Induction Of Mouse Embryonic Stem Cells To Neuron-like Cells With RNAi

PurposeIn recent years, RNA interference (RNAi) becomes a highly efficacious means to control gene expression, it can reduce or silience specific gene expression in mammalian cells according to its dose , which is more convenient than other traditional methods such as antisense RNA technology and knocking out of gene etc. So it is a more effective tool to study genes of stem cells.Embryonic 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 neither 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. Its also a perfect model in vitro of the mammal neural system development. Because of their 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 disappointing, 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. Our group has successed to induce ESCs into neuron — like cells by sequential neural induction and analyzed it by genechip assay. Dased on analysis of genechip and filtering the key gene of neuronal dif-ferentiation, here we focused on the mechanism of ESCs differentiation and reported a simple and effective method to induce ESCs to neurons - like cells with RNAi.Methods1. Culture and induction of ESCsESCs line(AB2. 1) were routinely propagated on feeder cells. To initiate induction, undifferentiated ESCs were dissociated from feeder cells and replanted on gelatin - coated tissue culture plastic dishes in ESC medium without LIF after transfecting dsNRSE/RE - 1 by electroporation.2. Identification of the undifferentiated ESCs and neuron - like cells. 2. 1 Immunostaining of neural specific antigens;NSE2. 2 Identification of neural related genes by semi - quantitive RT - PCR a-nalysis.Results1. ESCs can be induced to neuron -like cells with RNAi.The undifferentiated ESCs grew in colonies, with clear boundaries. After the RNAi 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 immunopositive for neural marker NSE(82. 3 ±8.1% positive) .2. Gene expression of neural related genes was identified by semi - quantitive RT - PCR analysis and confirm the results of genechip analysis.Genechips showed that NRSF/REST discreased during the neural differentiation of ESCs, whereas the expression of mature neurons specific and related genes increased. Semi - quantitve RT - PCR confirmed the above results.Conclusion1. It is confirmed that NRSF/REST is main negative modulatory factor during the process of ESCs into neuron - like cells.2. The RNAi neural induction can convert ESCs into neuron -lile cells effectively.