| PurposeEmbryonic stem cells (ESCs) which are from the inner cell mass (ICM) of preimplantation blastula are pluripotent. Neurons are considered as terminally differentiated cells that can not divide nor proliferate after injury. The study of induction of ESCs into neurons is valuable for the future application of cell transplantation , which may be a useful treatment for the patients of CNS disorders, such as the Pakinson's disease, epilepsy, stroke. It's also a perfect model in vitro of the mammal neural system development. Because of there easy genetic manipulation, gene expression and regulation can be studied conveniently.However, the utility of ES cells as a neural developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is disappointed, because their differentiation in vitro is poorly controlled. Specification of ESCs is not well - understood and differentiation protocols are empirical, producing variable and heterogeneous cells. The common inducing protocols , including specific inducers, five - step induction, stromal cell coculture and gene transfection, are not very effective, with the shortcomings of complicated manipulation and high costs. Here we report a simple and effective method to induce ESCs into neurons, and with the help of genechip analysis we can confirm the induced cells as neurons and find some useful or specific genes in neural conversion as well.Methods1. Culture and induction of ESCsESCs line(AB2. 1) were routinely propagated on feeder cells. To initiate induction, undifferentiated ESCs were dissociated and replanted on none - coated tissue culture plastic dishes in NSC medium. The medium was changed ever-y other day into serum - free NSC medium.2. Identification of the undifferentiated ESCs and neural induced cells.2.1 Alkaline phosphatase test of undifferentiated ESCs by Ca - Co.2.2 Immunostaining of neural specific antigens; nestin, NSE, NCAM1, NF-L, Tau and GFAP.2. 3 Gene expression identification of neural related genes by semi - quanti-tive RT - PCR analysis.2. 4 Affymetrix MOE430A genechip analysis of expression pattern differences of undifferentiated ESCs and neural induced cells.2.5 Telomeric repeat ampliation protocol (TRAP) assays.Results1. ESCs can be induced into neuron - like cells by sequential neural inductionThe undifferentiated ESCs grew in colonies, with clear bound. Alkaline phosphatase staining showed dark brown positive particles were distributed in every ESC colony. After the sequential neural induction, the cells converted into neuron - like cells, with homogeneous forms, which had round bright cell bodies and thin long bipolar or multipolar processes. The differentiated cells were im-munopositive for many neural markers, such as NSE, NCAM1, nestin, Tau and NF - L, and 81 +9.1% induced cells were NSE - positive. Few GFAP - positive cells were detected.2. Gene expression of neural related genes was identified by semi - quanti-tive RT - PCR analysis and genechip assay.4 and 10 days after neural induction, gene expression pattern was analysed by genechips, which showed the expression of some neural stem cells and mature neurons specific and related genes, repectively. Especially the expression of GABAr and Glutamate dehydrogenase ( GAD) , which meant the induced cells could be GABAnergic neurons. The astrocyte marker GFAP and oligodendrocyte marker Mbp were not detected. Nor the endoderm and mesoderm markers. Semi - quantitve RT - PCR also confirmed the above results.3. The telomerase activity was decreased during differentiation The undifferentiated ESCs showed strongest telomerase activity. It decreased during differentiation. Until on the tenth day, it was almost invisible, but the telomerase activity of the MEF was still lower than that.Conclusion1. The sequential neural induction can convert ESCs into neurons ( GABA nergic) effectively.2. With the help of genechip analysis, more neural differentiation related or specific g...
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