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Objective During neural development, neural stem cell (NSC) undergoes a precise proliferation and differentiation process.This process is directed by various factors from the NSC niche.In order to investigate the effect of spatial signal on NSC differentiation, in the present study, the survival and differentiation of CM-DiI labeled NSCs were observed on the organotypic slice cultures from neonatal rat cortex.Methods SD rats on 3 to 5 postnatal days were sacrificed according to the ethical guidelines of the NIH Regulations for Experimentation on Laboratory Animals and set out by the Xi'an Jiaotong University.Brain slices with 200-μm thickness were cut on a vibrotome microtome and cultured in modified DMEM/F12 serum free medium at 37 C with 95% O2 and 5% CO2.Organotypic slice cultures were observed under inverted phase-contrast microscope every day and the medium was half refreshed every 3 to 4 days.NSCs were isolated from the cortex of rat embryos on embryonic day 14.5 and cultured in DMEM/F 12 serum free medium supplemented with B27 and N2.NSCs on passage number 3 were labeled by CM-DiI for 20 min in dark and cultured for 3 to 4 days.The labeled ceils were transplanted onto the organotypic slices in 2 weeks culture.The survival of transplanted NSCs was confirmed by visual CM-DiI labeled cells on the slices.The differentiation of transplanted NSCs was identified by immunofluorescence staining and the DiI/GFAP or DiI/β tubullin Ⅲ double labeled cells were considered to be astrocytes or neurons.Results The organotypic slice cultures survived well for at least 4 weeks in the modified medium and became clear vision on 10 days later, which was possibly due to the elimination of microglia.CM-DiI labeled NSCs were observed on the slice cultures 4 days after transplantation.These labeled NSCs differentiated into GFAP+ glia and β tubullin Ⅲ+ neurons.Conclusion Our results suggest that the organotypic slice culture might produce some specific signals to induce the transplanted NSCs to differentiate into a certain cell type.The further study about NSCs differentiation on organotypic slice cultures from different brain regions will reveal the effect of niche in NSC behavior.