| [Objective] To establish the method on differentiation of human embryonic stem cells (hESCs) into dopaminergic neurons in vitro and acquire more dopaminergic neurons that are pure and have function . At the same time , to develop new perspective for the study on cell transplantation treatment of Parkinson's disease.[Methods] HESCs were cultured on mouse embryonic fibroblast (MEF) cells and were passaged when cell fusion . They were cryopreserved by programmed freezing method and were resuscitated by quick thawing. The recovered rates of frozen clumps, the levels of proliferation and differentiation of hESCs after freezing, thawing and culture were determined. The biological characteristics of hES cells after thawing were identified.HESCs were used to differentiate into neural stem cells (NSCs) by means of three-step differentiation and was evaluated by the morphological observation, immunocytochemistry assay and RT-PCR to detection of NSCs surface markers. The plasticity of NSCs was detected by the differentiating test.HESCs were induced into dopaminergic neurons by means of adding Sonic Hedgehog Peptide (SHH) and fibroblast growth factor eight (FGF8) early (group A) under the condition of mocked microenvironment and different development stage of dopaminergic neurons in vivo ,meanwhile made comparison with traditional method . The marker of dopaminergic neurons was tested by immunocytochemistry and RT-PCR, the content of dopamine (DA) and its derivation homovanillic acid (HVA) were measured by HPLC-ECD.Establishing PD rat model, and the rats were divided into two groups which are therapy group and control group. Cells after differentiation were transplanted into the rat's striatum using stereotaxis technique in therapy group and injected normal sodium in control group. Evaluating the therapeutic efficacy based on improvement of rat's rotation behavior and TH immunohistochemistry.[Results] HESCs were stable proliferation for 5 months and 20 passages. The cells accumulated as clumps on MEF feeder layer, which had large nuclei and transparent nucleoli. The recovered rate after programmed freezing was78.3%. The passage 12 of programmed freezing hESCs retained normal 46XX karyotype, expressed special protein and gene.The positive value of the nestin was up to 90% in the differentiated cells by the three-step differentiation. The differentiated cells expressed nestin gene and could keep the characteristics of NSCs after continuous passage culture and could be further induced into neurons, astrocytes and oligodendrocytes.The cells after differentiation expressed special gene and protein of dopaminergic neurons. The percentage of TH-positive cells (73.68±4.71 %) in group A was significantly higher than that (31.37±1.63%) in group B. The expressions of TH, Nurr1, Lmx1B mRNA in group A were more than that in group B. Furthermore, the content of DA and HVA of group A in differentiation cell and supernatant of culture medium were significantly higher than that of group B (P<0.01,P<0.05) .The results of rotation 1-4 weeks pretransplantation and 1-8 weeks post transplantation: 289±60 turns/30min pretransplantation decreased to 229±37 turns/30min (P<0.05) post transplantation in the therapy group, 290±47 change to 286±34(P>0.05)after transplantation in control group. TH immunohistochemistry showed there were TH positive cells in the rat's striatum of therapy group, there were no TH positive cells in control group.[Conclusions] The culture mode of hESCs in vitro is established successfully, which provides cell model and resource for the study of hESCs and the prevention ,therapy of related diseases. The hESCs could be induced into NSCs with high purification and keep the plasticity of stem cells by three-step differentiation. The effect of adding SHH and FGF8 early proves the differentiation of hESCs into dopaminergic neurons which could secrete DA. The induced dopaminergic neuron have the therapeutical effect on PD which provides a new clue for clinical treatment of PD.
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