| Objective: To explore the culture methods of neural stem cells from the mesencephalic flexure of embryonic mice on the base of successful cultivation of neural stem cells from the cortic of embryonic mice; And to investigate the difference of culture and differentiation between cortical neural stem cells (C-NSCs) and mesencephalic neural stem cells (M-NSCs); And to investigate the effects of ascroid acid (AA) and interleukin-l bate (IL-1β) on the induction to differentiation of M-NSCs and C-NSCs into TH positive (TH-ir) neurons in vitro, which establish a basis for further research in neural stem cells and cell transplantation therapy for Parkinson's disease..Methods: 1. Embryonic 12-13 mesencephalic flexure of mice were dissociated and single cell suspensions were achieved by trypsin digestion and mechanical dissociation in sterile conditions, And the cells were plated in uncoated culture dishes in DMEM/F12 supplemented with bFGF and B27, then they were maintained, expanded and passaged by mechanical methods. Immunocytochemical techniques were used to identify the stem cells and their progeny.2. We dissociated and cultured NSCs from the embryonic cortic and ventral mesencephalon respectively. These cells were induced to differentiate in DMEM/F12 medium with 10%FBS (without growth factors bFGF and B27). After examining the incubative features of C-NSCs and M-NSCs with reverse phase-contrast microscopy, immunocytochemical techniques were applied to detect the specific antigen (TH) of neurons differentiated from the two kinds of NSCs. We observed and compared the difference of the two kinds of NSCs.3. The two kinds of NSCs were cultured and passaged in vitro, then IL-1β and AA were used to induce differentiation in the condition of DMEM/F12 medium with 10%FBS. Immunocytochemical techniques and DAPI-cell-count assessment were used to detect the TH-ir cells from two NSCs differentiation.Results: 1. In the serum-free medium containing bFGF and B27 the cells from embryonic mesencephalon can be divided and expanded in vitro, in which nestin antigen was expressed; In the serum-containing medium which was removed bFGF and B27, the cells possessed the ability to differentiate into neurons and glia.2. The density of NSCs in cortic was higher than that in ventral mesencephalon and the culture condition of C-NSCs was simpler than that of M-NSCs. There were some TH positive cells differentiated from M-NSCs but none came from C-NSCs.3. In the inducing experiment, the proportions of M-NSCs differentiation into TH positive neurons were about 10%, 9% and 11% in AA group, IL-ip group and AA+IL-ip group, respectively, Which were much higer than the control group (1%, P <0.01). There were no statistical differences among the AA group, IL-ip group and AA +IL-ip group, but the morphology of TH-ir neurons were more mature in AA +IL-ip group than that in IL-ip group. No TH positive cells was differentiated from the C-NSCs under these experimental conditions.Conclusions: Neural stem cells, which present in the mesencephalon of embryonic mice, can be cultured, passaged and differentiated into neurons and glia in vitro. In compaison with the C-NSCs, the cultivation of the M-NSCs is more diffcult in vitro, but it had more ability in differentiation into TH positive neurons. IL-ip and AA can significantly increase the differentiation of M-NSCs into TH positive neurons. AA +IL-1P can enhance the mature phenotype of TH positive neurons, but the combination did not increase the rate of production of TH positive cells, and the C-NSCs can't differentiate into TH positive neurons under our experimental conditions. So the M-NSCs is the ideal cell sources for cell transplantation therapy for Parkinson's disease.
|
PDF Full Text Request