Bilogical Characters And Hematopoietic Support Of Bone Marrow Derived Mesenchymal Stem Cell (MSC)

Objective:To study the biological character, karyotype and cytokineexpression of bone marrow-derived mesenchymal stem cells (MSC) inpatients with systemic lupus erythematosus (SLE) and hematopoieticsupport of MSC, as well as to explore the role of these abnormalities in theoccurrence and development of SLE.Methods: MSC were isolated from bone marrow of both 11 SLEpatients and 6 healthy controls by density centrifugation and adhesiveculture in vitro. Briefly, mononuclear cells were isolated by 1.077 g/L Ficoll.The cells were plated in L-DMEM containing 10ï¼…fetal bovine serum andincubated in a humidified 5ï¼…CO₂ atmosphere at 37℃. Non-adherent cellswere removed and media was replaced 48 hours after initial plating andthen every third days thereafter. When the confluence of the fibroblasticcells became 90ï¼…, these cells were trypsinized with 0.25ï¼…trypsin andreplated. The morphological changes of MSC were observed in primary andpassage cultures. The surface markers were detected by flow cytometry. Thegrowth curves were assayed. The karyotype of MSC was detected byblocking cellular fission with colchicines. Total RNA was extracted fromMSC in two groups and semi-quantitative RT-PCR was used to detect themRNA expressions of IL-6, IL-7, IL-11, stem cell factor (SCF) andmacrophage colony stimulating factor (M-CSF). The MSC from SLEpatients and healthy controls were infused to ICR mice after high-dosechemotherapy. The changes of peripheral blood counts of the mice werecounted. Results were shown on the mean±standard deviation. Anunpaired t-test was used to compare the values and P＜0.05 was consideredas statistically significant.Results: After 3-12 days of primary culture, MSC in two groups adheredto the plastic surface presented a small population of single cells with aspindle shape. On days 8-37 after initial plating, the cells looked like long spindle-shaped fibroblastic cells, and began to form colonies and becameconfluent. Morphologically, MSC in SLE appeared very similar to normalcontrols. The primary passage-time in SLE group is 26±7.76 days comparedwith 17±5.64 days in normal group (t=2.507, P=0.029). Meanwhile thegrowth curves showed that the growth of MSC in SLE was slower thanwhich in normal group (P＜0.01). MSC in SLE and normal group werepositive for CD29, CD44 and CD105, and negative for CD14, CD34, CD45and HLA-DR. Karyotype of MSC was normal in SLE. MSC in both groupsexpress IL-6, IL-7, IL-11, M-CSF and SCF at mRNA level. IL-6 and IL-7were down-regulated in MSC from SLE patient (P＜0.01). MSC infusions ofboth groups were accompanied by no adverse events and the recovery ofwhite blood cell, red blood cell, hemoglobin and platelet was more rapidthan the controls (P＜0.05).Conclusion:Method isolating and expanding of MSC from patients withSLE was established. MSC from SLE behaved the abnormalities inexpansion in vitro. MSC from SLE have a normal karyotype. The cytokineexpression was abnormal in MSC from SLE and may play an important rolein the disease pathogenesis. Ex vivo MSC infusions from SLE similar tonormal MSC could support hematopoiesis. Objective: To explore the biological characters and to compare thedifferent regulations of bone marrow mesenchymal stem cells (MSC)derived from both lupus (MRL/lpr) and normal (C57BL/6) mice on Tlymphocyte in vitro.Methods: MSC from MRL/lpr and C57BL/6 mice bone marrow wereisolated and expanded. The surface phenotypes were detected by flowcytometry (FCM). The morphological changes of MSC were observed inprimary and passage cultures. The growth curves were assayed. CD3⁺ Tlymphocytes from spleen of C57BL/6 mice were isolated by nylon woolcolumns and stimulated by ConA and co-cultured with or without the twostrains of MSC or supernatant for 72h.The proliferation of T cells stainingby CFSE and activation of T cells were detected. The apoptosis wasassessed by flow cytometry using Annexin V/PI.Results: The growth of lupus MSC is quicker than normal controls(P＜0.05). Both groups were positive for CD106 and CD105, and negativefor CD34 and CD45.Both of two MSC can inhibit the proliferation of Tcells whereas supernatant can not. The expression of CD69 and CD25 onCD3⁺ T