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.).
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Nature Medicine (2019)
Nature Methods (2019)
Nature Neuroscience (2019)