Differentiation of neural stem cells isolated from subventricular zone and hippocampus of neonatal rats in serum-free medium

Dejin SHI; Ying GUO; Chaofeng LIANG; Liping HU; Yan LI

ABSTRACT

BACKGROUND:

Many scholars have paid attentions on neural stem cells (NSCs), which are regarded as the cell source to repair the injured nervous tissues. And it is becoming a key point that NSCs will be obtained in an effective way.

OBJECTIVE:

To observe the characteristics of isolation, proliferation and differentiation of in vitro NSCs from subventricular zone (SVZ) and hippocampus of neonatal rats.

DESIGN:

Single-sample trial.

SETTING:

Department of Neurosurgery in the Third Affiliated Hospital of Sun Yat-sen University.MATERIALS Ten SD rats aged 3 days, of either gender, were offered from the Experimental Animal Center of Sun Yat-sen University. Nestin antibody (rabbit-anti-rat), neurofilament (NF-200) antibody (rabbit-anti-rat), and glial fibrillary acidic protein (GFAP) antibody (mice-anti-rat) were all purchased from Sigma Company.

METHODS:

The experiment was carried out in the Laboratory of Histology and Embryology, Basic Medical College of Sun Yat-sen University from September to December in 2006. According to the ethical requirement, the serum-free DMEM/F12 medium containing basic fibroblast growth factor and epidermal growth factor was utilized to isolate and incubate NSCs from SVZ and hippocampus of neonatal rats. Then the proliferation and differentiation of NSCs in vitro were observed.MAIN OUTCOME

MEASURES:

Fluorescence immunocytochemistry was applied to detect the antigen of nestin expressed from NSCs, NF expressed from neuron cells and GFAP expressed from astrocytes. The differentiation of NSCs was identified.

RESULTS:

The cells isolated from SVZ and hippocampus of neonatal rats possessed the ability of proliferation and self-renewal. Through inverted microscope, nestin positive cells could be found in the cell clone sphere at passage 20 of cells. Cells induced to differentiate could express antigen of NF and GFAP by the attached cell clone sphere.

CONCLUSION:

The NSCs successfully isolated from SVZ and hippocampi of neonatal rats possess the ability of self-renewal, proliferation, and differentiation into neuron-like cells and astrocytes.