Carbon Nanotube Composites as Multifunctional Substrates for In Situ Actuation of Differentiation of Human Neural Stem Cells

Despite the promise of human neural stem cells (hNSCs) as an emerging cell source for neural tissue engineering, hNSC applications are hindered by the lack of advanced functional biomaterials that can promote cell adhesion, survival, and differentiation while also integrating neuronal stimulatory cues, specifically electrical stimulation. Electrospun fibrous substrates with controlled fiber architectures provide topographical cues to cells by presenting three dimensional (3-D) geometries that are representative of the extracellular matrix (ECM),1 defined by a high surface-to-volume ratio and porosity, and are thus better suited for differentiation studies of neural stem cells than standard two dimensional substrates. The architecture of ECM is of special importance because it supports 3 -D cellular networks together to form a tissue, allows for the proliferation and growth of cells, and regulates cellular processes capable of enhancing neurite outgrowth and neuronal differentiation of several cell types, including embryonic stem cells and hESC-derived NSCs2-6. Furthermore the inherently high surface to volume ratio of electrospun polymer substrates can facilitate mass transfer of nutrients and waste, promote cell attachment, and enable drug loading, properties that are inherent to bioactive matrix microniches. For stem cells, conductive substrates can promote neuronal maturation by providing electrical shortcuts between developing cells, while also permitting application of electrical stimuli that can mimic the electrophysiological environment experienced by cells in a variety of biological processes, including muscle contraction, wound healing, and synaptic transmission7-12. In addition to enhancing neurite outgrowth and neuronal maturation, applied electrical stimulation may also direct neural stem cell migration, opening the possibility to guide these cells towards injured sites8. Various methods of delivering electrical stimulation to cells in culture include 2 -dimensional (2-D) substrates such as etched ITO glass13 and conductive polymers1, 14-19 like polypyrrole17. However, the use of conductive polymers alone is impeded by their poor processability, electroactive stability, and mechanical properties after doping20, 21. Another alternative has emerged involving nanocarbon materials such as carbon nanotubes (CNT)22, 23 and graphene24. In particular, single-walled carbon nanotubes (SWNT) have been employed due to their inherently high conductivity and the ability to regulate neuronal behavior both structurally and functionally25. Along with their biocompatibility at low concentrations, SWNT are ideal candidates for biomedical composites26, 27. It has been shown that SWNT interfaced with neural cells can promote neuron growth28-30 and enhance differentiation of NSCs into neurons31, 32. This is likely a result of a combination of topographical cues, enhanced signal transmission from the tight contacts formed between the SWNTs and the neuron membranes, and differential production of ECM proteins that modulate synaptic stability7, 32-34. While multi-walled carbon nanotubes (MWNTs) have been incorporated into electrospun fibers35, the incorporation of SWNTs into fibrous composite substrates that mimic the ECM has proved challenging36. The use of SWNTs during the electrospinning process could be circumvented altogether if the SWNT incorporation or deposition can be designed post facto, thus avoiding any bulk modification of the substrate properties and thereby retaining the SWNT bio-interfacial features. Doing so yields the additional benefit of using insulating polymers, already extensively used for differentiation studies, and thereby providing grounds for the use of highly biocompatible substrates. Other methods to do so have included spraying SWNT onto substrates37, layer-by-layer deposition (LbL)31, 38 and the attachment of SWNT to self assembled monolayers (SAM)39. In the first method, the growth of SWNT on the substrates can leave behind unwanted catalyst particles detrimental to cell viability. Regarding the latter two methods, enhancement of the differentiation kinetics has been reported with mouse embryonic stem cells by the LbL31 approach and with immortalized human neural stem cells via SAM40 approach, neither of which makes use of electrical stimulation. This study is the first demonstration of electrically actuated SWNT-based composites for differentiation of human neural stem cells. We fabricated extracellular matrix-mimetic, composites by vacuum impregnation of electrospun poly(lactic-co-glycolic acid) (PLGA) membranes with single-walled carbon nanotubes (SWNTs) and investigated the ability of these substrates to enhance differentiation of induced pluripotent stem cell (iPSC)-derived NSCs. The SWNT-polymer substrates are electrically conductive, mechanically robust, and highly biocompatible with human NSC cultures in vitro and showed enhanced levels of electrically responsive cells. Notably, changes in the expression of two major neuronal markers, Neurofilament M (NFM) and microtubule -associated protein-2 (MAP2) of 14-day cultures showed that the composite enhanced neuronal differentiation of NSCs compared to PLGA controls without SWNT. To further utilize the multifunctional nature of SWNT-PLGA to affect neurogenesis, early NSC cultures on SWNT-PLGA were subjected to a 10 minute, 30μA direct current regimen of electrical stimulation. The electrical stimulation markedly increased neuronal differentiation after 14 days. These results highlight the multifunctionality of SWNT-PLGA, which afford a fibrous topography with high surface area to volume ratios to help to organize neuronal networks, along with the ability to exploit electro-conductivity to stimulate neuronal induction and neuronal maturation.