Cultivation,Proliferation And Differentiation Of Human Embryonic Neural Stem Cells And Brain Tumor Stem Cells In Vitro

[Objective] To explore the methods of isolation, cultivation and expansion in vitro of human embryonic neural stem cells and brain tumor stem cells and to observe the characteristics of proliferation and differentiation in vitro. To establish the techniques of isolation, cultivation and in vitro differentiation of human embryonic neural stem cells and brain tumor stem cells for further research.[Methods] Neural stem cells :The cortex tissues of human embryonic brain were isolated from human embryo at 8-12 weeks of gestation by mifepristone abortion. Unicellular suspension was obtained from the embryonic brain tissues by dissociating enzymatically and mechanically. A serum free medium(DMEM/F12) containing EGF and bFGF(both 20ng/ml) was used to culture , expand and passage neural stem cells continuously in vitro. Cell morphology was studied with phase contrast microscope. Immunocytochemistry are used to identify expression of Nestin(marker for the neural stem cells), NSE(marker for the neurons), GFAP(marker for the astrocytes) and CNP(marker for the oligodendrocytes). Brain tumor stem cells: Human glioma cell line SHG-44 maintained for long term and cell suspensions from glioma samples collected shortly after operation, were cultured in serum-free DMEM/F12 containing bFGF, and EGF respectively. Marker CD133 were used to isolated tumor stem cells by magnetic cell sorting. The cells cultured in condition described above were also detected with immunohistochemistrical staining with antibodies against Nestino CD133+ cells were acquired and induced to differentiate with medium containing 10% fetal bovine serum, and immunocytohistologically examined with differentiation related cell surface markers, such as nestin, NSE and GFAP before and after differentiation.