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

Figure 1

Expression of zinc homeostasis genes during stem cell differentiation into motor neurons. (a) Quantitative evaluation of mRNA expression levels normalized to HMBS of selected zinc homeostasis and the zinc-dependent SHANK genes. Analyses were performed in triplicate () and the mean normalized expression is shown. Neurofilament H (NEFH) expression was used to control successful differentiation and increases significantly in NRs and rises again after a significant decrease in NSC in suspension (iPSC versus NR: ; NR versus NSC: ). Several zinc transporters show developmental stage-dependent expression as well as altered expression levels during differentiation. Expression of ZnT1 is significantly increased in motor neurons at d42 compared to the iPS cell stage (). ZnT2 expression in turn was only detected above background in NSC. Expression of ZnT3 is very low in early stages (iPS, EB, and NR) but increases in NSC and becomes significant in motor neurons (iPSC versus MN d21: ) in which ZnT3 levels remain elevated. No expression of ZnT4 was detected in iPS cells, but mRNA levels gradually increase from EB to motor neurons (EB versus MN d42: ; NR versus MN d21: ). This is also observed for ZnT5 expression; however it is only seen as trend. Additionally, the expression of ZnT6 was found to be significantly higher in motor neurons compared to iPS cells (iPS versus MN d42: ). Along with the increase in expression of some zinc transporters during motor neuron differentiation, the mRNA levels of MTF1 are significantly higher in motor neurons compared to iPS cells (iPSC versus MN d42: ; NSC versus MN d42: ; MN d21 versus MN d42: ). While ZIP1 and ZIP3 are expressed on similar level through all stages of motor neuron differentiation, the expression of MT2A shows some variance in iPSC but otherwise is significantly higher in motor neurons compared to most previous stages and significantly increases in motor neurons between d21 and d42 (EB versus MN d42: ; NSC versus MN d42: ; MN d21 versus MN d42: ). The expression of MT3 in turn could only be detected in NSC and motor neurons but not in earlier stages. Although SHANK proteins are associated with synapses that only occur in motor neurons, their expression can be already detected in iPS cells and later stages of motor neuron differentiation. SHANK1 and SHANK2 expression increases towards later stages (SHANK1: iPSC versus NR: ; iPSC versus MN d21: ) (SHANK2: EB versus MN d42: ; NR versus MN d42: ; MN d21 versus MN d42: ), although the expression of SHANK2 in iPS cells shows high variability. SHANK3 is expressed on similar level throughout differentiation. (b) Representative images of hiPSC undergoing differentiation to motor neurons (MN) via the generation of embryoid bodies (EB), neural rosettes (NR), and neural stem cells (NSC). (c) Comparison of mRNA expression levels across different zinc homeostasis genes. In general, expression levels of ZnT1 and ZnT6 were the highest compared to other ZnTs across all developmental stages. Similarly, ZIP1 expression was high throughout all phases of motor neuron differentiation, while MTF1 and MT2A expression increased in old motor neurons. ; .

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