The Biologic Characteristics Of Mesenchymal Stem Cells Derived From Mouse Thyroid

Objective Isolation mesenchymal stem cells(MSCs) from mouse thyroid and comparing its biological characteristics to mouse bone derived MSCs; this will be laid a theoretical and experimental foundation for the mechanism of auto-immune thyroiditis.Methods1. Isolation and culture of MSCs from murine thyroid by adherent culture method,and then comparing cell characterization with murine bone derived MSCs. The Morphology of adherent cells was observed by inverted microscope and the cell growth curve was analyzed by CCK8 method. The immunophenotype and cell cycle of the murine thyroid adherent cells was determined by flow cytometry and the induction ability was determined by different induction reagents. The adipogenic differentiation potential ability of murine thyroid adherent cells was assessed by oil-red-O staining, and osteogenic differentiation identified by immunocytochemical stain with alkaline phosphatase. To assess mineralization, deposit calcium in cultures was stained with silver nitrate(Sigma) by the method of Von Kossa. The key transcription factor expression of adipogenic and osteogenic differentiation for murine thyroid MSCs detected by quantitative real-time polymerase chain reaction(Q-PCR).2. Base on the murine thyroid MSCs(TMSCs), T-cell proliferation by coculture with TMSCs was evaluated by lymphocyte transformation test(LTT) and mixed lymphocyte reaction(MLR). Furthermore, the dendritic cells from mouse spleen monocyte were cocultued with TMSCs and study their morphology by inverted microscope, phenotype by flow cytometry and the inflammation cytokine expression by Q-PCR.3. Established the model of mouse tail skin flap allotransplantation, and then assessed the immunoregulation ability of murine TMSCs in vivo. The murine TMSCs were injected into the lateral tail vein of mouse tail skin flap allotransplantation mice,and then the inflammation and intertion status was observed everyday. Seven days later, the lymphocyte infiltration in the skin flap transplantation was analyzed by pathological section method.Results1. The murine thyroid adherent cells displayed fibroblastoid morphology; was CD29, CD140, CD105, and Sca-1 positive; but was negative for CD34, CD45, CD31,CD11 b and MHCII. Functionally, it could be induced into adipocytes, and osteocytes.Adipogenic differentiation of adherent cells was indicated by accumulation of oil-red-O staining lipid-rich vesicles. Under osteogenic conditions, they also could form aggregates or nodules displaying alkaline phosphatase activity. Calcium deposits,evaluated by the Von Kossa method. In agreement with the results of differentiation assays, Q-PCR also demonstrated that the adherent cells displayed corresponding transcriptional expression of paroxysm proliferation activated receptor gamma(PPARγ), C/EBPα, Run X2, and osteocalcin, respectively. The morphologic,immunophenotypic, and differentiation assays described strongly indicated that the adherent cells isolated from mouse thyroid were MSCs. Moreover, the growth characteristic of TMSCs was showed S-shaped growth curve, and more than 80%cells were in G0/G1 by flow cytometry analysis. These data suggest that the adherent cells were stem cells.2. The immuno-modulatory activity of MSCs derived from mouse thyroid was investigated using lymphocyte transformation experiment and mixed lymphocyte culture test. The results showed that the immuno-modulatory activity was similar among TMSCs and BMSCs. All of the groups inhibited T-lymphocyte proliferation stimulated by mitogen and allogeneic antigens in a dose-dependent manner(P<0.05).In both assays, the T-lymphocyte count gradually decreased with increasing concentrations of MSCs(P<0.05).3. Dendritic cells(DCs) are specialized antigen-presenting cells(APCs) with a unique ability to prime effective immune response. MSCs play a major role in the immune response for DCs. To test the immunoregulatory effect of TMSCs on DC, the DCs were cocultured with TMSCs, showed significantly reduced the numbers of protruding veils in MDCs. In parallel, these cells displayed decreased expression of CD11c, CD80, CD86 and MHCII. Real-time PCR was performed to detect the m RNA expression of inflammation cytokines, the results showed that there was significantly decreased expression of immune-related cytokines, including IFN-γ, TNF-α and IL-12(P<0.05).4. To determine the immunoregulatory effect of TMSCs in vivo through lateral tail vein in mouse tail skin flap allotransplantation model. The results demonstrated that TMSCs had repaire capacity for mouse tail skin damage, good healing of the flap by TMSCs was better than that in control group. The lymphocyte infiltration degree of TMSCs treatment flap group was lower than those of control group. These data suggest that TMSCs could promote the repair effect for skin flap damage and decrease the inflammation infiltration in injury position.Conclusion1. We successfully isolated MSCs from mouse thyroid tissue, their morphology,phenotypes, and induction differentiation ability was similar to that of bone derived MSCs. The growth curve and cell cycle showed the stem cell characterization.2. TMSCs harbor an immunoregulatory activity by LTT and MLR assay, they can inhibit T lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli in a dose-dependent manner. Also, TMSCs could inhibit the function and mature of DCs.3. In vivo, TMSCs could inhibit allograft rejection, induce the immune tolerance,and repair the damaged tissue. Moreover, they can reduce inflammation reaction, and decrease the lymphocytic infiltrates in damage position.These results demonstrated that TMSCs has the similar biological properties with BMSCs, these will be provided the theory and experimental foundation for TMSCs to achieve the mechanism of autoimmune thyroiditis.