Effects of different differentiation times on mouse embryonic neural stem cells differentiation in vitro

Abstract

Objective To study the effects of different differentiation times (one, 3 and 5 d) on mouse embryonic neural stem cells (NSCs) differentiating into astrocytes and neurons. Methods (1) Fetal cortices of embryonic 14 d (E14) C57BL/6 mice were isolated, digested and cultured. The nestin and Sox2 expressions in the second passaged neural spheres and monolayer cultured NSCs were detected by immunofluorescent staining. (2) And then second passaged neural spheres were digested into NSCs; they were inoculated in the slide and overnight cultured, and then, they were changed into the differential medium the next day; immunofluorescence assay was used to observe the glial fibrillary acidic protein (GFAP) and Tuj1 expressions; the averaged numbers, mean longest length and mean branches of the neuritis were analyzed one, 3 and 5 d after differentiation. (3) Real time fluorogenic quantitative PCR was used to detect the nestin, GFAP and Tuj1 mRNA expressions one, 3 and 5 d after differentiation of NSCs. Results (1) NSCs were successfully cultured and almost of all cells were both nestin and Sox2 positive NSCs. (2) Immunofluorescence assay showed that as the differentiation time increasing, numbers of differentiated astrocytes and neurons became larger, and their morphologies became more complicated. The cell counting results showed that: as compared with one d group, 3 and 5 d groups had significantly high GFAP+ astrocyte percentages (P<0.05); as compared with one and 3 d groups, 5 d group had significantly higher Tuj1+ neuronal percentage (P<0.05); as compared with one d group, 3 and 5 d groups had significantly larger averaged neurite numbers (P<0.05); the averaged longest neurite length was increased as differentiation time increasing and there was obvious difference between each two time points (P<0.05); as compared with one d group, 3 and 5 d groups had significantly higher averaged neurite branching levels (P<0.05). (3) And PCR results mainly showed that nestin mRNA expression in one d group was significantly higher than that in 3 and 5 d groups (P<0.05); GFA P and Tuj1 mRNA expressions in 5 d group were statistically higher than those in one and 3 d groups (P<0.05). Conclusion With the differentiation time increasing, the numbers of differentiated astrocytes and neurons become large, their morphologies become complicated, and their percentages are gradually increased; more mature neurites are noted; nestin mRNA expression gradually decreases while GFAP and Tuj1 mRNA expressions gradually increase in monolayer cultured mouse embryonic NSCs. Key words: Neural stem cell; Differentiation; Astrocyte; Neuron; Neurite