Study On Therapy Of Multiple Sclerosis Using Human Bone Marrow Stromal Cells, Gingko Biloba Extract And Electroacupuncture

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system. The pathological hallmark of MS is the sequential development of demyelination. There are many symptoms related to MS such as paralysis and loss vision. The MS is characterized by affecting young adults, having long course and relapsing-remitting. Till now there is no reliable means for therapy of MS.Since the mechanisms of myelin breakdown are still unknown, immunosuppression and hormones are commonly applied to MS patients. However, there are possible long term side effects from hormone replacement therapy. Accordingly,to explore the pathogenesis of MS and to successfully treat MS are required.In present study, immunohistochemistry, molecular biology, electron microscopy and FACS assay were performed. Firstly, we investigated whether human bone marrow stromal cells (hBMSCs) can be induced to become oligodendrocytes. Secondly, we studied the effect of ginkgo biloba extract (GBE) on plptg/- mice, an animal model for late onset chronic demyelination disease. Lastly, we explored the mechanism of electroacupuncture treatment of MS.1. Differentiation of Human Bone Marrow Stromal Cells into Oligodendrocyte-Like Cells through Activation of F3/Notch SignalingMethods: hBMSCs were purified through a cell adhesion approach and preinduced forβ-ME (24h) and RA (3d)). Cells were randomly divided into two groups treated with either multiple factors (MFS) or F3 another 3 days. The MFS group (positive control) was cultured in DMEM medium contained 5mM forskolin, 10ng/ml bFGF, 5ng/ml PDGF, and 200ng/ml HRG. The F3 group was cultured in DMEM medium contained 10%FBS and 20nM F3. FACS assay, immunofluorescence and Luciferase reporter assay were performed to characterize the nature of hBMSCs under different culture conditions. Subsequently eight-week-old mice were injected with the predifferentiated cells induced by RA/β-ME, MFS/RA/β-ME, or F3/RA/β-ME. Tow months after intravitreous injection, animals with transplanted cells were analysed by immunostaning and electron microscopy.Results: CD90 (+) cells were isolated from hBMSCs through a cell adhesion approach and immunostaining indicated that 40.88% cells expressed Sox2, an early stage stem cells marker. After treatment with RA plusβ-ME, CD 90 (+) hBMSCs were changed into oligodendrocyte-like cells (OLLCs) expressing more NG2 (54.43%), an early oligodendrocytes marker. After further treatment with F3 or MFS the cells expressed O4, a mature OLs marker. The population of O4 (+) cells (31.55+5.8% for F3 and 32.61+6.8% for MFS) significantly increased under both culture conditions, there was no significant difference between F3 group and MFS group. The expression of F3/Notch signalling component Notch1/NICD and DTX1 at all stages of induced differentiation was detected using antibodies, and immunofluorescence analysis showed that the intensity of Notch1/NICD and DTX1 in nucleus was increased after treatment by F3. Luciferase reporter assays showed that the relative reporter activity of Hes1 was significantly inhibited by F3 (0.33±0.08; p<0.01), but not by MFS (0.9±0.13). Notably the F3, but not RA and MFS, -induced transactivation of Hes1 was inhibited by over-expressed DTX1 and activated by a deletion mutant of Flag-tagged DTX1 mutant (dnDTX1). These observations demonstrated that F3/Notch signalling pathway may play a role in promoting maturation of immature OLLCs, derived from CD90 (+) hBMSCs, thought inhibiting classical Notch/Delta/Hes1 signaling pathway. Two months after transplantation of pre-differentiated cells, O4 (+) OLLCs send out more branches and processes (p<0.01) in mouse retina, as compared to in vitro. Interestingly, the number of processes per branch from the OLLCs predifferentiated by F3 (1.7+0.13), as well as by MFS (1.8+0.1), was significantly increased as compared to the RA group (1.13+0.13; p<0.001). To investigate this further, whole mounted retinas of F3 group were prepared for double immunocytochemistry using antibodies O4 andβ-tublin III. O4 (+) cells sent out their processes which landed onto the surface of retinal ganglion cell, and formed axoglial contacts and single myelin sheaths in retina.Conclusion: In the present study, we have demonstrated that hBMSCs have the capability to trans-differentiate into cells with oligodendrocytic characteristics. F3 triggers F3/Notch signaling in the CD90 (+) hBMSCs and induces them to differentiate into OLLCs with a high degree of morphological and functional maturation.2. Effects of ginkgo biloba extract on plp transgenic miceMethods: Three-month-old plp transgenic mice (plptg/- mice) were devided into PLP