A Mechanistic Study Of Regulation Of Neural Stem Cell Differentiation On Graphene Substrates

The neural stem cells(NSCs)are a self-renewing and multipotent cell population in the central nervous system,which exhibit promising prospects in developing cell therapies for neural regeneration.In order to govern cell-specific behaviors in tissue engineering for neural repair and regeneration,we use material to regulate proliferation,differentiation and migration of neural stem cells.Graphene has been reported to be a potential candidate for use as a neural interfacing material.It has been shown that the graphene can promote stem cells regeneration and induce stem cell to preferentially differentiate into specific lineage.The research discovered the possibility of using graphene as a neural interfacing material because it could promote neurite sprouting and outgrowth.These pioneering studies clearly demonstrate the great potential of graphene-based materials in cell therapies.However,the underlying mechanisms behind the altered cell behaviors,such as enhanced differentiation and promoted cellgrowth,remain largely unknown.Therefore,we explore trascriptomic profiling differentiation of neural stem cells on graphene substrates.Firstly,gaphene samples were synthesized according to the previously published chemical vapor deposition method and then transferred to a tissue-culture polystyrene(TCPS)substrate(TCPS alone was used as the control substrate in the following experiments).The graphene films exhibited good characteristics of light transmission.The surfaces of the graphene films were shown by SEM that consist of many ripples and wrinkles on the micrometer scale.To further validated by transmission electron microscope images and Raman spectroscopy of the graphene films,we estimated that the graphene films contained 3~5 layers.NSCs were identified by several methods.Free-floating neurospheres can be formed at DIV5,which shows NSC ability to proliferate.The immunostaining against nestin,a NSC marker,shows that most of the cells were nestin positive.After a week,SEM showed that the cells adhesion by spreading extensively and forming strong filopodia/graphene interactions.This showed that the cells grown on graphene substrates displayed typical cell body morphology and size accompanied by neurite growth and extension.Immunostaining of NSC-differentiated neural network grown on TCPS and graphene substrates with anti-MAP-2,it showed that graphene promote differentiation of NSCs.Lastly,the RNAseq results showed that NSC differentiated 21 days on graphene substrates have genes highly enriched and differentially expressed compared with TCPS in vitro.The results indicated that graphene regulate the gene of NSC differentiation.This study might provide new therapeutic method and targets for graphene as a material for neural interfacing in biomedical applications.