Optical control of network formation in stem cell-derived neurons

 描述(申请人提供)：神经元丢失是全球数百万患者神经损伤和疾病造成的深远破坏性影响的原因，而且中枢神经系统几乎没有自我修复的能力。用干细胞来源的神经元再生受损的神经回路是解决这个问题的一种有前途的方法，特别是考虑到发现多能干细胞可以通过对患者自己的皮肤细胞(诱导的多能干细胞，IPSCs)进行重新编程来获得。然而，干细胞来源的神经元与宿主组织的功能整合仍然是一个挑战，但几乎没有成功，该领域需要新的更好的工具来控制干细胞的命运和连接。我们提出了新的光学方法来构建定义的神经网络，其中光被用来设计特定的神经元亚型，并选择性地将它们与目标细胞类型连接。该方法使用最近描述的光敏细菌转录因子来驱动基因表达，以及对神经元尖峰的光遗传控制，以选择性地加强或削弱特定神经元群体之间的联系。为了演示概念验证，该项目从大鼠IPSC来源的神经元的功能特征开始，随后生成试点数据，以证明使用光激活基因表达的时空模式和通道门控来构建神经网络的可行性。对于连接的光学控制，采用了文献中的刺激频率和模式，证明了光遗传刺激后成熟神经元中突触的消除或稳定。对于光驱动的基因表达，光敏转录因子通过带有广谱神经启动子的重组复制缺陷逆转录病毒来传递。结果通过全细胞电生理学、突触标记与免疫组织化学的鉴定以及荧光报告基因表达的空间模式光学诱导相结合来验证。这些概念验证研究将建立新的协议来控制特定神经元群体与光之间的连接，未来有可能使用修改的光激活转录因子来确定亚型规范。这将为利用光刺激来确定干细胞来源的神经元的身份和连通性奠定基础，为未来的工作提供新的工具，在体外和体内构建和重建神经网络。

 DESCRIPTION (provided by applicant): Neuronal loss is responsible for the profoundly devastating effects of neurological injury and disease for millions of patients worldwide, and the central nervous system has little capacity for self-repair. Regeneration of damaged neural circuitry with stem cell-derived neurons is a promising approach to the problem, particularly given the discovery that pluripotent stem cells can be derived by reprogramming a patient's own skin cells (induced pluripotent stem cells, iPSCs). However, functional integration of stem cell-derived neurons with host tissue continues to be a challenge met with few successes, and the field requires new and better tools to control stem cell fate and connectivity. We propose new optical methods to enable the construction of defined neural networks, where light is used to pattern specific neuronal subtypes and selectively connect them with target cell types. The approach uses a recently-described photosensitive bacterial transcription factor to drive gene expression as well as optogenetic control of neuronal spiking to selectively strengthen or weaken connections between specific populations of neurons. To demonstrate proof-of-concept, the project begins with a functional characterization of rat iPSC-derived neurons and subsequently generates pilot data to demonstrate the feasibility of using spatiotemporal patterns of light-activated gene expression and channel gating to build neural networks. For optical control of connectivity, the frequencies and patterns of stimulation are adopted from literature demonstrating either elimination or stabilization of synapses in mature neurons following optogenetic stimulation. For light-driven gene expression, the photosensitive transcription factor is delivered via recombinant replication-defective retroviruses with broad-spectrum neural promoters. Results are validated through a combination of whole-cell electrophysiology, identification of synaptic markers with immunohistochemistry, and spatially patterned optical induction of fluorescent reporter gene expression. These proof-of-concept studies will establish new protocols to control connectivity between specific neuronal populations with light, with the future potential to use a modified light-activated transcription factor to determine subtype specification. This will lay the foundation to use optical stimulation to define the identity and connectivity of neurons derived from stem cells, giving new tools to construct and reconstruct neural circuitry in vitro and in vivo in future work.