Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA.
Stanford Brain Organogenesis, Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA.
Nat Protoc. 2022 Jan;17(1):15-35. doi: 10.1038/s41596-021-00632-z. Epub 2022 Jan 6.
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.
神经回路的发育涉及神经元在局部的布线，遵循其生成和迁移的过程，以及在大脑区域之间建立长程连接。由于难以获得可在体外随时间维持功能的组织，因此研究人类神经系统中的这些发育过程仍然具有挑战性。我们之前开发了一种将人类多能干细胞转化为脑区特异性类器官的方法，这些类器官可以融合和整合形成 assembloids 并研究神经元迁移。与在 2D 培养物中混合细胞谱系或工程微芯片的方法不同，assembloids 利用自组织来实现复杂的细胞-细胞相互作用、电路形成和长期培养中的成熟。在本方案中，我们描述了在人类大脑 assembloids 中模拟长程神经元连接的方法。我们介绍了如何生成类似于神经系统特定区域的 3D 球体，然后如何将它们物理集成以允许轴突投射和突触组装。此外，我们还描述了一系列测定方法，包括病毒标记和逆行追踪、3D 轴突投射活体成像以及光遗传学与钙成像和电生理记录相结合，以探测和操纵 assembloids 中的电路。这些测定需要 3-4 个月的时间才能完成，并且需要干细胞培养、成像和电生理学方面的专业知识。我们预计这些方法将有助于解析神经回路组装中的人类特异性方面，并使用患者来源的细胞模拟神经发育障碍。
