In Vitro Differentiation Of Embryonic Stem Cells Into Neural Cells And Correlated Research

Injury to the adult central nervous system (CNS) is devastating because of the inability of central neurons to regenerate correct axonal and dendritic connection. The consequences of injury are not just break in communication between healthy neurons, but a cascade of events that can lead to neuronal degeneration and cell death. So in most cases of CNS trauma and disease, both neurons and glia are lost. Cell replacement is a vital step in CNS lesions, in which spared systems cannot supplant the function of list cells. An excellent example is the loss of motor neurons in the case of injury to the cervical spine. No matter how well the lesion is bridged or reinnervated, the target motor neurons that project to the muscles are irrevocably lost, recognizing the need for cell replacement, several laboratories have taken advantage of fetal tissue grafts to replace cells after a variety of CNS insults, these studies have even led to clinical trials for humans. However, the inherent mechanical, physical and ethical hurdles of using fetal tissue limit its large scale use. Ideally, a cell replacement source should be more homogeneous, readily obtainable and syngeneic. Stem cells have great promise as a source for introducing new neurons or gilal cells to the damage CNS, Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro , when reintroducedback into mouse blastOcyste, they rethe the ability to contribute to ai1 celllineag6s of the develoPing mouse,including the gu line. ES cells canform three-dbosionai sAnCtUrs described as embryedc bodies. FromWhich they differeniate spOnaneously into a larg variety of ceil tyPes.Establishing the mode of RA-induced ES cells differeniatinn to neuralcells is the base of cell theaPy the injwr of CNS. The firSt steP isNed ES cells differenthen into neural cells using RA. The secon4ES cells was transfected by pEGFP--Nl plaSInid WAn integratedGFPMA. Makin it becom the marker ofES cell.Then we first cultUre basic fibroblast cell of fetus mouse. After thecell was treated with whomycin C, it becomes the feeder cell ofES cell.ES cell was put on the feeder cell and survivals and proliferation. Thegood state ofES ceU Will be dissociated with tryPsin. According the tAneof plating is diffeent contrasting with basic flbOblasL The ES cell willbe sCParate and transfer to no adhesiVe substrate bacteriai dishes in thepresence of RA and withut LIF, aggregate forIning eInbryonic bodics.After inducing 4 dap the cell will be tryPsin again and plated asmonlayer Wes on an adhsive substrat in the absence of RA. After4-5 days, neuronal-lilie cel1s @peared. IInInunOhistochendsny wereperfOnnd to identify the proPertes of the differeniated cells in differentiIne after inducing. According the time order these cells wer positivefOr wt procurer and giia(including astrocyte or oligodendrOcyte)andneuron. Some neuron was positive fOr GAD and GABA antigens. Itmeans the neuron deriVed frOIn ES cell is fimctional neurons. So thesereaChin provide evidene that embryedc stem cells can diffeentiate firstinto neuron-giia progentors, and later into giial cells and thectionalll6us, in vitrOThe secOnd steP, we amPlify pEGFP--Nl plasndd. Extract it usingkit and trallsfeCt the ES cell. GFP-exPressing the ES cell were detected byFinOescen InicroscoPy, after G4l8 selecting and ctond CUlture. Thenestablishing of stable green fluorescellt protein-exPressing ES cell line.in sunnnny we establish the model of indUing ES cell differentiatedinto neural cen and mta ES cell exPress GFP ms reSUit is the base ofned wO4 Which is abOu aPPlicate ES theraPy damage of CNS...