Cultivation In Vitro Of Rat Neural Stem Cells And Its Conditioned Medium Effects On The Differentiation Of Bone Marrow-derived Mesenchymal Stem Cells Into Neural Stem Cells

Objective（1）To establish the isolation, differentiation，identification and long-termcultivation in vitro of the neural stem cells (NSCs) from neonatal SD rats, and toinvestigate the effect of the serum concentration on the differentiation of NSCs.（2）To observe the effect of the conditioned medium from neural stem cells on thedifferentiation of bone marrow derived-mesenchymal stem cell(BMSCs) into neuralstem cell-like cells.Methods（1）Brain tissue was isolated from neonatal SD rats and NSCs were culturedin serum free medium.To identify NSCs and detect Nestin expression in NSCs byimmunofluorescence. In the condition of DMEM/F-12containing differentconcentrations (2%,5%,10%) of FBS to induct the differentiation of NSCs, toidentify the differentiated cells with the antibodies of neuron specificmicrotubule-associated protein2(MAP-2) and glial cell specific glial fibrillary acidicprotein(GFAP) by immunofluorescence. In the differentiation conditions containingdifferent concentrations of FBS, to detect the expression of MAP-2and GFAP byWestern blot.（2）The concentrated conditioned medium, which contained of a varietyof factors secreted by neural stem cells (NSCs), delivered to DMEM/F12withfibroblast growth factor(bFGF) and epidermal growth factor(EGF) according to anappropriate proportion. By observing the morphology of BMSCs and Western blotanalysis of the protein expression of Nestin,the differentiation of neural stem cell-likecells between the experimental groups with conditioned medium of NSCs and thecontrol groups without conditioned medium was compared. At last, theimmunoeytochemistry was used to investigated whether the neural stem cell-like cellsderived from BMSCs were able to differentiate into nerve cells. Results（1）A mass of undifferentiated neurospheres were obtained and cultured insuspension, those NSCs could differentiate into neurons and astrocytes. Withincreasing serum concentrations, the expression of MAP-2in each group graduallyincreases (P<0.05), and the expression of GFAP gradually decreases (P<0.05).（2）When cultured with inducing medium, the BMSCs from both experimental and controlgroups lost fibroblast-like morphology within24h.At5th days, a number ofneurosphere-like structures were formed and were able to express the protein of Nestinby immunocytochemical examination. Through the analysis of Image J, the quantity ofneurospheres in experimental groups were significantly more than that in controlgroups(P<0.05),the diameter and square of neurospheres in experimental groups wereslightly more but without statistical difference(P>0.05). Analysis of western-blotshowed that the expression of Nestin in experimental groups were higher(P<0.05).Theneural stem cell-like cells derived from BMSCs were able to differentiate into nervecells after exchanging the medium into DMEM/F12contained of2%B-27and5%FBS.Conclusion（1）We successfully obtained the fetal rat NSCs in vitro, which have thecapacities of proliferation, self-renew and pluripotentiality with the application ofserum free cultivation. In the differentiation conditions containing differentconcentrations(2%,5%,10%) of FBS, NSCs differentiate into neurons and glial cells,the conditioned medium containing low concentration of serum promote thedifferentiation of NSCs into neurons, the high concentration of serum is conducive tothe differentiation of NSCs into neural glial cells（.2）Without the NSCs themselves, thedeliver factors secreted by NSCs in the form of concentrated conditioned mediumpromoted the differentiation of BMSCs into neural stem cell-like cells.