Abstract: Objective To observe proliferation, apoptosis, and differentiation of cultured neural stem cells（NSCs）from the hippocampus of fetal rats by laser scanning confocal microscopy. Methods NSCs from the hippocampus of fetal rats were isolated and cultured, and the resultant neurospheres grown in petri dishes prior to preparation for confocal microscopy. The nuclei were stained with Hoechst 33258, and an anti-nestin antibody was employed to identify NSCs. Neuron-and astrocyte-specific markers β-III tubulin and glial fibrillary acidic protein（GFAP）were detected to assess NSCs differentiation. Optical continuous tomography of the neurospheres was performed using a laser scanning confocal microscope, and a software was employed to perform three-dimensional reconstruction in order to dynamically observe the neurospheres. Results Nestin-positive neurospheres were observed under a laser scanning confocal microscope. The NSCs gradually developed neurites that intertwined with each other. At the end of 7 days in vitro and twice-passaged, the apoptosis rate was (8.60±2.20)%. After serum-induced differentiation, we observed radial migration of NSCs neurites, which eventually intertwined into a mesh. At the end of 7 days when NSCs from twice-passaged culture were induced to differentiate，the proportions of NSCs that had differentiated into astrocytes and neurons were (68.60±7.64)% and (29.90±8.22)%, respectively（EM>P/EM><0.01）. Conclusion Laser scanning confocal microscopy allows the simultaneous observation of rat neurospheres proliferation, apoptosis, and differentiation, which provides a simple and feasible experimental method for basic NS

Key words: Hippocampus, Neural stem cell, Proliferation, Differentiation, Laser scanning confocal microscopy, Rat