Proportion and difference of neural stem cells and neurons from different embryonic mouse brain tissues / 中国组织工程研究

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.