cells stimulated by ConA was lower when co-cultured with twoMSC or supematant than that in control group (P＜0.05). A decrease of Tcell apoptosis was seen when MSC or supernatant of MRL/lpr or C57BL/6were added (P＜0.05). No difference was seen between two groups.Conclusion: MSC from lupus mice behaved abnormalities inexpansion in vitro. There is no apparent defection in someimmunoregulatory functions of lupus MSC on T cells, such as proliferation,activation, apoptosis, when compared with normal controls. Objective: To investigate whether the immunoregulatory effects ofbone marrow derived mesenchymal stem cells(MSC) in systemic lupuserythematosus (SLE) on B lymphocytes is abnormal.Methods: Human bone marrow MSC were isolated from SLE patientsand normal controls and expanded ex vivo. B lymphocytes were purifiedfrom normal peripheral blood mononuclear cells by using CD19microbeads. Purified B cells were cocultured in 96-well flat-bottom plateswith MSC.B cell apoptosis and surface markers were analyzed by flowcytometry. B cell proliferation was assessed by [³H]-thymidineincorporation. ELISA was used to measure supernatant levels of IgG, IgMand IgA.Results:â‘ B cell proliferation in normal MSC coculture group(4306.8±307.9 cpm ) was significantly inhibited compared with simple Bcell group (7058.7±346.1) (P＜0.05);â‘¡B cell apoptotic percentage innormal MSC coculture group( 13.5％±6.7ï¼…)was lower than in simple B cellgroup (24.8％±8.0ï¼…) (P＜0.05);â‘¢The percentage of CD86 moleculeexpression on B cells in normal MSC coculture group (52.6％±5.1ï¼…) waslower than in simple B cell group (66.2％±10.2ï¼…) (P＜0.05);â‘£The levelsof IgG, IgM and IgA secreted by B cell decreased when cocultured withMSC (P＜0.05);⑤There were no significant differences of B cellproliferation, apoptosis, CD86 expression and immunoglobulin productionregulated by MSC from lupus patients and normal controls.Conclusion: MSC shows an inhibitory effects on B cells.Both SLEpatient MSC and normal MSC have similar regulations to B cellproliferation, activation and immunoglobulin production. Objective: To investigate the therapeutic effects and mechanism ofhuman bone marrow mesenchymal stem cells(MSC) transplantation inMRL/lpr mice.Methods: Human MSC were expanded ex vivo. MRL/lpr mice weredivided into cyclophosphamide (CTX) group, MSC group, CTX+MSCgroup, and control group. At the age of 16 weeks, the mice in CTX groupand MSC+CTX group received intraperitoneal injection of CTX100mg/kg×2d. 24 hours after injection, the MSC were transplanted in MSCgroup and CTX+MSC group through tail vein. The 24-hour proteinuria wasdetected with Coomassi brilliant blue method. Indirect immunofluorescencewas used to detect the antinuclear antibody (ANA) and anti-double-strandedDNA (ds-DNA) antibody. Flow cytometry was used to detect the changes ofCD4⁺ T cells and Th1/Th2 subpopulation. The expression of complementC3 of the murine kidney was estimated by immunofluorescence.Results:â‘ At 24, 28, 32 weeks, the 24 hours proteinuria in MSC group(4.3±0.8mg, 5.4±0.8mg, 5.8±0.8mg) and MSC+CTX group (2.8±1.0mg,3.9±0.4mg, 4.5±1.0mg) was significantly decreased than in control group(9.0±1.3mg, 9.3±0.6mg, 10.0±1.6mg) (P＜0.05), and it was also significantly decreased in MSC+CTX group (2.8±1.0mg, 3.9±0.4mg,4.5±1.0mg) than in CTX group (5.1±1.0mg, 7.7±1.1mg, 9.6±1.9mg)(P＜0.05).â‘¡At 28 weeks, the anti ds-DNA antibodies were significantlydecreased in MSC+CTX group (2.7±0.8) than in control group (4.6±0.9)(P＜0.05).â‘¢At 32 weeks, the body weight of MSC group (38.6±3.3g) andMSC+CTX (35.8±3.7g) group increased significantly than the controlgroup (32.8±3.0g) (P＜0.05).â‘£At 32 weeks, serum creatinine decreasedsignificantly in MSC group (13.5±2.2μmol/L) and MSC+CTX(12.7±4.2μmol/L) group than in control group (20.8±5.1μmol/L) (P＜0.05).⑤The number of CD4⁺ T cells and Th2 subpopulation decreasedsignificantly in MSC group (76.4％±1.9ï¼…, 58.4％±1.8ï¼…) and MSC+CTXgroup (73.9％±3.2ï¼…, 57.6％±3.2ï¼…) than in control group (82.7％±2.9ï¼…,64.2％±2.8ï¼…) (P＜0.05).Conclusions: Human MSC transplantation is effective treatment forMRL/lpr mice. The regulation of Th1/Th2 balance and inhibition theantibodies of B cells, mediated by human MSC may be the mechanisms ofeffective treatment with MSC transplantation.