Human neural stem cell differentiation and electrical stimulation on a novel single walled carbon nanotube-polymer composite

Increasing control of human neural stem cell (hNSC) differentiation is critical to development of cellular models for neurodegenerative diseases such as Parkinson's disease because current methods do not result in the required fully developed cells. In addition, existing cell culture and differentiation regimen are inefficient due to lengthy differentiation times and low yields of functional cells. The use of carbon nanotubes (CNTs), particularly in 3D geometries, offers a possible solution by improving the kinetics and efficiency of NSC differentiation. Electrical stimulation through conductive substrates, such as CNTs, can cause increased rates of NSC differentiation. In this work, a combination of a three dimensional, in vivo mimetic, single walled CNT substrate and electrical stimulation is used to investigate survival and differentiation behaviors of hNSCs derived from induced pluripotent stem cells (iPSCs). First, fibrous poly(lactic-co-glycolic acid) (PLGA) substrates, with an average fiber diameter of 1.11?m, are manufactured via electrospinning onto a flat plate collector. A novel vacuum driven impregnation technique forces an aqueous dispersion of CNTs to coat the PLGA fibers while maintaining the microscale features of the fibers' architecture. The CNTs provide increased electrical conductivity, >0.1 S/m up to 25 S/m, and nanosurface roughness, which can increase neurite interfacial interactions, resulting in improved differentiation of NSCs to neurons. Immunocytochemistry of hNSC differentiated on these surfaces reveal an 18% rise in the number of cells staining positive for neurofilament M (NFM), a marker of maturing neurons, on CNT versus control PLGA substrate after 14 days of differentiation. When a 10 minute, 30?A direct current stimulation is applied on the 3rd day of differentiation, there is a further 4% improvement in the number of cells staining positive for NFM on the 14th day of differentiation. Calcium imaging indicates that on day 14, 0.3% of cells on PLGA scaffolds compared to 5.9% of cells on the CNT composite substrate had an electrical event in response to electrical stimulation. These results strongly support the use of electrically conductive CNT substrates for neural differentiation and suggest electrical cues could be more systematically investigated for directing the differentiation process to sub-type specific and functional human neuronal systems.