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Generation and assembly of human brain region–specific three-dimensional cultures


The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both healthy individuals and patients. In this protocol, we describe how to generate and assemble subdomain-specific forebrain spheroids, also known as brain region–specific organoids, from human pluripotent stem cells (hPSCs). We describe how to pattern the neural spheroids toward either a dorsal forebrain or a ventral forebrain fate, establishing human cortical spheroids (hCSs) and human subpallial spheroids (hSSs), respectively. We also describe how to combine the neural spheroids in vitro to assemble forebrain assembloids that recapitulate the interactions of glutamatergic and GABAergic neurons seen in vivo. Astrocytes are also present in the human forebrain–specific spheroids, and these undergo maturation when the forebrain spheroids are cultured long term. The initial generation of neural spheroids from hPSCs occurs in <1 week, with regional patterning occurring over the subsequent 5 weeks. After the maturation stage, brain region–specific spheroids are amenable to a variety of assays, including live-cell imaging, calcium dynamics, electrophysiology, cell purification, single-cell transcriptomics, and immunohistochemistry studies. Once generated, forebrain spheroids can also be matured for >24 months in culture.

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We thank the Pașca lab for experimental support. This work was supported by a US National Institute of Health (NIH) BRAINS Award (R01MH107800), the California Institute of Regenerative Medicine (CIRM), an MQ Fellow Award, and a Donald E. and Delia B. Baxter Foundation Award (to S.P.P.); NIMH T32GM007365, F30MH106261, and a Bio-X Predoctoral Fellowship (to S.A.S.); a Physician Scientist Development in Pediatrics Award (PSDP) and a Child Health Research Institute Postdoctoral Fellowship (to A.M.P.); Child Health Research Institute Postdoctoral Fellowship UL1-TR001085 (to F.B.); a Walter V. and Idun Berry Postdoctoral Fellowship (to J.A.); and Stanford Medicine’s Dean’s Fellowships (to F.B. and J.A.).

Author information

S.A.S., J.A., A.M.P., and F.B. collected data and contributed to the optimizations of the protocols. S.A.S. and S.P.P. wrote the manuscript with input from all authors. S.P.P. supervised this work.

Competing interests

The authors declare no competing interests.

Correspondence to Sergiu P. Pașca.

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Further reading

Fig. 1: General schematic for the generation of human forebrain spheroids from hPSCs.
Fig. 2: Outline of the human forebrain spheroid protocol.
Fig. 3: Images of forebrain spheroid formation.
Fig. 4: Expression of region-specific markers and fusion of dorsal and ventral neural spheroids into assembloids.


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