Generation and assembly of human brain region-specific three-dimensional cultures
Open accessAbstract
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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Region-specific spheroids or organoids can be fused in vitro to generate 3D assembloids that model complex cellular interactions during human brain development.
“We have previously developed an approach to model complex cellular interactions during human brain development by specifying region-specific spheroids or organoids and then fusing them in vitro to generate three-dimensional (3D) assembloids”
From Generation of Functional Human 3D Cortico-Motor Assembloids (2020) · cites this paper as Sloan et al., 2018
The hiPS cell lines used were validated by standard methods.
“The hiPS cell lines used in this study were validated using standard methods as previously described (Paşca et al., 2011; Sloan et al., 2018).”
From Generation of Functional Human 3D Cortico-Motor Assembloids (2020) · cites this paper as Sloan et al., 2018
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- Authors
- Sloan, S. A., Andersen, J., Pașca, A. M., Birey, F., & Pașca, S. P.
- Affiliations
- Stanford Medicine (US)
- Corresponding author
- Pașca, Sergiu P.
- Venue
- Nature Protocols
- Published
- 2018-09
- Volume
- 13
- Issue
- 9
- Pages
- 2062-2085
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC6597009
- OpenAlex
- W2890597376
- Cited by
- 490
- References
- 52
- Citations by year
- 2026: 69 · 2025: 93 · 2024: 90 · 2023: 60 · 2022: 66 · 2021: 48 · 2020: 37 · 2019: 25 · 2018: 2
- FWCI
- 10.05
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (UL1TR001085)
- National Institute of Mental Health (F30MH106261, R01MH107800)
- National Institute of General Medical Sciences (T32GM007365)
- National Center for Advancing Translational Sciences
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Life Sciences › Neuroscience › Developmental Neuroscience › Neurogenesis and neuroplasticity mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
- Keywords
- human pluripotent stem cells, dorsal forebrain, ventral forebrain, assembloids, glutamatergic neurons, GABAergic neurons, astrocyte maturation, human brain development
- MeSH terms
- Humans, Models, Biological, Organ Culture Techniques, Organoids, Prosencephalon, Tissue Engineering, Organogenesis, Pluripotent Stem Cells
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