Construction Of Engineered Neural Tissue By Regulating Human Neural Stem Cells Through Electrical Fields

Objective:Human Neural Stem Cells(hNSCs)were used as seed cells and plated into Matrigel to construct a engineered neural tissue.Physiological Electric Fields(EFs)were applied as a stimulator to regulate the differentiation,protuberant growth and maturation of hNSCs,this 3d model provides a novel method for the treatment of central nervous system diseases.Methods:hNSCs were amplified and cultured in vitro by suspension culture,and hNSCs were stimulated by EF stimulation to construct an ordered human engineered neural tissue(hENT)in 3D Matrigel.Immunofluorescence staining,3D tissue co-culture technology and calcium imaging technology were applied to detect the cell composition and tissue structure,formation and distribution of synapses and myelin sheath,as well as neural network activity of hENT at cellular,tissue structural and functional levels.Results:(1)hNSCs were cultured in suspension and formed neurospheres,which expressed NSCsspecific markers such as Musashi1 and Nestin.(2)The results showed that the ratio of Tuj1 positive cells increased from 18.67 ± 2.69 % to68.53 ± 4.28 % at day 14 after EF stimulation.After 28 days of culture,MAP2 positive cells reached 72.48 ±7.28 %;With 24 d culture,35.3% of cells within the hENT expressed the early marker of Glutaminergic neuron,Tbr1.On day 45,a large number of terminal-differentiated mature neuron subtypes VGLUT1-positive Glutaminergic neurons and GABA-positive gamma-aminobutyric acid(GABA)neurons were detected in hENT.(3)3D hENT cultured for 28 days in vitro was detected under laser confocal microscope.MAP2-positive neurons were distributed evenly and synaptophysin expression could be observed over the whole tissue.Under electron microscope,synapses and myelin sheaths in neurons could be further clearly observed,indicating the formation of complex neural networks in 3D hENT.(4)When hENT was co-cultured with EGFP-mouse engineered neural tissue(EGFP-mENT),EGFP+ /MAP2+ mouse neurons were detected in hENT.EGFP-/ MAP2+ human neurons and synaptophysin were also detected in EGFP-mENT.(5)In the co-culture system of EGFP m Cerebral Slice and hENT,EGFP+ /MAP2+ double positive mouse neurons were detected in hENT.EGFP-/ MAP2+ human neurons and synaptophysin were also detected in the EGFP-m Cerebral Slice.(6)Calcium imaging results showed that there was calcium ion conduction activity between neurons in the 3D hENT constructed in vitro.Conclusion:(1)EF stimulation can induce the neuronal differentiation of hNSCs and promote the maturation of different neuronal subtypes in hENT;(2)hENT is a living tissue with certain vitality that can survive in vitro for at least 45-48 days;(3)EF stimulation can guide the orderly growth of neuronal processes in hENT with a large number of synapses and myelin sheaths;(4)3D hENT induced by EFs can form synaptic connections with other neural tissues,and axons project to each other to generate neural network activities.