Isolation And Identification Of Neural Stem Cells From The Bovine Fetus

Neural stem cells were successively isolated and cultured from the corpus striatum andhippocampus of the adult mouse by Reynolds et al. and Richards et al. in1992, which brokethe traditional concept that the adult mammalian neurons cannot be regenerated. NSCs havethe promising application in treating nervous system diseases as the substituted cells. Inaddition, it’s a shortcut that the stem cells are used as seed cells in animal genetic breedingand transgenic animal cloning, which have been aimed at by the researchers in the world. Ithas been confirmed that there are NSCs in the neural system of the human being, rat andmouse fetus. The suitable fetal age of NSCs isolation, NSCs growth characteristic, culturalconditions and induced differentiation methods in vitro have been studied. At present, thereare still no report about bovine NSCs. Bovine NSCs can be used as donor cells in cloning cowby nuclear transfer. Furthermore, NSCs are of great significance in transgenic animalsproducing and premium animals breeding.In this experiment we isolated NSCs from the brain tissues of bovine fetus, and observedthe growth, proliferation and differentiation characteristics of NSCs in vitro. We used themechanical separation method and the enzyme digestion method to isolate and cultivate NSCsfrom the brain tissues of bovine fetus separately, and comparing the effects of the twomethods. Based on whether or not adding bFGF, EGF and serum in the culture medium, wecultivated NSCs in the mediums containing different components. Then the suitable culturemedium was selected. In order to identify the biology characteristic of NSCs and thedifferentiated cells, the morphological observation method and the immunofluorescencemethod were used.The results are as following: NSCs derived from the bovine fetus can proliferate inculture medium consisting of Neurobasal Medium,20ng/mL bFGF,20ng/mL EGF,20μL/mLB27,100IU/mL penicillin and100IU/mL streptomycin. The number of neurospheresobtained using mechanical separation method is higher than that of neurospheres obtainedusing enzyme digestion method, and the cells activity obtained using mechanical separation method is better. NSCs could express Nestin which is a specific surface marker of NSCs. AndSox2, Nanog, Oct4and SSEA4which are cell surface markers of stem cells could be detectedin NSCs, too. NSCs could be induced into astrocyte (GFAP+) and neuron (βIII-tubulin+).The results suggest that we can isolate NSCs from the bovine brain tissues. The bovineNSCs can be cultured for a long time, and they have the function of differentiation. BovineNSCs can be used as donor cells in cloning cow by nuclear transfer, and also can be used asreceptor cells in transgenic cattle producing.