Abstract:

Objective  To explore the optimal method for the isolation of primary neural stem cells (NSCs) from mouse, and the effects of bone morphogenetic protein-2 (BMP-2) on the proliferation and differentiation of NSCs. Methods  NSCs were isolated from mouse (S129 background) embryos at E12.5 by mechanical blowing combined with nylon membrane filtration. The nestin immunofluorescent staining technique was used to identify the isolated NSCs. Then NSCs were divided into two groups: control group and BMP-2 (10ng/mL) treated group.The proliferation and differentiation of NSCs were determined by BrdU labeling and immunofluorescent staining of microtubule associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP). Results  ① The isolated NSCs were round and existed in single or pairs in different sizes observed under the inverted microscope. At day 3 to 5 after culture, the neurospheres in different sizes appeared. The nestin-positive cells accounted for more than 90 percent in the cultured neurospheres. ② The ratio of BrdU-positive cells against total NSCs treated with BMP-2 was significantly decreased in comparison with that in control group (P<0.05). ③ The percentage of MAP2-positve cells in BMP-2 treated group was lower than that in control group(P<0.05), while the GFAP-positve cells in NSCs treated with BMP-2 was higher than control group(P<0.05). Conclusion   ① Primary neural stem cells can be successfully isolated and cultured from mouse using a modified mechanical blowing-nylon membrane filtration method, which is highly steady and feasible. ② BMP-2 (10ng/mL) can inhibit the proliferation of NSCs in vitro and inhibit its neuronal differentiation, while promoting its astrocytic differentiation. 

Key words:  Bone morphogenetic protein-2, Proliferation, Differentiation, Neural stem cells, Mice