| Objective: To investigate the prevention and treatment for multiple sclerosis by mouse embryonic stem cell-derived thymic epithelial progenitors expressing mylin oligodendrocyte glycoprotein and the mechanism of inducing antigen-specific tolerance.Methods: 1) Using gene clone and transfection technology, we constructed recombinant plasmid p EF1α-IRES-Ac GFP with or without MOG to transfect TC-1 m ESCs, and then the cell line MOG/m ESCs expressed MOG stably was screened by 200μg/ml of G418. We demonstrated the characateristic of MOG/m ESCs by AP-staining, immunofluorescence staining, RT-PCR, western blot technology.2) MOG/m ESCs were induced and differentiated into MOG/TEPs by combination of 50ng/ml of EGF, 20ng/ml of KGF, FGF10 and BMP4, respectively. The number and ratio of Ep CAM+K5+K8+ were analized by FACS and m RNA quantity of Pax1、Pax9、Plet1、Fox N1 and Hox A3 that all express on TEPs was detected by quantitative Real-Time PCR.3) We tested the quantity and ratio of MOG, GFP, K5 or K8 in thymii on 2nd, 30 th and 90 th respectively by western blot, immunofluorescence and FACS after MOG/TEPs were intrathymic injected into 129S6 Sv Ev Tac mice. Intrathymic injection using p EF1a/TEPs、MOG/PEI and p EF1a/ PEI is as control.4) Induction and assessment of EAE. A total of 500 mg or 300 mg of mouse encephalitogenic peptide MOG35-55 in 100 ml of PBS was emμlsified in 100 ml of complete Freud’s adjuvant supplemented with 100 ml of 400 mg Mycobacterium tubercμlosis H37 Ra. 129S6 Sv Ev Tac mice were injected s.c. with a 500 mg of MOG35-55 in 200 ml of CFA at 4 points in the dorsal flank on days 0 and 7, and B6 mice were injected with 300 mg of MOG35-55 in 200 ml of CFA on day 0 after B6 mice that had received 1000 c Gy total body irradiation from a 137 Cs and T cell-depleted(TCD)-BM cells were injected intravenously(i.v.). In addition, both 129S6 Sv Ev Tac and B6 mice were injected intraperitoneally(i.p.) with 500 ng of purified Bordetella pertussis toxin on days 0 and 2. The mice were observed for clinical scores up to 42 days.5) Histopathologic changes including inflammatory infiltration, demyelinationand axon loss from spinal cords were observed by HE, Luxol Fast Blue and Bielschowski silver immersion.6) The numbers of total T cells, CD4-CD8-, CD4+CD8+, CD4+ or CD8+, TECs(CD45-Ep CAM1+), c TECs(CD45-Ep CAM1+ Ly51+), m TECs(CD45-Ep CAM1+ Ly51-) were analyzed by FACS.7) Splenocyte proliferation assays. Splenocytes were cμltured in 96-well plates, and stimμlated with 2.5, 5 or 10 mg/ml of MOG35-55 peptide or 5 mg/ml of plate-bound anti-CD3 and anti-CD28 antibodies at 37°C. Cell proliferation was analyzed by Brd U Labeling and Detection Kit III according to the manufacturer’s instructions. Absorbance(measured as OD), proportional to Brd U uptake, was measured at 405 nm using an ELISA microplate reader. The stimμlation index was calcμlated as the ratio of the OD from stimμlated cells over the OD from unstimμlated cells.8) Cytokine production detection. Splenocytes were stimμlated with MOG35-55 in vitro for 3 days. Samples of supernatant were collected and measured for cytokine content using the Cytometric Bead Array(CBA)-Mouse Th1/Th2 cytokine kit for IL-4, IL-5, IFN-r and TNF. IL-10 content was measured by the CBA Mouse IL-10 Flex Set and Mouse/Rat Soluble Protein Master Buffer Kit, and IL-17 A content by the CBA Mouse IL-17 A Enhanced Sensitivity Flex Set and Mouse Enhanced Sensitivity Master Buffer Kit. These assays were performed according to the manufacturer’s instructions and analyzed using a FACScalibur flow cytometer and FCAP Array software.9) MOG-specific antibody detection. Anti-MOG special antibody detection in serum samples was done by ELISA.10) Transplantation