Culture and identification of monoclonal neural stem cells derived from cerebral cortex

Abstract

To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice, new-born mice cerebral cortex was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. Immunocytochemistry was used to dtect the specific marker of neuroepithelial stem cells (Nestin) of the primary and monoclonal neurospheres. In the differentiated cells we detected the specific antigen of NF-200 and GFAP. Our results showed that the primary neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin and had capabilities of self-renewal, proliferation and the potentiality of differentiation into neurons and glial cells. It is concluded that monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new-born mice by limiting dilution.

Keywords

neural stem cells / monoclone cell / culture

References

[1] TempleS.. The development of neural stem cells. Nature, 2001, 414(6859): 112-117

[2] ReynoldsB. A., WeissS.. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, 1992, 255(5052): 1707-1710

[3] GageF. H.. Mammalian neural stem cells. Science, 2000, 287(5457): 1433-1438

[4] AmarA. P., ZlokovicB. V., ApuzzoM. L.. Endovascular restorative neurosurgery: a novel concept for molecular and cellular therapy of the nervous system. Neurosurgery, 2003, 52(2): 402-412

[5] ModoM., RezaieP., HeuschlingP., et al.. Transplantation of neural stem cells in a rat model of stroke: assessment of short-term graft survival and acute host immunological response. Brain Res, 2002, 20958(1): 70-82

[6] BenedettiS., PirolaB., PolloB., et al.. Gene therapy of experimental brain tumors using neural progenitor cells. Nat Med, 2000, 6(4): 447-450

[7] ReynoldsB. W., WeissS.. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol, 1996, 175(1): 1-13

[8] GrittiA., ParatiE. A., CovaL., et al.. Multipotential stem cells from the adult mice brain proliferate and self-renew in response to basic fibroblast growth factor. J Neurosci, 1996, 16(3): 1091-1100