The Investigation Of The Neural Differentiation Of Mouse Embryonic Stem Cells

IntroductionEmbryonic 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 and the spinal injury. It's also a perfect model in vitro of the mammal neural system development. But we have not understood mechanism of differentiation of neural cells.We have make use of sequential neural induction which is builded all by ourselves, induce embryonic stem cells to neuron, and initially investigate the mechanism of neuron induced from embryonic stem cells.Based on the above results, we optimize the conditions of induction, use Immunostaining and Western Blotting to identification of the neural induced cells, use FCM to determine ratio of differentiation of induced cells.Methods1,Preparation of PMEF , culture and induction of ESCsmESCs line(AB2.1) were routinely propagated on PMEF-P_2-4 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 every other day into serum-free NC medium containing bFGF-N2-B27 medium. 2,Optimization of the conditions of induction(1) Gradient of density of ESCs(2) Gradient of concentration of NSC medium of first time(3) Gradient of time to change into serum-free NC medium3,Identification of the neural induced cells.(1) Immunostaining of neural specific markers: Nestin,NSE,NeuN,TUJ1,NCAM1 and GFAP.(2) Western Blotting of neural specific marker: TUJ14,FCM detects the percent of NeuN positive cells, and determines the ratio of induced differentiation.Results1,Optimization of the conditions of inductionOptimized of concentration of NSC medium of first time is 12.5%, optimized of density of ESCs is 1.0×10⁵/ML, and optimized of the day to change into serum-free NC medium is D5.2,ESCs can be induced into neuron-like cells effectively by optimized "sequential neural induction"The Undifferentiated ESCs grew in colonies, with clear bound. After the optimized 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.3,The neural induced cells'specific markers, Nestin,NSE,NeuN,TUJ1 and NCAM1 are positive , and GFAP is negative.4,FCM determines the ratio of induced differentiation is 89.91% ±2.03%.Conclusions1,The optimized "sequential neural induction" can induce mESCs into neuron-like cells effectively, and these neuron-like cells can express mature neurons' specific markers. The expression of these specific markers may relate to mechanisms of directed differentiation.2,FCM determines the ratio of differentiation of induced cells is 89.91 %±2.03%.