Inhibition of vitamin C transport impairs neuronal differentiation of hiPSCs

Abstract

Vitamin C is an antioxidant that acts as a free radical scavenger and cofactor for many important biological reactions. It plays an important role in stem cell homeostasis, proliferation and differentiation. However, the role of vitamin C transporters in the neuronal differentiation of human induced pluripotent stem cells (hiPSCs) has not yet been investigated. Therefore, we investigated if vitamin C transporter knockdown impairs neuronal differentiation in hiPSCs. We differentiated hiPSCs into neurons from undifferentiated hiPSCs using established protocols and employed siRNA‐mediated knockdown experiments. Validation of results was performed by RT‐qPCR, Western blot analysis, RNA‐Sequencing (RNA‐Seq) and confocal imaging. First, we determined the expression of the human sodium‐dependent vitamin C transporters, hSVCT1 and hSVCT2 (products of the SLC23A1 and SLC23A2 genes, respectively) using RNA‐Seq analysis. RNA‐Seq analysis showed that both SLC23A1 and SLC23A2 were expressed during differentiation of neuronal hiPSCs. As expected, the immunofluorescence results showed that the hSVCT2 is predominantly expressed on the plasma membrane during neuronal hiPSCs differentiation. We validated the effectiveness of SLC23A1, SLC23A2and SCAMP2 (a vitamin C transporter interacting protein) knockdown by gene specific siRNA using RT‐qPCR analysis. RT‐qPCR analysis found that the expression of all three genes were significantly reduced. We also demonstrated that knockdown of SLC23A1, SLC23A2 and SCAMP2 was accompanied by a significant decrease in the number of Tuj‐1 (early neuron marker)‐positive cells during neuronal hiPSCs differentiation. Reciprocally, we demonstrated vitamin C supplementation dose‐dependently increases Tuj‐1 positive cells during neuronal hiPSCs differentiation. In conclusion, we demonstrate that knockdown of vitamin C transporter impairs neuronal differentiation of hiPSCs. These findings suggest that vitamin C and its transporters play an important role in neuronal differentiation, development, and function in hiPSCs.