| It has been known as "ectomesenchyme" when cranial neural crest migrates into facial process. These neural crest-derived ecto-mesen chymal stem cells may be pluripotent stem cells which have the self -renew ability, In vivo experimental approaches have reinforced this view that EMSCs can differentiate into cells of the osteogenic, chondrogenic, tendonogenic, and myogenic lineages which forming . the oro-facial organ. Few information were documented on cell lineage diversity is available from in vitro experiments. During the differentiation process, precise cell-growth factors, cell-matrix, and growth factors-matrix interactions are responsible for triggering Multi-lineage differentiatiate direction were not elucidated. Previous report show the neural crest cell which had been induced to differentiate to neuron by BMP-2/4, to glial cell by GGF, to smooth muscle cell by TGF-p in vitro. Moreover, the other mesenchymal stem cells such as marrow sesenchymal stem cell could trans- differentiation from one tissue to another tissue which has paved the way for potential use of stem cells in treatment of diseases.The tissue engineering product was invoving in biomaterial, seed cells and growth factors. The aging and resource of seed cells iscells possess the self-renew potentiality and the ability to proliferate rapidly in vitro, are expected to be the new source for t issue engineering seed cells. The aim of the present study was to test the hypothesis that ecto-mesenchymal stem cells show properties of pluripotent stem cells, and constrct a new methods and technology in the stem cell differentiation research as well as specific gene and proteins molecular regulation to simulate microenvironment defined their ability to differentiate. The clinical application of stem cell bioengineering will also be discussed.Part 1: Isolation and Biologic Characterization of Undifferentiated Ecto-mesenchymal Stem Cells from the First Branchial Arch of Rat Embryo.Ecto-mesenchymal stem cells were enzymatically isolated from the rat embryo; ecto-mesenchymal stem cells were isolated and purified because of the difference of the tolerance to trypsin and characteristic of stem cells. The HNK-1, VIM, NSE, S-100 and MG are expessed in the cells, while GFAP, NF, CK presented negative. These cells maintained in an undifferentiated state while cultured with leukemia inhibitor factor (LIF). bFGF could stimulate EMSCs proliferate activity.Part 2: Induction Study of Rat Ecto-mesenchymal Stem Cells Differentiation into Multi-lineage CellsUndifferentiate state of stem cell was maintained by inhibitor, ecto-mesenchymal stem cells will differentiate spontaneously without inhibitor. Differentiating to smooth muscle cells from ecto-mesenchymal stem cells could be enhanced by TGF- . Chondrocyte could be induced by cartilage promoting culture medium whichcontain TGF- , IGF, Vitamin C and Dexasone. These cells could differentiate into Skeletal muscles like cells through Dimathyl sulfoxide, adding the DMSO, cells appear the expression of myoblast relation proteins such as myoD, myogenin and myosin. For detect whether cells have apility to differentiate into neuron, we construct the TC10 eukaryon expression plasmid. The TC10 cDNA derived from rat facial nuclear with RT-PCR. When cells transfected with TC10, cells began to expressed NF and nestin. The BPE and forskolin could stimulate cells to differentiate into glial-like cells which express GFAP. These result indicated that EMSCs is a special kind of cell that has a unique capacity to renew itself and to give rise to specialized cell types.Part 3: The Application of EMSCs on the Tissue EngineeredNerve Repairing Sciatic Nerve DefectThe aim is to establish a protocol of constructing tissue engineering peripheral nerve. The method is to mix the EMSCs which induced into glial-like cells with BPE and forskolin, bovine acellular matrix (BAM), fetal bovine serum and DMEM on the special ratio and inject them into the PLGA tube, the compound form gel construct when incubated 24 hours,... |
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