor-kappaB ligand(RANKL),transforming growth factor-beta(TGF-?),and interleukin-10(IL-10)in BMMSCs from patients and control was detected using real-time PCR.Telomere lengthThe telomere length of BMMSCs from 12 MM and 8 controls was valued by Real-time PCR.Flow-Fish was also used to measure the telomere length of BMMSCs from age-matched patients and controls.The effect of the myeloma associated condition medium on the gene expression and cell cycle of MM-BMMSCs.The BMMSCs from three MM patients were cultured with the MCCM for 24 hours.Then the gene expression and cell cycle of BMMSCs were detected by Real-time PCR or Flow Cytometry,respectively.ResultsCompared with the primary BMMSCs from the control groups,the primary MM-BMMSCs grew more slowly but had normal morphology.The passaged BMMSCs from both groups had no difference in the morphology and the rate of proliferation.Flow Cytometry showed that BMMSCs both from MM and the control groups had the same immunophenotype,with the expression of CD73,CD90 and CD 105 but rarely expression of CD34,CD45 and HLA-G.Cell cycle anylysis displayed that MM-BMMSCs had more cells in G0/G1 phase,but less in phase of S and G2/M,compared with those from control groups.The BMMSCs from myeloma patients and control groups had no difference in the expression of DKK1,RANKL,TGF-? and IL-10.The former showed higher expression of IL-6 and MIP-1?,but lower expression of IDO.The telomere length of MM-BMMSCs(0.98 ± 0.11)was significantly longer than that of contorl BMMSCs(0.98 ± 0.11)measured by Real-time PCR.Flow-Fish confirmed that the telomere length of MM-BMMSCs was longer compared with that of control BMMSCs derived from three age-matched patients and controls.The hTERT mRNA expression were undetable in BMMSCs from both groups.The expression of IL-6 and MIP-1? in the MM-BMMSCs was further increased but the expression of IDO was further decreased when cultured with the MCCM for 24 hours.Additionally,percentage of the MM-BMMSCs in G0/G1 phase was increased and that in S phase was significantly reduced.Interestingly,our results also showed that telomere length was positively associated with the expression of IL-6 and MIP-la at mRNA level in MM-BMMSCs.Furthermore,after comparing telomere length with several selected clinical parameters,we found that the telomere length of BMMSCs from MM patients with bone lesions was much shorter than that of the ones from the patients without bone lesions.However,our results did not reveal an association between the expression of IL-6 and MIP-la in MM-BMMSCs and the patients'clinical characteristics including bone lesion severity,disease stage,serum ?2 microglobulin(?2-MG)concentration,or the percentage of plasma cells in bone morrow,respectively.ConlusionBMMSCs from MM patients had the similar growth characteristics,immunophenotype compared with those from control groups.MM-BMMSCs had more cells in G0/G1 phase,much longer telomere and upregulation of IL-6 and MIP-1? but downregulation of IDO.The number of BMMSCs in G0/G1 and the increase in the expression of IL-6 and MIP-la or the decrease in the expression of IDO by BMMSCs were further induced by the MCCM,which indicated that myeloma cells may influence the function of BMMSCs through paracrine style.Correlation analysis showed that the telomere length was positively associated with the expression of IL-6 and MIP-la at mRNA level in BMMSCs of MM.Moreover,the telomere length of BMMSCs from patients with bone lesions was much shorter than that from patients without bone lesions.Our result indicated that BMMSCs might be involved in pathothegenesis of MM and MM related bone diseases.