Optogenetic stimulation of neural stem cells (NSCs) enables their activity-dependent photo-modulation. This provides a spatio-temporal tool for studying activity-dependent neurogenesis and for regulating the differentiation of the transplanted NSCs. Currently, this is mainly driven by viral transfection of channelrhodopsin-2 (ChR2) gene, which requires high irradiance and complex in vivo/vitro stimulation systems. Additionally, despite the extensive application of optogenetics in neuroscience, the transcriptome-level changes induced by optogenetic stimulation of NSCs have not been elucidated yet. Here, we made transformed NSCs (SFO-NSCs) stably expressing one of the step-function opsin (SFO)-variants of chimeric channelrhodopsins, ChRFR(C167A), which is more sensitive to blue light than native ChR2, via a non-viral transfection system using piggyBac transposon. We set up a simple low-irradiance optical stimulation (OS)-incubation system that induced c-fos mRNA expression, which is activity-dependent, in differentiating SFO-NSCs. More neuron-like SFO-NCSs, which had more elongated axons, were differentiated with daily OS than control cells without OS. This was accompanied by positive/negative changes in the transcriptome involved in axonal remodeling, synaptic plasticity, and microenvironment modulation with the up-regulation of several genes involved in the Ca²⁺-related functions. Our approach could be applied for stem cell transplantation studies in tissue with two strengths: lower carcinogenicity and less irradiance needed for tissue penetration.

中文翻译：

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在具有高灵敏度光遗传学的神经干细胞中驱动神经发生。

神经干细胞（NSCs）的光遗传学刺激使它们能够进行活动依赖性的光调制。这提供了一个时空工具，用于研究活动依赖的神经发生和调节移植的神经干细胞的分化。当前，这主要是由病毒视紫红质通道2（ChR2）基因的转染驱动的，该基因需要高辐照度和复杂的体内/体外刺激系统。另外，尽管光遗传学在神经科学中得到了广泛的应用，但是尚未阐明由光遗传学刺激NSC引起的转录组水平的变化。在这里，我们制作了经转化的NSC（SFO-NSC），它通过非病毒稳定表达嵌合通道性视紫红质的步进功能视蛋白（SFO）变体之一ChRFR（C167A），它比天然ChR2对蓝光更敏感。转染系统使用piggyBac转座子。我们建立了一个简单的低辐照度光刺激（OS）孵育系统，该系统可诱导c - fos mRNA表达，该表达依赖于活性，以区分SFO-NSC。与没有OS的对照细胞相比，每天OS可以分化出更多具有更长轴突的神经元样SFO-NCS。这伴随着与轴突重塑，突触可塑性和微环境调节有关的转录组的正/负变化，以及与Ca ²⁺相关功能有关的几个基因的上调。我们的方法可用于组织的干细胞移植研究，具有两个优点：较低的致癌性和较少的组织穿透所需的辐照度。