Physiological Electric Fields Regulated Neural Stem Cells To Construct Mice 3D Engineered Neural Tissue

Objective: The construction of in vitro three-dimensional(3D)neural tissue has to overcome two main types of challenges: How to obtain enough number of functional neurons from stem cells in 3D culture;how to wire those lately developed neurons into functional neural networks.Here,we describe the potential of using direct current(DC)electric fields(EFs)together with basic fibroblast growth factor(b FGF)synergistically in promoting neural stem cell(NSCs)neuronal differentiation following by directing neurite outgrowth in the 3D neural tissue construction.Methods: NSCs were grown in two different systems: BNb system(b FGF(20ng/ml),B27(1:50),N2(1:100))and 10%FBS system.There are four groups were set up according to the culture systems and the presence or absence of EFs: BNb + EF group,BNb group,FBS + EF group,FBS group.By adjusting the electrical stimulation setup in this study,long-term electrical stimulation could be present in vitro.At an EF strength of 150 m V/mm,cell responses,including cell viability,neuronal differentiation,cell morphology,the length of neuronal processes,synaptic structure and neural network formation,were quantified and analyzed.Results:(1)Neural stem cells were cultured under the gradient cell density and Matrigel thickness for 7days,we found the condition that NSCs were planted in 300μm Matrigel with a density of 20 neurospheres /mm3 is a proper system to achieve more developed and outstretched neural network.were.(2)When 3D cultured neural tissues were stimulated with 150 m V/ mm EFs for 1 h per day for three consecutive days,electrical stimulation led to only 5.47% ± 0.74% of total cell death(mean ± SEM)determined by a fluorescent live/dead cell double staining assay.A TUNEL assay of engineered neural tissues showed that very few apoptotic(TUNEL-positive)cells were found in both EF-treated neural tissues and controls without EF-stimulation.(3)Cells grown in 10% FBS medium but without EFs,the percentage of Tuj1-positive cells was only 6.21% ± 0.94%.When cells were stimulated by 150 m V/mm EF stimulation,the percentage of Tuj1-positive cells was dramatically increased to 74.80% ± 2.25%(mean ± SEM).(4)The average length of neuronal processes in the EF-treated group was 42.6 ± 4.37 μm(n = 36).Neuronal processes not exposed to electrical stimulation were only 18.87 ± 1.57 μm(n = 28).(5)When applying BNb system along,the average length of the neuronal processes reached 47.55 ± 2.29 μm,which was much longer compared with 18.87 ± 1.57 μm in the group with FBS only.When applying 150 m V/mm EF-stimulation along(10% FBS),the average length of neural outgrowth was 42.6 ± 4.37 μm.When BNb was combined with EF stimulation,it achieved the highest Tuj1-positive rate(82.2% ± 5.65%)and neurite length(67.13 ± 3.11 μm).(6)For BNb group and FBS +EF group,the rate of neuronal differentiation was 60.2% ± 2.89% and 74.8 ± 2.25%.While the rate of neuronal differentiation in BNb group was under that of FBS+EF group.However,the rate of neuronal differentiation in BNb+EF reached to the peak(82.2%+5.65%).(7)Morphological analysis of Tuj1 positive cells showed that Tuj1 positive cells were composed of elliptic cells,parallel cells,neurons with short neurites(< 30μm)and neurons with long neurites(≥ 30μm).we counted the proportions of all kinds of cell shapes: for FBS + EF group,the proportion of band cells reached to31.47 ± 5.18%.the number of elliptic cells in the BNb group decreased significantly(25.7 ± 2.18%).While the number of neurons with long neurites increased significantly(15.9 ± 1.27%).The percentage of spherical cells and neurons possessing long neurites under 10% FBS control conditions was 92.4 ± 4.58% and 1.83 ± 0.93%,respectively.When the BNb system was combined with EF stimulation,the majority of the cells retained multipolar morphologies or was linearly arranged,and cells with long filopodia extensions were dominant in the EF-treated BNb system.(8)Electron microscopy images showed that typical neurons grown in the EF + BNb system branched repeatedly and spread out over a more extensive area.These neurons showed increased presynaptic membrane thickness and postsynaptic density.The EFs + BNb system was found to stimulate the spreading out of cells over a more extensive area and more organized cellular network.highly developed rough endoplasmic reticulum was observed in 3D tissue.We also observed typical synaptic structure.Conclusion:(1)The survival rate of NSCs will not be affected by the 150 m V/mm of EF stimulation in 3D constructed Matrigel;(2)EFs could induce neuronal differentiation and neurite extension in 3D constructed Matrigel;(3)b FGF and EFs act synergistically to promote neuronal differentiation and neurite extension in 3D constructed Matrigel;(4)By combined the EF stimulation with BNb system in 3D constructs,we obtained numbers neurons with long extending neurites and the EF + BNb system could be a modulating factor to stimulate the formation of neuronal networks(5)EFs-induced engineered neural tissue formed mature neuronal network which contained numbers of synapses and myelin sheath.