Electrical stimulation boosts differentiation and maturation of neural stem cell by improving microenvironment on carbon nanotube scaffold

Abstract

Electrical stimulation (ES) has been applied in the cell culture system to enhance neural stem cells (NSCs) proliferation, migration, and neuronal differentiation. The underlying mechanisms, however, remain thoroughly understood. In this study, we presumed that ES might provide an effective cue for NSCs that combined with a carbon-nanotube topographical cues to initiate biological activations of NSCs. The microenvironment in the neurogenesis of NSCs following ES was a matter of interested research in the current study. Methods:NSCs was isolated from brain of embryonic day 14.5(E14.5) C57 mice. The following experiments can be divided into three groups: PDL, carbon nanotube scaffold (CNTs) and CNTs-ES. After seeding two days, we applied ES to NSCs and the parameters are frequency at 20 Hz, current at 1mA for 2h. Immunfluorescence assay, RT-PCR and Western bolt were used to test the neural markers, such as DCX, Tuj1, MAP2 and NeuN. The neurite growth of differentiated NSCs was constructed by using the neurolucida software. Whole-cell patch clamp recording was performed using an EPC-9 amplifier. Calcium imaging was measured with the indicator Fura-2 AM in living cell system. RNA-seq was used to detect the change of transcriptional level during the development of NSCs to neurons, cytokines protein arrays and hierarchical clustering analysis will be done to check the change of microenvironment.Results:ES could promote elongation of neurite growth from NSCs compared with the CNTs condition. ES also increased the expression of neural markers during the differentiation and maturation of NSCs, including DCX, Tuj1, MAP2 and NeuN on day 7 compared with no-ES group. ES could enhance the expression of PSD95 and BDNF on day 14, which meant the differentiated neurons of CNTs-ES group could be more mature and better functionality. Then, Electrophysiological results showed that the frequency and peak amplitude of action potential in the CNT-ES group were higher than no-ES CNTs group. ES was performed during live cell monitoring, and intracellular calcium concentration was rapidly increased and quickly decreased after ES was withdrawn. RNA-seq results and cytokines protein arrays suggested that multiple pathways associated with NSCs growth, differentiation and maturation, especially about the calcium channels. Conclusion: ES could exert a positive effect on the growth, differentiation and maturation of the whole process of NSCs. This article reveals a new mechanisms that ES combined with CNTs regulates the cytokines and trophic factors, thus improved the microenvironment for NSCs differentiation and maturation.