group (control group) and GBE group (experimental group). The GBE group received daily injection of 70mg/kg GBE, as well as the same volume of saline was injected into PLP group. Two months after injection, animal were sacrificed and histopathological changes were analyzed against untreated plptg/- mice. We also observed the influence of GBE on OLN cells (oligodendrocyte precursor cell lines) using MTT analysis and immunofluorescence.Results: Two months after treatment, the survival rate of GBE group was 76%, an increase of 23% as compared to PLP group. Behavioral test showed GBE group displayed longer latency interactions (33.0±4.36 sec). Double staining for MBP andβ-tublinin in optic nerve indicated that the expression level of MBP was decreased and some axons were naked in PLP group. However high expression of MBP was detected in GBE group and MBP (+) myelin was found along the surface of axons, which indicated well myelination in GBE group. We then analyzed the sagittal brain section in fimbria of hippocampus and found that the numbers of both OX-42 (+) microglia and CD4+T in GBE group were decreased as compared to PLP group. However the number of GFAP positive astrocytes was no different between GBE group and PLP group. To further confirm the role of GBE, OLN cells were cultured in DMEM medium containing GBE for 24h. MTT analysis indicated that the viability of OLN cells was increased in a dose-depend manner from GBE concentration of 0.07 mg/ml to 0.138mg/ml. GBE concentrations of 0.175mg/ml, 0.28 mg/ml and 0.35mg/ml promote increases in OD values by 0.091, 0.107 and 0.138 respectively. After treating OLN cells with 0.28mg/ml GBE for 24h, 48h and 72h, NG2 immunostaining found the OLN cells still were dipolar at all time points, absent the cells with multi-processing.Conclusion: Based on these observations we demonstrate that GBE can block demyelination process in plptg/- mice by inhibiting cytotoxin, which is released from activated microglia and T cell, suggesting that GBE has potential to treat MS in future.3. Effects of electroacupuncture stimulation-derived serum on microglia activity in vitroMethod: Baihui, Fengfu acupuncture points on the rats were stimulated with 100HZ high frequency electroacupuncture. Two weeks later blood was drawn, and serum was collected from the stimulated rats. Subsequently we divided the microglia into three groups: normal group cultured with normal rat serum; control group added with normal rat serum and microglia activator LPS ( 1μg/ml); and the experimental group added with the electroacupuncture stimulation-derived serum and LPS(1μg/ml). Immunostaining, MTT assay and Griess assay were used to detect the morphology, cell viability and NO release of microglia at 8h, 12h, 24h and 48h.Results: After treatment with 1μg/ml LPS, microglia was activated, which was indicated by the increased soma and the up regulation level of OX-42. Meanwhile Griess assay showed that the level of NO was higher in control group than in normal group at all time points, especially at 48h when the level of NO peaked to 16μM. However, MTT assay indicated that there was no change in cell number. After treatment with LPS for 12h, the viability of microglia in experimental group was less than that of control group. NO released from experimental group was significantly lower than that released from control group at 12h, 24h and 48h (p<0.05). NO release was also dose-dependent.Conclusion: In present study we indicate that the mechanisms of electroacupuncture protection of oligodendrocytes may be through inhibiting the proliferation of microglia and NO release from activated microglia.Conclusion:1. CD90 (+) hBMSCs can be trans-differentiated into OLLCs after treatment with F3/RA/β-ME. F3/Notch signaling promotes OLLCs maturation both in vitro and in vivo. 2. After transplantation, predifferentiated OLLCs have the capacity to become further mature OLLCs and send out processes, forming one layer of myelin in retina.3. GBE can promote plptg/- mice survival and improve behavior.4. GBE prevents plptg/- mice demyelination through inhibition of microglia activity and T cell infiltration.5. GBE promotes the proliferation of OLN cells, but cannot induce them to differentiate into oligodendrocytes.6. Electroacupuncture stimulation-derived serum inhibits the activity and the proliferation of microglia. Electroacupuncture stimulation-derived serum also reduces the level of NO released from activated microglia.At present, we have developed an approach to selective induce hBMSCs differentiation into OLLCs by F3/RA/β-ME, this may offer new strategies for cell therapies in MS. We also studied the effect of GBE on plp transgenic mice, and suggesting that GBE has potential to treat MS in future.