Non-gene Transfer/Non-invasive Light Regulation Of Neural Stem Cells Proliferation And Differentiation And Its Molecular Mechanism

This study found that the neural stem cells of mice express various light-sensitive proteins(photoreceptors).The experiments of cell and molecular genetics series have proved that blue light(455nm,300?w/cm2)irradiation could significantly improve the proliferation and differentiation of neural stem cells by using the signaling pathway(Melanopsin-TRPC6-Jabl).Based on the in vitro results,endogenous neural stem cells in the deep brain regions of adult mice were found to express this photoreceptive/phototransduction pathway and could be activated by the blue light irradiation in the SGZ region.Upconversion nanoparticles(UCNPs)converted deep-penetrating near-infrared(NIR)light into specific wavelengths of visible light.Accordingly,we demonstrated that UCNP-mediated NIR light could be used to modulate in vivo NSC differentiation in a less invasive manner.ObjectiveIt is essential to develop new optogenetic manipulation techniques to extend the range of responses of stem cells to light-based sensory stimulation.In this study,we explicitly addressed the issues of NSC behaviors and nongenetic optical modulation.Methods1?RT-PCR was used to detect the expression of light-sensitive proteins in NSCs;2?Dissociate and culture the NSCs from forebrain of E13.5 C57BL/6 wild mice.After stimulating NSCs with monochromatic blue light(455nm,300?w/cm2),red light(635nm,300?w/cm2)and blue-red light,the proliferation,self-renewal capacity and differentiation index changes of neural stem cells were detected;3?Immunofluorescence technology was used to detect the expression of the light-sensitive protein of Melanopsin on the surface of neural stem cells;Whole-cell patch clamp and calcium ion probe technology were used to detect the activation of Melanoopsin-TRPC6 after irradiated blue light.Western Blot was used to verify the photoreceptive/light transduction pathway of Melanopsin/TRPC6/Jabl;4?Immunofluorescence technology was used to detect the expression of Melanopsin and TRPC6 in vivo NSCs;After irradiating NSCs in the SGZ region of C57BL/6 wild mice with 25mW/10Hz 200?m blue light fiber,we detected index of neuronal differentiation by immunofluorescence,Western Blot and RT-PCR;5?UCNPs were injected into the SGZ region of C57BL/6 wild mice and irradiated by NIR,and then we detected index of neuronal differentiation by immunofluorescence,Western Blot and RT-PCR.Results1?NSCs expressed a series of light-sensitive proteins;2?Monochromatic blue light(455nm,300?w/cm2)can promote the proliferation and self-renewal of NSCs.The light could promote differentiation to astrocytes and inhibit differentiation to neurons of NSCs;3?Monochromatic red light(635nm,300?w/cm2)can inhibit differentiation to astrocytes and promote differentiation to neurons of NSCs;4?We found that the light-sensitive protein Melanopsin(Opn4)is expressed of NSCs.Blue light can activate the calcium channel of Melanoopsin-TRPC6 by whole-cell patch clamp technology.We have initially identified the photoreceptor/phototransduction pathway of Melanopsin/TRPC6/Jabl;5?Immunofluorescence technology was used to verify that NSCs of E13.5 fetal mice and SVZ and SGZ of adult mice simultaneously expressed Melanopsin and TRPC6;6?We directly modulated the zone of SGZ in the hippocampus of the brain through 200 ?m blue light fiber,and found that it can promote the differentiation of NSCs into glial cells;7?Near infrared light(980nm)and up-converted nanoparticles(UCNPs)could stimulate neural stem cells in vivo.ConclusionsThis study found that neural stem cells express the light-sensitive protein Melanopsin(Opn4),which is used as a scientific basis to regulate the behavior of neural stem cells in vitro using monochromatic light(455nm,635nm).Then we found that blue light can affect the proliferation,self-renewal and differentiation of neural stem cells through the photoreceptive/phototransduction pathway of Melanopsin/TRPC6/Jab1.On this basis,this study used blue fiber and near infrared light irradiation to activate endogenous neural stem cells in vivo.