Engineering brain assembloids to interrogate human neural circuits
Engineering brain assembloids to interrogate human neural circuits
复制标题
构建工程化脑类器官以探究人类神经回路
DOI：
10.1038/s41596-021-00632-z
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发表时间：
2022-01-06
期刊：
影响因子：
14.8
通讯作者：
Pasca, Sergiu P.
中科院分区：
文献类型：
--
作者：
Miura, Yuki;Li, Min-Yin;Pasca, Sergiu P.
神经回路的发展涉及神经元在其产生和迁移后的局部布线，以及在大脑区域之间建立长距离连接。研究人类神经系统中的这些发育过程仍然很困难，因为在体外可以随着时间的推移保持功能的组织有限。我们之前已经开发了一种方法，将人类多能干细胞转化为脑区域特异性类器官，这些类器官可以融合和整合以形成类神经元并研究神经元迁移。与在2D培养中混合细胞谱系或设计微芯片的方法相反，类胶质细胞利用自组织来实现复杂的细胞间相互作用，在长期培养中形成回路和成熟。在这个协议中，我们描述的方法来模拟长距离的神经元连接在人脑类胶质细胞。我们提出了如何生成类似于神经系统的特定域的3D球体，然后如何将它们物理地整合以允许轴突投射和突触组装。此外，我们还描述了一系列检测方法，包括病毒标记和逆行追踪，轴突投射的3D实时成像和光遗传学结合钙成像和电生理记录来探测和操纵类胶质细胞中的回路。这些检测需要3-4个月才能完成，需要干细胞培养、成像和电生理学方面的专业知识。我们期望这些方法将有助于破译人类特定方面的神经回路组装和建模神经发育障碍与患者源性cells。一个协议，用于产生人脑类胶质细胞和进行病毒标记和逆行追踪，三维实时成像轴突投射和光遗传学与钙成像和电生理记录，以模拟神经回路。
The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into brain region-specific organoids that can be fused and integrated to form assembloids and study neuronal migration. In contrast to approaches that mix cell lineages in 2D cultures or engineer microchips, assembloids leverage self-organization to enable complex cell-cell interactions, circuit formation and maturation in long-term cultures. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. The assays take 3-4 months to complete and require expertise in stem cell culture, imaging and electrophysiology. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.A protocol is described for generating human brain assembloids and performing viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics with calcium imaging and electrophysiological recordings to model neural circuits.