of MOG/TEPs resulted in long-term remission in an established model of EAE. C57BL/6 mice were immunized with MOG35-55 to induce EAE. Fifteen days later, the mice were lethally irradiated, injected i.v. with TCD-BM from CD45.1-B6 mice, and i.t. with MOG/TEPs, MOG/Ep CAM- cells, or PBS. The clinical benefits of MOG/TEPs were investigated further by assessing the clinical scores after being rechallenged with the MOG35-55 on day 44. The mice were observed for clinical scores day by day.Results: 1) Both MOG/ m ESCs and p EF1a/m ESCs expressed GFP, confirming that they were stably transfected with MOG-p EF1a-IRES-Ac GFP vector with or without MOG. Both MOG/m ESCs and p EF1a/m ESCs were positive for alkaline phosphatase(AP) activity and expressed the pluripotent markers Oct4 and SSEA1, indicating the m ESCs were in an undifferentiated state.2) After differentiation, MOG/m ESC- and p EF1a/m ESC-derived cells contained Ep CAM positive cells that co-expressed K5 and K8, a phenotype of TEPs. Furthermore, purified m ESC-TEPs(Ep CAM+ cells) expressed significantly higher levels of the TEP-related genes Hoxa3, Fox N1, Plet1, Pax1 and Pax9 than purified m ESC-Ep CAM- cells.3) MOG protein coμld be expressed over time in the thymi after MOG/m ESC-derived TEPs(MOG/TEPs) were transplanted into mice. 33-78% of CD45-Ep CAM+TECs were GFP+ donor-derived cells. GFP+K5+, GFP+K8+ and GFP+K5+K8+ cells were detected. After transplantation of MOG/TEPs, the number of total thymocyte and thymocyte subsets increased obviously.4) Transplantation of MOG/TEPs prevents EAE development in 129S6 Sv Ev Tac and C57BL/6 mice. Mice that received MOG/TEPs showed a significant reduction in the mean clinical scores and the number of mice with a disease-free condition was increased in the MOG/TEP-transplanted group. There were fewer inflammatory cell infiltrates and significantly less axonal loss in MOG/TEP-treated mice. MOG/TEP transplantation also restricted the devel- opment of demyelination. The histological scores were significantly reduced in the MOG/TEP-treated mice, as compared to those in the control groups.5) Splenocytes from control-(p EF1a/TEPs, MOG/Ep CAM- cells, MOG/PEI, or PBS)-treated mice proliferated robustly in response to p MOG35-55 stimμlation in a dose- dependent manner, whereas splenocytes from MOG/TEP-treated mice did not. In contrast, splenocytes from all animal groups were able to mount an immune response to TCR-mediated signals(anti-CD3/CD28 antibodies).6) Mice that received MOG/TEPs showed the number of Tregs(CD4+CD25+Fox P3+) increased robustly.7) Analysis of proinflammatory cytokines in MOG/TEP-treated mice. The content of Th1 cytokines IFNr and TNF, as well as Th17 cytokine was significantly reduced by the splenocytes from the MOG/TEP-transplanted mice as compared to the control mice. We also analyzed anti- MOG autoantibody and found that there was no significant differ- ence among the groups that had been injected with MOG/TEPs, p EF1a/TEPs, MOG/Ep CAM- cells, MOG/PEI, or PBS, followed by EAE induction by MOG.8) Transplantation of MOG/TEPs resμlts in long-term remission in an established model of EAE. a significant remission in clinical signs and histopathologic changes was observed in mice that had undergone HSCT in combination with MOG/TEPs, MOG/Ep CAM-cells, or PBS.Conclusion: In this study, we show that i.t. injection of m ESC-TEPs expressing autoantigen MOG in mice resμlts in generation of the m ESC-derived TECs that support T cell regeneration, long- lasting MOG expression in the thymus, induction of MOG antigen-specific tolerance, prevention of EAE development, and long-term remission of established EAE when combined with HSCT. |
PDF Full Text Request