Cellular Zinc Homeostasis Contributes to Neuronal Differentiation in Human Induced Pluripotent Stem Cells

Figure 2

Zinc deficiency alters the expression of zinc homeostasis genes during stem cell differentiation into motor neurons. Quantitative evaluation of mRNA expression levels normalized to HMBS of selected zinc homeostasis and the zinc-dependent SHANK genes. Data shows the average normalized gene expression based on measurements. Neurofilament H (NEFH) expression was used to control successful differentiation. For statistical analysis, a 1-way ANOVA was used followed by Tukey’s multiple comparison test. Several zinc transporters show expression levels depending on the availability of zinc in the medium. Comparing cells grown in zinc depleted medium with cells grown in medium which was resupplemented with zinc in the amount used in control medium after zinc depletion, a significant difference in ZnT1 expression was found in d42 motor neurons (one-way ANOVA: ; MN d42 Ctrl versus −Zn: ). No significant difference was detected regarding the expression of ZnT2, ZnT3, ZnT4, ZnT6, and MTF1 comparing controls and zinc deficient conditions. The expression of ZnT5 was significantly increased in motor neurons at d21 under zinc deficient conditions (one-way ANOVA: ; MN d21 Ctrl versus −Zn: ). The expression of ZIP1 was significantly reduced in neuronal stem cells under zinc deficient conditions (one-way ANOVA: ; NSC Ctrl versus −Zn: ). The expression of ZIP3 was not found to be altered, similar to the expression levels of MT2A. MT3 expression was significantly higher in neuronal stem cells under zinc deficient conditions (one-way ANOVA: ; NSC Ctrl versus −Zn: ). The expression of SHANK1 was increased in motor neurons at d21 under zinc deficient conditions (one-way ANOVA: ; MN d21 Ctrl versus −Zn: ) and the expression of SHANK3 in neural rosettes under zinc deficient conditions (one-way ANOVA: ; NR Ctrl versus −Zn: ). No change was observed for SHANK2 expression. ; .