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ISOLATION, CULTIVATION AND IDENTIFICATION OF HIPPOCAMPUS NEURAL STEM CELLS FROM NEWBORN RATS |
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Saturday, 24 December 2005 |
Jian Yang, Xian-Di Zhang, and Li-Dong Pan
China Rehabilitation Research Center, Beijing, 100068 China
The central nervous system is generated from neural stem cells during embryonic development. These cells have self-renewal capacity and generate multiple differentiated neural cells types. In last few years it has been found that there are neural stem cells also in the adult mammalian brain and spinal cord. In the present study, we investigated the proliferation and multipotential properties of hippocampus neural stem cells from newborn rats in vitro.
The hippocampal tissues from newborn rats were minced and enzymatically dissociated in trypsin. The obtained cells were cultured in tissue culture flask in DMEM/F12 medium, containing 15ng/ml EGF, 15ng/ml bFGF. The majority of the dissociated cells in the cultures died after 3 days. However, some cells survived and in about 2 weeks small aggregates of cells which had divided became apparent in cultures of hippocampal tissue from newborn rats. These aggregates had typical neurosphere morphology and continued to proliferate over several weeks. Immunofluorescence staining confirmed that the neurosphere cells were nestin-expressing neural stem cells. When the neural stem cells were induced to differentiate in culture medium with 10% fetal calf serum and without growth factors, the cells attached to culture dish and spread out and cells of different morphologies were apparent. Cell-type-specific antibodies against NSE (neuron), GFAP (astrocytes) and CNP (oligodendrocytes) were used to test for multipotency. It was found that the differentiated cells expressed the phenotypes of neurons, astrocytes and oligodendrocytes.
In this study we demonstrated the hippocampal tissue from newborn rats retain neural stem cells which are able to proliferate and differentiate in vitro.
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