Isolation, culture and identification of neural stem cells in new-born rats

Abstract

The cortexes were obtained from new-born rats and dissociated to single cells by triturating. The cells were cultured in neural stem cell (NSC) culture medium (DMEM supplemented with bFGF, EGF and B27) and formed primary neurospheres after 7 days. Single cells dissociated from neurosphere were cultured in 96-well plates and formed single-cell cloning neurosphere 7 days later. The primary and single-cell cloning neurospheres were both positive for the immunofluorescent staining of nestin and were identified as NSC. It was proved that NSC can be expandedin vitro and provide seed cells for neural tissue engineering.

Keywords

neural stem cell / cell culture / tissue engineering

References

[1] TempleS. Division and differentiation of isolated CNS blast cells in microculture. Nature, 1989, 340: 471-471

[2] DavisA A, TempleS A. Self-renewing multipotential stem cells in embryonic rat cerebral cortex. Nature, 1994, 372: 263-263

[3] ReynoldsB A, WeissS. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, 1992, 255: 1707-1707

[4] GageF H. Mammalian neural stem cells. Science, 2000, 287: 1433-1433

[5] ReynoldsM. Stem cell in the central nervous system. Science, 1997, 276: 66-66

[6] UrbanL, ZimmermanL B, ReynoldsM, et al.. CNS stem cells express a new class of intermediate filament protein. Cell, 1990, 60(23): 585-585

[7] HulspasR, TiarksC, ReillyJ, et al.. in vitro cell density-dependent clonal growth of EGF- responsive murine neural progenitor cells under serum-free conditions. Exp Neurol, 1997, 148(1): 147-147

[8] RichardsL J, KilpatrickT J, BartlettP F, et al.. De novo generation of neuronal cells from the adult mouse brain. Proc Natl Acad Sci USA, 1992, 89(18): 8591-8591

[9] WeissS, DunneC, HewsonJ, et al.. Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci, 2004, 6(23): 7599-7599