Gold Nanostrip Array-Mediated Wireless Electrical Stimulation for Accelerating Functional Neuronal Differentiation

Abstract

Neural stem cell (NSC)-based therapy holds great promise for the treatment of neurodegenerative diseases. Presently, however, it is hindered by poor functional neuronal differentiation. Electrical stimulation is considered one of the most effective ways to promote neuronal differentiation of NSCs. In addition to surgically implanted electrodes, traditional electrical stimulation includes wires connected to the external power supply, and an additional surgery is required to remove the electrodes or wires following stimulation, which may cause secondary injuries and infections. Herein, a novel method is reported for generation of wireless electrical signals on an Au nanostrip array by leveraging the effect of electromagnetic induction under a rotating magnetic field. The intensity of the generated electrical signals depends on the rotation speed and magnetic field strength. The Au nanostrip array-mediated electric stimulation promotes NSC differentiation into mature neurons within 5 days, at the mRNA, protein, and function levels. The rate of differentiation is faster by at least 5 days than that in cells without treatment. The Au nanostrip array-based wireless device also accelerates neuronal differentiation of NSCs in vivo. The novel method to accelerate the neuronal differentiation of NSCs has the advantages of wireless, timely, localized and precise controllability, and noninvasive power supplementation.

Keywords: NSC-based therapy; electromagnetic induction; mature functional neuron; neuronal differentiation; wireless electrical stimulation.

Figure 1

Wireless device for electrical stimulation of NSCs. a) Schematic illustration of the generation of electrical signals on the Au nanostrip array for stimulating the differentiation of NSCs into mature, functional neurons. Top b) and side view (c) SEM images of the Au nanostrip array. d) AFM image of the Au nanostrip array. e) Magnetic field intensity at the specific position (20 mm below the magnet) of the rotating magnetic field at different speeds (0, 100, 200, 300, 400, and 500 rpm). f) Generated induced voltages on the Au nanostrip array upon rotation of the magnet at 0, 100, 200, 300, 400, and 500 rpm. g) Viability of NSCs after culture on the TCP, Si, and Au nanostrip array for 1, 2, or 3 days. h) Survival rate of NSCs after culture on the Au nanostrip array for 3 days with the magnet rotating at 0, 100, 200, 300, 400, and 500 rpm. In panels g) and h), data are presented as the mean ± standard deviation (n = 4). ^ns p > 0.05.

Figure 2

RT‐qPCR analysis of the expression of neural‐related genes a) Tuj1, b) MAP2, and c) GFAP for NSCs seeded on TCP, Si, or the Au nanostrip array and cultured without or with rotating magnetic field (300 rpm) for 3, 5, or 7 days. All data are presented as the mean ± standard deviation (n = 3). ^ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Au nanostrip array‐based wireless device accelerates neuronal differentiation of NSCs at the protein level. a) Confocal microscopy images of NSCs seeded on TCP or Au nanostrip array and cultured without or with the rotating magnetic field (300 rpm) for 3, 5, or 7 days. Tuj1 and GFAP were stained red and green, respectively. Cell nuclei were stained blue with 4',6‐diamidino‐2‐phenylindole (DAPI). b) Quantitative mean immunofluorescence intensity of Tuj1. c) Confocal microscopy images of NSCs seeded on TCP or Au nanostrip array and cultured without or with the rotating magnetic field (300 rpm) for 3, 5, or 7 days. MAP2 and GFAP were stained red and green, respectively. Cell nuclei were stained blue with DAPI. d) Quantitative mean immunofluorescence intensity of MAP2. b,d) At least 30 cells were analyzed in each group using Image J software. The data are presented as mean ± standard deviation; ^ns p > 0.05, ***p < 0.001. e) Images of 2D‐reconstructed neuronal morphologies that were traced and visualized based on the Tuj1 protein expression of NSCs seeded on TCP, Si, or Au nanostrip array, and cultured without or with the rotating magnetic field (300 rpm) on day 5. f) Sholl analysis of the neurite complexity of the 2D‐reconstructed neuronal morphologies presented in e). Neurites of ten neurons on each group were randomly measured. g) Western blot analysis of the Tuj1 and MAP2 protein expression of NSCs seeded on TCP, Si, or Au nanostrip array, and cultured without or with the rotating magnetic field (300 rpm) on day 5. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as the housekeeping gene.

Figure 4

Evaluation of the functional neurons. a) Calcium dynamics within the differentiated neurons seeded on Au nanostrip array and cultured with rotating magnetic field (300 rpm) for 5 days (calcium ion indicated in green). Left panels show images of neurons after calcium dye (Fluo‐4 AM) loading. Right panels show time‐series imaging of relative fluorescence intensity change (%ΔF/F) for individual neurons after stimulation by acetylcholine (ACh) or g‐aminobutyric acid (GABA) neurotransmitters. The inset shows the fluorescence intensity expression of the cell body in the selected region at different time points. b) SEM images of the differentiated neurons on TCP, Si, or Au nanostrip array, cultured without or with rotating magnetic field (300 rpm) for 3 days.

Figure 5

Mechanism underlying NSC differentiation promoted by the Au nanostrip‐based wireless device. a) GO functional enrichment analysis of differentially expressed genes between the Au nanostrip array+ and TCP‐ groups. b) KEGG pathway classification of differentially expressed genes between the Au nanostrip array+ and TCP‐ groups. c) Western blot analysis of the ChAT, GAD65, and c‐Fos protein expression of NSCs seeded on TCP, Si, or Au nanostrip array and cultured without or with rotating magnetic field (300 rpm) on day 5. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. Quantitative analysis data obtained using Image J software are presented as mean ± standard deviation (n = 4); ^ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. d) Western blot analysis of ChAT, GAD65, and c‐Fos protein expressions by NSCs seeded on TCP, Si, or Au nanostrip array and cultured without or with rotating magnetic field (300 rpm) on day 5. The NSCs in the Au nanostrip array+ group were treated with 3 × 10⁻³ m CoCl₂ before the rotating magnetic field was applied. GAPDH was used as the housekeeping gene. Quantitative analysis data acquired by Image J software are presented as mean ± standard deviation (n = 4); ^ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. e) Diagram of the mechanism of NSCs differentiation promoted by the Au nanostrip‐based wireless device.

Figure 6

Au nanostrip array‐mediated electrical stimulation promotes neuronal differentiation of NSCs in vivo. a) H&E staining and b) Nissl staining results in the cerebral cortex section obtained at the interface of substrates (PI or Au nanostrip array) seeded with NSCs and without or with rotating magnetic field (300 rpm) on day 7. Black arrows point to neuron‐like cells. c) Immunofluorescent images of the cerebral cortex section obtained at the interface of substrates (PI or Au nanostrip array) seeded with NSCs and without or with rotating magnetic field (300 rpm) on day 7. In the upper row of panels, Tuj1 and cell membrane were stained red and green, respectively. Nuclei of cells were stained blue by DAPI. In the lower row of panels, MAP2 and cell membrane were stained red and green, respectively. Nuclei of cells were stained blue by DAPI. Quantitative mean immunofluorescence intensity of Tuj1 or MAP2 in each group are presented as mean ± standard deviation (n = 4); ^ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.