Investigation of the mechanism of enhanced and directed differentiation of neural stem cells by an atmospheric plasma jet: A gene-level study

Cold atmospheric plasmas (CAPs) have been shown to be capable of enhancing stem cell differentiation, especially directed differentiation of neural stem cells (NSCs). Consequently, one-step CAP treatment shows promise as an aid to tissue transplantation. However, the mechanisms involved in the enhancement of NSCs differentiation by CAP treatment are not yet fully understood. We have previously shown that in atmospheric helium plasma jet treatment, nitric oxide (NO) is the main factor involved in promoting NSC differentiation. This article further investigated the possible signaling pathways stimulated by NO in the neuronal differentiation of C17.2-NSCs after plasma treatment. Extracellular and intracellular NO concentrations were measured at different time points of incubation to monitor NO production. Meanwhile, the expressions of related genes and proteins were detected by quantitative real-time polymerase chain reaction and western blot, respectively. It is found that plasma treatment could both generate extracellular NO and increase extracellular NO concentration by inducing inducible nitric oxide synthase expression. The synergetic effect of extracellular and intracellular NO then downregulated Notch1 and Id2, and upregulated Ngn2 and Ascl1, thereby activating downstream NeuroD expression and finally enhancing and directing differentiation of NSCs into neurons.