Electrical Stimulation Induced Structural 3D Human Engineered Neural Tissue with Well-developed Neuronal Network and Functional Connectivity

Three-dimensional (3D) neural tissue engineering is expected to provide new stride in developing neural disease models and functional substitutes to aid in the treatment of central nervous system injury. We have previously detailed an electrical stimulation (ES) system to generate 3D mouse engineered neural tissue (mENT). However, ES-induced human ENT (hENT) has not previously been either investigated or identified in structural and functional manner. Here, we applied ES as a stimulator to regulate human neural stem cells in 3D Matrigel, explored the components and functional properties of hENTs.By immunofluorescence chemical staining and electron microscope imaging, we evaluated the effects of ES on (1) neuronal differentiation and maturation, (2) neurites outgrowth and alignment in hENT, (3) formation of synapses and myelin sheaths in hENT. We further investigated the formation of synaptic connections between-fused mouse and human tissue. We used calcium imaging to detect activities of neurons in hENT culture.ES could induce neuronal differentiation, the orderly growth of neurites and the maturation of neuron subtypes to construct a well-developed neuronal network with synapses and myelin sheaths. Most importantly, we discovered that raising extracellular Kconcentration resulted the increasing neuronal excitability in the hENT, indicating electrical activities in neuronal cells.We applied ES to generate the organised 3D hENTs and identified them in both structural and functional manner.