| [objective] To detect the intracellular zinc level change during the process of induced pluripotent stem cells(iPSCs)differentiating into neural stem cells(NSCs)and explore the effects of different extracellular zinc level on iPSCs neural differentiation;To preliminarily explore the mechanism of zinc-promoted neural differentiation of iPSCs and provide new ideas for neural regeneration therapies based on patient-derived induced pluripotent stem cells.[Methods] iPSCs were reprogrammed from human gingival fibroblasts(hGF)and cultured in vitro.iPSCs were induced into NSCs using PSC Neural Induction Medium(Gibco)and identified by flow cytometry and immunofluorescence.During the process of neural induction,zinc fluorescent chemosensor BTA was used to detect the relative intracellular zinc level under confocal microscope.Different concentrations of Zn Cl2(2.5-20?M)and zinc chelator TPEN(0.5-2?M)was added into the medium to establish different zinc conditions.Cell proliferation and apoptosis were analyzed using CCK-8 assay and Apoptosis Detection Kit respectively.According to the results of cell proliferation and apoptosis assay,2.5?M?5?M?10?M Zn Cl2 and 1?M TPEN was used in following experiments.The neurite length of cells in different groups was measured using Image J software.NSCs marker Nestin expression and phosphorylation level of ERK1/2,STAT3 were detected by western blot and the gray scale analysis was performed using Image J software.Nestin expression was also detected by ELISA.Then we used ERK inhibitor VX-11 e to block the activation of ERK,expression of Nestin,ERK1/2,p-ERK1/2,STAT3,p-STAT3 was analyzed by western blot.[Results] 1.hGF-derived iPSCs were induced into NSCs successfully.After neural induction,the morphology of cells is different from that of iPSCs.The cells no longer grow in the form of cell colonies,but grow in a monolayer adherent to the plates.The shape of the single cell is irregular or radial,with neurite-like structures observed;Immunofluorescence showed that the cells expressed NSCs markers Nestin,Pax6,Sox2 positively.Flow cytometry analysis showed that CD 133 were expressed positively and CD45,CD34 were expressed negatively;2.Fluorescent detection showed that after changing the zinc level in medium,intracellular zinc level changed correspondingly.During the process of neural induction,intracellular zinc level showed an increasing trend.After neural induction and passaging,intracellular zinc level in NSCs was significantly higher than iPSCs(p < 0.05);3.Comparing with the control group,when Zn Cl2 added into the medium reached 20?M,cell proliferation was significantly inhibited(p < 0.05).In cell apoptosis assay,when the concentration of TPEN reached 2?M,the apoptosis rate increased.4.At day 7 of neural induction,Nestin expression of cells that added Zn Cl2 increased significantly(p < 0.05),however,among different concentrations(2.5?M,5?M,10?M),no significant difference was observed(p > 0.05);Nestin expression of cells that added TPEN decreased significantly comparing with the control group(p < 0.05);Cells that added 5?M Zn Cl2 at day 3 and cells in control group at day 7 showed no significant difference in Nestin expression(p > 0.05),suggesting that the addition of Zn2+ accelerated the differentiation of iPSCs into NSCs.5.Phosphorylation levels of ERK1/2 and STAT3 are similar to the Nestin expression level,which increased after Zn Cl2 addition and decreased after TPEN addition;After blocking ERK phosphorylation using VX-11 e,phosphorylation of STAT3 was also inhibited significantly.Among groups treated with VX-11 e,no significant difference of Nestin expression was observed.[Conclusions] 1.Zinc addition of chelation in medium can cause corresponding changes of intracellular zinc level.During the process of iPSCs neural differentiation,the intracellular zinc level has an increasing trend;2.Zinc can promote differentiation of hGF-derived iPSCs into NSCs.3.Zinc may play a role in iPSCs neural differentiation via ERK-STAT signaling. |
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