[DIFFERENTIATION AND PROLIFERATION POTENTIAL OF NEURAL STEM CELLS IN SUBVENTRICULAR ZONE OF MICE IN VITRO].

OBJECTIVE

To establish the system of isolation, cultivation, and identification of the neural stem cells (NSCs) from subventricular zone (SVZ) of neonatal mice so as to seek for the appropriate seed cells for potential therapeutic interventions of neurological disorders.

METHODS

NSCs were isolated enzymatically and mechanically from SVZ of neonatal mice and cultured. The cellular morphology was observed by inverted microscopy. Immunocytochemical stainings of anti-Nestin and anti-SOX-2 were used to identify NSCs of passage 3. To study the differentiation of NSCs, NSCs were plated into 24-wells in the medium supplemented without epidermal growth factor (EGF) and basic fibroblastic growth factor (bFGF) for 3 or 7 days. To compare the differentiation and proliferation potential of NSCs with different cultivation time, the BrdU pulse-labeling method and MTT test were used. To identify neurons and astrocytes, the anti-β-tubulin III (Tuj-1) and anti-glial fibrillary acidic protein (GFAP) staining were used.

RESULTS

The cells of the SVZ can be isolated and cultured in vitro, and these cells began to form neurospheres after cultured for 3 days at primary passage. While cultured for 7 days, these cells formed more neurospheres, and the volume of the neurospheres became bigger than neurospheres cultured for 3 days. In addition, after cultured for 7 days, the phenomena of fusion of neurospheres and adherent differentiation of neurospheres were observed under inverted microscope. These cells were provided with the typical phenotype of NSCs. The immunofluorescence staining results revealed that these cells showed positive immunoreactivity to Nestin and SOX-2. During the 4 hours BrdU pulse, the number of proliferated NSCs cultured for 3 days (75.817 ± 2.961) was significantly higher than that of NSCs cultured for 7 days (56.600 ± 4.881) (t = 3.366, P = 0.028). The results of MTT assay revealed that the absorbance (A) value of NSCs cultured for 3 days (0.478 ± 0.025) was significantly higher than that of NSCs which were cultured for 7 days (0.366 ± 0.032)(t = 2.752, P = 0.011). After cultivated without EGF and bFGF, the percentage of Tuj-1 and GFAP positive cells in NSCs was 23.1% ± 3.7% and 23.7% ± 3.8% for 3 days and was 40.1% ± 3.6% and 37.1% ± 4.5% for 7 days, respectively, all showing significant differences (t = 3.285, P = 0.030; t = 3.930, P = 0.017).

CONCLUSION

The NSCs from SVZ of neonatal mice have potentials of self-renewal and multipotential differentiation in vitro. With different cultivation time, the potentials of proliferation and differentiation of NSCs are different.