Biological Characteristics Of Neural Stem Cells Modified By Human Neurotrophin-3 Gene Mediated By Lentivirus In Vitro Culture

Background and Objective:Neural stem cells(NSCs) transplantation and gene therapy are the most important approaches to novel strategies for ischemic stroke therapy.In recent years,NSCs,for their pathotropism and their longterm persistence in target tissue have,been used as a promising vehicle for targeted gene deliverys.The innate capacity of NSCs to release protective molecules can further be increased by genetically transfecting NSCs to secrete additional neuroprotective peptides or molecules that can play an important role in hypoxic-ischemic brain injury repair and regeneration.Combined gene therapy and NSCs transplanation brings a novel thinking and pathway for ischemic stroke therapy.NSCs,transduced by hNT-3 gene mediated by LV,were grown,expended and propagated in serum-free medium.We investigated the optimal MOI of LV.The study observed the expression of NSCs modified by NT-3 gene after transfection,as well as tested the neuronal differentiation under the influence of expression of the neuron-inducing factor,NT-3,by genetically modified NSCs.Methods:The study consisted of three parts.PartⅠ:Neural stem cells were extracted from rat embryonic brain(E14),then grown,expended and propagated in serum-free medium.Passage 5～6 neural stem cells were seeded at a density of 1×105/500μl/well in 24-well plates and exposed to lentiviral vector(LV) coding for GFP reporter gene(LV/GFP) at multiplicities of infection(MOIs) of 0,1,5,10,15,20 respectively.There were six groups totally,and each group had six wells seeded.The cells were incubated in a humidified atmosphere with 5%CO2 at 37℃.2～3 days later,the cells were observed under inverted fluorescence microscope.And 3 days later,neurospheres were counted followed by flow cytometer examination to get the percentage of transgene positive NSCs.PartⅡ:The lentiviral vector(LV) coding for Flag-tagged hNT-3(LV/hNT3) gene was constructed by the co-transfection of 293T cells with transfer plasmid coding for hNT-3(pGC-E1/hNT-3) and two help plasmids (pHelper 1.0 and Helper 2.0) using Lipofectamine 2000.NSCs were transduced by hNT-3 gene mediated by LV,grown as partⅠ.Immunofluorencence staining was used to label NSCs-hNT3(MAP2,GFAP,GalC) and newly proliferated cells.The proportion of neuronal differentiation was gotten.We collected the cells and medium at 48h,72h,96h,and observed the expression of NT-3 by ELISA.Results:PartⅠ:7～8 days after isolated from rat embryonic brain,NSCs grew as free-floating nestin-positive neurospheres.2～3 days after GFP gene transduction, GFP-expressing cells cound be seen except in the control group,Over 90%cells were GFP-positive at 3 days after transduction by LV at MOI of 10.A significant dose-response was observed with increasing virus titer for MOI 0 to 10(P＜0.05), whereas the amount of neurospheres decrease with increased virus load for MOI 10 to 20.PartⅡ:When cultured in serum containing media,NSC-NT3 cells,like the parent NSCs,still differentiated into all three fundamental neural cell types((neurons, astrocytes and oligodendrocytes).However,NSCs-NT3's percentage of neurons was higher than the parent NSCs'.In NSCs-NT3,neuronal differentiation always predominated.The neurons that derived from the NSCs-NT3 cells bore more numerous and longer processes than non-engineered NSCs assayed at the same time in culture.Conclusion:1) LV is an ideal vector for gene transduction and it can transduce a foreign gene into NSCs with high efficency of over 85%at the MOI of 10.2) NSCs-NT3's percentage of neurons is higher than the controls.The neurons that derived from the NSCs-NT3 cells born more numerous and longer processes than non-engineered NSCs.