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Engineering brain assembloids to interrogate human neural circuits

Abstract

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.

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Fig. 1: Overview of generation and functional assays in brain assembloids.
Fig. 2: Generation of region-specific brain organoids and assembloids.
Fig. 3: Neural circuit formation in cortico-striatal assembloids.
Fig. 4: Optogenetics and calcium imaging in cortico-striatal assembloids.
Fig. 5: Electrophysiological recordings in assembloids.

Data availability

The data presented in this protocol are available on request from the corresponding author. Source data are provided with this paper.

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Acknowledgements

We thank members of the Pașca laboratory at Stanford University for experimental support, as well as the Stanford WuTsai Neurosciences Institute Virus Core for production of AAVs. This work was supported by the US National Institutes of Health (NIH) BRAINS Award (MH107800) (to S.P.P.), Stanford Bio-X, the NYSCF Robertson Stem Cell Investigator Award (to S.P.P.), the Stanford Wu Tsai Neurosciences Big Idea Project on Human Brain Organogenesis (to S.P.P.), the Kwan Research Fund (to S.P.P.), the Coates Foundation (to S.P.P.), the Senkut Research Fund (to S.P.P.), the Chan Zuckerberg Initiative Ben Barres Investigator Award (to S.P.P.), the Stanford Medicine Dean’s Postdoctoral Fellowship (to Y.M.) and the Stanford Maternal & Child Health Research Institute (MCHRI) Postdoctoral Fellowship (to Y.M. and O.R.).

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Authors and Affiliations

Authors

Contributions

Y.M. collected data and optimized the protocols for generating hStrSs and assembloids, clearing of assembloids, retrograde viral tracing and optogenetics coupled with calcium imaging. M.-Y.L collected data and optimized the protocols for whole-cell recordings. O.R. optimized the protocols and analyzed optogenetics experiments combined with calcium imaging. S.-J.Y. optimized the protocols for generation of hCSs. G.N. collected data and optimized the protocols for clearing of assembloids. Y.M. and S.P.P. wrote the manuscript with input from all authors. S.P.P. supervised this work.

Corresponding author

Correspondence to Sergiu P. Pașca.

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Competing interests

Stanford University has filed and holds patents for the generation of brain region–specific spheroids/organoids and assembloids. Y.M. and S.P.P. are listed as inventors on some of these patents.

Additional information

Peer review information Nature Protocols thanks J. Gray Camp, Madeline A Lancaster and Alysson R. Muotri for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Miura, Y. et al. Nat. Biotechnol. 38, 1421–1430 (2020): https://doi.org/10.1038/s41587-020-00763-w

Yoon, S.-J. et al. Nat. Methods 16, 75–78 (2019): https://doi.org/10.1038/s41592-018-0255-0

Sloan, S. A. et al. Nat. Protoc. 13, 2062–2085 (2018): https://doi.org/10.1038/s41596-018-0032-7

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Miura, Y., Li, MY., Revah, O. et al. Engineering brain assembloids to interrogate human neural circuits. Nat Protoc 17, 15–35 (2022). https://doi.org/10.1038/s41596-021-00632-z

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