Isolation and Differentiation of Neural Stem/Progenitor Cells From Subventricular Zone of One Adult Rat

In adult mammalian brain, neural stem cells are isolated from both the dentate gyrus and subventricular zone. This study aimed to isolate neural stem cells from adult rat subventricular zone and differentiate them into neurons and astrocytes.
In this study, the whole brain was removed after full anesthesia and creating cervical dislocation. Under a microscope, subventricular zone was dissected by a coronal incision in optic chiasm zone. Enzymatic digestion was performed using trypsin-EDTA. The isolated cells were cultured in serum free DMEM/F12 medium, containing bFGF (basic Fibroblast Growth Factor) and EGF (Epidermal Growth Factor) growth factors.
Neurospheres were observed five days after culturing. Immunocytochemistry was used to investigate nestin gene expression and identify neural stem cells. Neural stem cells were differentiated in poly-L-lysine coated plates in the absence of growth factors. The expression of GFAP, β tubulin III, and nestin genes were analyzed by RT-PCR. The results of immunocytochemistry confirmed nestin gene expression in the neural stem cells. Phenotype of neurons and astrocytes were observed 5 days after cell culture in differentiation medium. RT-PCR analysis revealed the expression of GFAP and β tubulin III genes.
The results of this research show that only one rat brain is needed for neural stem cells isolation and differentiation to neurons and astrocytes.