| Embryonic stem cells (ESCs) are pluripotent stem cells derived from inner cell mass of blastocysts of mammals. ESCs employ a complex signaling network to maintain self-renewal, in which pluripotency related molecules play critical roles. Currently, knowledge of ESC pluripotency related molecules is limited. So it is quite important to identify new pluripotency related molecules for ESC study. While the progress of ESC theoretical research would doubtlessly push forward the development in clinical application of ESCs.According to some existing knowledge on ESC self-renewal, we could effectively search for new factors and their roles in ESC pluripotency. In our research we supposed that Midkine (MK) a heparin-binding developmentally regulated growth factor play important roles in ESCs, based on the research of some other heparin-binding growth factors such as Fibroblast growth factor (FGF) and Pleiotrophin (PTN). To confirm this hypothesis the expression of MK in ESCs was evaluated by reverse transcription-PCR (RT-PCR) and immunocytochemistry (ICC). The effects of MK on the self-renewal of ESCs were measured using alkaline phosphatase assays, ICC, RT-PCR and colony-forming assays. The mechanism of the growth-promoting effect of MK in ESCs was assessed by cell cycle analysis and western blot analysis. The results show that MK is expressed in both mouse ESCs (mESCs) and human ESCs (hESCs). Furthermore MK promotes proliferation of mESCs by inhibiting apoptosis while accelerating progression toward the S phase and enhances mESC self-renewal through the PI3K/Akt signaling pathway. This finding of a previously unrecognized MK signaling pathway in ESCs extends our knowledge of pluripotency control in ESCs.Antibody is able to recognize and bind to the antigen with great specificity. So it is a good tool for exploring new ESC-specific molecules. To identify new ESC-specific molecules, we generated a panel of rabbit monoclonal antibodies (rMabs) against mESCs by immunizing rabbits with live mESCs. To our knowledge, it is the largest monoclonal antibody panel generated against stem cells. Twenty monoclonal antibodies were selected for preliminary characterization. Most of the antibodies generated distinguishable staining patterns, suggesting that they recognize different antigens. We identified the antigen of the rMab ZjuESrMab29 as granulocyte macrophage colony-stimulating factor receptor a (GM-CSFRa) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and found that the expression of GM-CSFRa decreased upon mESC differentiation. This study demonstrated that rabbit monoclonal antibody production via whole-cell immunization could be a practical method for the discovery of new ESC-specific molecules. The decreased expression of GM-CSFRa upon ESC differentiation implied its functions in ESCs.ROCK inhibitor Y-27632 has been reported to decrease the dependence of hESC on cell-cell contact and permit survival of single hESC. In our study we developed a novel, simplified method of hESC treatment with Y-27632 to obtain morphologically homogeneous hESC-derived mesenchymal stem cells (hESC-MSCs). The hESC-MSCs obtained with this method were remarkably similar to MSCs derived from other sources, including a homogenous fibroblastic morphology, an immunophenotype similar to adult MSCs and multilineage differentiation potential. Furthermore, our hESC-MSCs exhibit strong immunosuppressive effects toward lymphocytes in vitro co-culture system and can not only survive into damaged tissue but also ameliorate inflammatory infiltration of injured area in CC14-induced liver injury mice. Our study on immunomodulative effects of hESC-MSCs offered information essential to the application and mechanism studies of hESC-MSC-based therapy.
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