Engineering brain assembloids to interrogate human neural circuits

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.

Automated summary

This article describes a method for generating human brain assembloids and studying neural circuits. By converting human pluripotent stem cells into brain region-specific organoids, which are then fused and integrated to form assembloids, complex cell-cell interactions, circuit formation, and maturation can be studied using self-organization. A series of assays, including viral labeling and retrograde tracing, 3D live imaging, and electrophysiology, are used to model and manipulate neural circuits in the assembloids. These approaches are valuable for understanding human-specific aspects of neural circuit assembly and modeling neurodevelopmental disorders.