Generation Of Induced Pluripotent Stem Cells And Differentiation

Dendritic cells (DC) are professional antigen presenting cells, they can uptake, process, handle and present antigen, and present the information of antigen to T lymphocytes, trigger a series of immune responses, and the efficiency is the strongest of all such cells. DC participate in a variety of pathophysiological processes and have a strong immune function, DC-based immunotherapy has become a new model of cancer treatment. Acquiring autogeneic DC is difficult through the separated cultivation and amplification of autogeneic bone mesenchymal stem cells, and the technology is difficult, and the patient is unbearable, its efficacy remains controversial, and is not suitable for clinical widely used. Preparing DC through embryonic stem cells (ESC) directional differentiation has ethics limits and the resulting DC is not autologous cells, there is the possibility of immune rejection. Induced pluripotent stem cells (iPSCs) technique is the new way to DC tumor immunotherapy. iPSCs are the stem cells induced from somatic cells, and have the developmental pluripotency of ESC, are very similar to ESC in terms of morphology, gene expression status and epigenetic modifications. iPSCs value is the method for obtaining relatively simple and stable, more advantages than other methods in the technical and ethical, plays a very important role in regenerative medicine.This study induces mature cells in mice to iPSCs through multi-factor reprogramming, and immunohistochemistry, fluorescence, embryoid body (EB) experiment, RT-PCR and Western blot are used to analyze and identify iPSCs. We add different cell growth nutritional factors of DC that promoted the iPSCs to differentiate into DC in vitro.ObjectiveThe retro viral vector system is used to induced the mice fibroblast to iPSCs by viral packaging technology, and the iPSCs are identified. And the iPSCs are induced to differentiate into DC in vitro.Materials and MethodsThe first part, the induction and identification of iPSCs. As follows:the retroviral transfection method was used to transfect exogenous pluripotent factor Oct4, Sox2, c-Myc, KLF4plasmid into virus that was used to package Plat E cells, supernatant with virus was collected and used to infect the primary cultured mouse tail fibroblasts, the process was observed and recorded, iPSCs were analyzed and identified. Mainly, the following identification methods were used,1) alkaline phosphatase (AP) staining was used to observed whether undifferentiated iPSCs had the characteristics of ESC, at the same time, the observation of morphology and immunocytochemistry was used to identify iPSCs totipotent;2) immunofluorescence method was used to observed the expressions of Oct4, Sox2, SSEA1in iPSCs surface of the expression;3) EB experiment was used to detect whether iPSCs can differentiate and develop into the embryo, and the immunofluorescence method was used to detect the information of3mesoderm-derived cells in embryo body;4) Western blot was used to detect the multi-gene protein expressions in the iPSCs. Western antibodies were immunohistochemical antibodies;5) analysis of gene expression (RT-PCR):RT-PCR was used to detect Oct4, Sox2, c-Myc, Klf4pluripotent gene levels in successfully induced iPSCs, ESC M1and mice fibroblasts, and their levels were comparatively analyzed.The second part, iPSCs were induced to differentiate into DC. The method as follows:in the culture media of iPSCs, the type of cytokines, such as interleukin IL-3(10ng/mL) induced IL-7(10ng/mL), IL-15(20ng/mL) were added, every2to3days, half the amount of liquid was changed, the fresh cytokines were added. After12to14days, the cytokine granulocyte cell-macrophage off the stimulation factor (GM-CSF)(4ng/mL) and tumor necrosis factor (TNF)(50ng/mL) were added, and the cells were cultured for3-5d, cell morphology was observed under an inverted microscope.Results1Cultivation of rat tail fibroblasts and induction of iPSCsTrypsin digestion method was used to culture the primary cultured rat tail fibroblasts, the next day, the inverted phase contrast microscope was used to observed the typical characteristics of fibroblasts; iPSCs was induced, pluripotent stem cell factor virus was transfected into the fibroblast cells, the next day, the morphology changes of fibroblast cells can be observed, cells were flattened, more rounded, while the small colony-like proliferation of groups were observed. Two weeks later, a large number of dense projections clone was observed, cells were closely arranged, the boundaries was clear, the reflected light of colony edge was strong. The clones were mechanically picked and passaged.2Identification of iPSCsThe AP staining, the EB experiment, Western blot and immunofluorescence staining used to identify the successfully induced iPSCs, the identification results were that iPSCs was similar to ESC; RT-PCR was used to detect multi-gene mRNA levels, the results were that iPSCs was similar to ESC, the expressions of endogenous Oct4, Sox2, c-Myc, K1f4were observed, and no stem cell pluripotency gene Oct4was observed in the differentiated fibroblast cells; exogenous gene RNA levels were higher in the created iPSCs cell lines, while exogenous gene was almost completely silent in the mouse ESC and mouse embryonic fibroblasts. 3Differentiation of iPSCs into dendritic cells in vitroThe methods of differentiation of iPSCs into dendritic cells in vitro:several DC differentiation nutritional factors were added, such as IL, GM-CSF, TNF, stem cell growth factor and DC somatomedin FMS-like tyrosine kinase3ligand (FIt3L), lipopolysaccharide endotoxinand. The typical DC morphology was observed, in the induction of specific factors, iPSCs could be differentiated into DC.Conclusions1) The mouse tail fibroblasts are successfully induced into iPSCs by exogenous factor transduction, and the iPSCs features and morphological characteristics are observed;2) In the induction of specific factors, iPSCs can be differentiated into DC, the typical DC morphology was observed;3) A new source of seed cells required for the DC tumor immunotherapy can be used, the development and its role in immune regulation of DC can be comprehended during the development, this is very valuable experimental platform for the prevention and treatment of disease and the drug research.