ABSTRACT
Objective To understand and compare the proportion of neural stem cells (NSCs) in the whole brain and cerebral cortex of mice at different embryonic days, and provide quantitative data for the later optimization of NSCs isolation and culture.Methods The whole brains (at embryonic 12.5, 14, 16 and 18 days) and cerebral cortex (at embryonic14, 16 and 18 days) were isolated and digested into single cell suspension, and were adherently cultured for 3-4 h.Immunofluorescence staining of Nestin, a NSCs specific marker, was used to statistically analyze the proportion of NSCs in each group.Expression of Nestin mRNA in the cerebral cortex of mice at E12.5, E14, E16, and E18 was detected by real-time fluorescence quantitative PCR.Results The result of immunofluorescence assay showed that there were Nestin-positive cells in the whole brain and cerebral cortex of mice at different embryonic days.In the whole brain,the proportion of NSCs was highest at E12.5 (53.42±1.57%) and lowest at E18(25.96±1.31%), and the proportions at E14 and E16 were placed in the middle among the groups.In the cerebral cortex, the highest proportion of NSCs was at E14 (33.65±0.29%), and the lowest at E18(25.29±0.28%), and the middle at E16 (26.82±0.30%).The result of real-time PCR showed that when the mRNA expression of Nestin in the cerebral cortex was set to 1, the relative mRNA expression of Nestin was 0.83±0.04 at E14, 0.77±0.05 at E16, and 0.44 ±0.05 at E18.Thus, the mRNA expression level of Nestin in the mouse cerebral cortex was gradually decreasing with the increase of embryonic days.Conclusions During the brain development, the proportion of NSCs is gradually decreasing in the whole brain and cerebral cortex of mice with the increase of embryonic days.
ABSTRACT
BACKGROUND:At present, mouse embryonic neural stem cells (NSCs) culture has been skillfully operated by many labs, but there are differences existing about which part are dissociated to get NSCs. Embryonic 14 days (E14) mouse brain tissues are widely used for culturing NSCs, but there are less studies about the detailed percentage and difference of NSCs separated from different brain tissues. OBJECTIVE: To test the proportion and difference of NSCs and neurons percentage from E14 mouse whole brain, cortex and forebrain, providing quantized data for optimizing the isolation of high-purity NSCs. METHODS:E14 C57BL/6 mouse whole brain, cortex and forebrain tissues were separated and dissociated into single cells that were adherently cultured for 3.0-4.0 hours and labeled by DAPI. Then the cells were immunostained with NSCs specific marker, Nestin, and neuron specific marker, Tuj1, to identify NSCs and neurons percentage by calculating Nestin+/DAPI and Tuj1+/DAPI. In addition, real-time PCR assay was used to test Nestin and Tuj1 mRNA expression in the E14 mouse whole brain, cortex and forebrain. RESULTS AND CONCLUSION: (1) Immunocytochemical results showed that there were a large amount of Nestin+ and Tuj1+ cells in the whole brain, cortex and forebrain of E14 mice. NSCs percentage in the forebrain was obviously higher than that in the whole brain (P 0.05); the Tuj1 mRNA expression in the forebrain was significantly lower than that in the whole brain (P < 0.05) and in the cortex (P < 0.05). These findings indicated that the forebrain had the most NSCs and the least neurons compared with the whole brain and the cortex. In summary, E14 mouse forebrain has the highest percentage of NSCs compared with the whole brain and cortex, which is a better source to obtain NSCs for the following cell culture experiments.