Self-organizing brain organoids provide a promising tool for studying human development and disease. Here we created human forebrain organoids with stable and homogeneous expression of channelrhodopsin-2 (ChR2) by generating AAVS1 safe harbor locus-targeted, ChR2 knocked-in human pluripotent stem cells (hPSCs), followed by the differentiation of these genetically engineered hPSCs into forebrain organoids. The resulting ChR2-expressing human forebrain organoids showed homogeneous cellular expression of ChR2 throughout entire regions without any structural and functional perturbations and displayed consistent and robust neural activation upon light stimulation, allowing for the non-virus mediated, spatiotemporal optogenetic control of neural activities. Furthermore, in the hybrid platform in which brain organoids are connected with spinal cord organoids and skeletal muscle spheroids, ChR2 knocked-in forebrain organoids induced strong and consistent muscle contraction upon brain-specific optogenetic stimulation. Our study thus provides a novel, non-virus mediated, preclinical human organoid system for light-inducible, consistent control of neural activities to study neural circuits and dynamics in normal and disease-specific human brains as well as neural connections between brain and other peripheral tissues.

中文翻译：

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AAVS1 靶向、稳定表达 ChR2 在人脑类器官中，用于一致的光遗传学控制。


自组织脑类器官为研究人类发育和疾病提供了一种有前途的工具。在这里，我们通过产生 AAVS1 安全港基因座靶向、ChR2 敲入的人多能干细胞 （hPSC），然后这些基因工程 hPSC 分化为前脑类器官，创造了通道视紫红质-2 （ChR2） 稳定和均一表达的人前脑类器官。所得的表达 ChR2 的人前脑类器官在整个区域显示出 ChR2 的均匀细胞表达，没有任何结构和功能扰动，并且在光刺激下表现出一致和强大的神经激活，允许对神经活动进行非病毒介导的时空光遗传学控制。此外，在脑类器官与脊髓类器官和骨骼肌球体连接的混合平台中，ChR2 敲入的前脑类器官在脑特异性光遗传学刺激下诱导强烈而一致的肌肉收缩。因此，我们的研究提供了一种新颖的、非病毒介导的临床前人类类器官系统，用于光诱导、一致的神经活动控制，以研究正常和疾病特异性人脑中的神经回路和动力学，以及大脑与其他外周组织之间的神经连接。