The Experiments On The In Vitro Differentiation Of Fetal Brain Derived Neural Stem Cells

Objective: To observe the fetal brain derived neural stem cells' differentiation in the normal adult cerebrospinal fluid and investigate the potential mechanism, and to investigate the role of ascorbic acid in neural stem cells' differentiation into dopaminergic neurons.Methods: The primary culture of fetal brain derived neural stem cells were transferred into the medium DMEM/F12+B27+30%CSF and DMEM/F12+B27+10%FBS. At different time points the differentiated cells are immuno-stained and the various types of cells are counted. The primary cultured fetal mesencephalic neural stem cells are passaged every 7days.After 7 days'culture ,upon mitogen withdrawal the neurospheres of different passages were differentiated in vitro in differentiation medium containing fetal bovine serum(DMEM+B27+10%FBS)or ascorbic acid (DMEM+B27+10%FBS+AA).Ten days later all the differentiated cells were immunohistochemically stained using rabbit anti-tyrosine hydroxylase(anti-TH) and the TH(+)cells were couted. Results:The neural stem cells can differentiated into neurons,astrocytes, and oligodendrocytes in normal CSF .The proportion of astrocytes is significantly increased compared to their differentiation in serum-containing medium; The yield of TH(+)cells dropped rapidly afterbeing passaged. The TH(+) cells derived from primary neurospheres(passage0,P0 was about 5.65% 1.39%.As to the P1 neurospheres the ratio of TH(+)cells is about 1.56 0.37%.After the third passage the ratio of TH(+)cells was less than 0.01%.10-4M ascorbic acid can increase the yield of TH(+)celIs the most effectively. Treatment with 10-4M ascorbic acid during the whole differentiation period lead to a final ratio of TH(+) cells about12.8% 1.9%.AA added during 1-3 days was more effective than during 5-10 days.Conclusion: In normal CSF the neural stem cells can survive and differentiated into neurons, astrocytes , and oligodendrocytes,which provides support of the neurotransplantation using neural stem cells, ascorbic acid can increase the number of dopampinergic neurons differentiated from fetal mesencephalic neural stem cells,which contribute to the in vitro generation of DA neurons.