Abstract: Objective To introduce a reliable method to induce neural stem cells directed differentiating into neurons EM>in vitro/EM>. Methods The rat neural stem cells were cultured in selected serum-free medium. After cultured for 2, 3 passages，the neurospheres or single-cells isolated from neurospheres were cultured in conditioned medium to induce directed differentiating into neurons. Besides morphological observation of those cells under inverted microscope, NSE immunocytochemistry was carried out to detect the differentiating ratio of those cells from neural stem cells into neurons. Results It was shown that the conditioned medium could induce neural stem cells differentiating into neurons effectively in both neurospheres culture mode or single-cells culture mode. Furthermore, compared with the neurospheres culture mode, the single-cells culture mode showed more synchronous in the differentiation process and higher in the percentage of NSEpositive cells. Conclusion This method can induce neural stem cells directed differentiating into neu

Key words: Neural stem cells, Neurons, Directed differentiation, Conditioned medium, Immunohistochemistry, Rat