MEF Derived Mesenchymal Stem Cell Induce The Differentiation Of HPC Into Regulatory Dendritic Cell Via IL-10Secretion

[Background] Mesenchymal stem cells (MSCs) have been isolated from many tissues and organs, and have generated a great amount of interest in the field of regenerative medicine due to their unique biological properties:(1) the ability to differentiate into various cell types;(2) the lack of immunogenicity and the ability to perform immunomodulatory functions;(3) the little teratoma formation. Previously, we isolated the Sca-1+CD117-Lin-MSCs from mouse embryo fibroblasts (MEF) population by the stem cell marker, stem cell antigen-1(Sca-1). This population of stem cells gave rise to adipocytes and osteoblast, and exhibited immune regulatory functions to inhibit the proliferation of lymphocytes. Moreover, Sca-1+CD117-Lin-MSCs could induce hematopoietic progenitor cells (HPCs) to differentiate into regulatory dendritic cells (DCs) without any exogeneous cytokines adding. Furthermore, IL-10played important roles on this induced process. However, the underlying mechanisms of IL-10mediated DC differentiation are not fully understood. Thus, in this study, we demonstrate that transcriptional signaling pathways and epigenetic mechanisms both function during the IL-10-dependent production of regulatory DCs.[Aims]1. Test the immune regulatory functions of Sca-1+CD117-Lin-MSCs in vivo, especially the effects on the differentiation of DCs;2. Explore the mechanisms of IL-10functions during MSCs inducing the generation of regulatory DCs.[Methods]1. Isolate Sea-1+CD117-Lin-MSCs from MEF with magnetic beads, analyze their biological properties, and immune inhibitory functions by mixed lymphocyte culture assay;2. Infuse Sca-1+CD117-Lin-MSCs in vivo to observe their regulatory effects on inflammatory reaction and the phenotype of produced DCs by flow cytometry;3. Coculture Sca-1+CD117-Lin-MEF-MSCs and HPCs in vitro to observe the generation of regulatory DCs by flow cytometry, the differences between induced DCs and traditional DCs by gene chip, the key functional cytokines in coculture medium by enzyme linked immunosorbent assay (ELISA) and neutralizing antibody assay, the key transcriptional factor in DC differentiation by siRNA interfering assay, and the histone modification on special gene promoter by Chromatin Immunoprecipitation (ChIP).[Results]1. MEF derived Sca-1+CD117-Lin-cells possess classic biological properties of MSCs:spindle-shaped morphology, typical "S"-shaped growth curve, most cells in G0/G1phase, express pluripotent transcription factors and stem cell markers, the potency of adipogenic and osteogenic differentiation;2. MEF-MSCs lack of immunogenicity and perform immunomodulatory functions:suppress the proliferation of allogeneic lymphocytes in vitro, and in DTH mice alleviate immune reaction, relieve the footpad swelling, increase the level of anti-inflammatory factor interleukin10(IL-10) in peripheral blood, up-regulate the percentage of regulatory DCs in spleen;3. Sca-1+Lin-CD117-MEF-MSCs can induce HPCs to differentiate into novel CDllbhhighIalowCD11clow regulatory DCs (Sca-1+Lin-CD117-MEF-MSCs-induced DCs, sDCs) when they are co-cultured in the absence of any exogenous cytokines. The induced sDCs, which are different from traditional DCs (imDCs and maDCs), can alleviate immune reaction both in vitro and in vivo.4. Sca-1+CD117-Lin-MEF-MSCs derived IL-10plays an essential role in generation of sDCs, while neutralizing IL-10in medium decreases the production of CD11bhighIalowCD11clow sDCs;5. IL-10activated suppressor of cytokine signal3(SOCS3) is fundamental for sDC generation. IL-10up-regulates SOCS3expression in sDCs through the JAK-STAT5activation and changes of histone modification.6. Compared with normal mice, the percentage of CDllbhighIalowCD11clowDCs in spleen of Trauma-hemorrhage mice is increased, while maDCs decreased, suggesting the immune regulatory functions of endosomatic MSCs after organism injury.[Conclusion] MEF derived Sca-1+CD117-Lin-mesenchymal stem cell can induce the differentiation of HPC into CDllbhighIalowCDl lclow regulatory dendritic cell via IL-10secretion, which activates the expression of SOCS3in HPC. These results shed new light on the role of MSCs in modulating regDC production, and support the clinical application of MSCs to reduce the inflammatory response in numerous disease states.