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Generating and characterizing human telencephalic brain organoids from stem cell-derived single neural rosettes

Nature Protocols volume 21, pages 718–748 (2026)Cite this article

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Abstract

We have developed a method for generating human telencephalic organoids from stem cell-derived isolated single neural rosettes. The use of single neural rosettes for generating organoids offers several important advantages. First, it mimics the development of neural tissue from a singular neural tube in vivo. Second, single neural rosette-derived organoids exhibit a relatively consistent and reproducible composition of telencephalic neural cells. Finally, single neural rosette-derived organoids demonstrate predictable organization of the identified neural cells around a single neural rosette-derived lumen and contain a large proportion of functionally mature neurons that generate action potentials and receive both excitatory and inhibitory synaptic inputs. These unique features of our protocol enable the study of the specification and organization of different neural cells in the developing human telencephalon, as well as modeling of neurodevelopmental disorders associated with disrupted neural networks. Here, we describe our protocols for generating CRISPR–Cas9-engineered human stem cells with a hemizygous SHANK3 deletion, stem cell-derived single neural rosettes and telencephalic brain organoids. We also offer insights on how to conduct single-cell RNA sequencing, immunohistochemistry and slice patch-clamp electrophysiology on these organoids. Completion of the protocols takes 5–6 months and requires experience working with cultured cells. We expect this protocol will prove useful for studies of human brain development and disease, as well as for advancing the development of new organoid-based biocomputers.

Key points

  • This protocol describes the generation and characterization of human telencephalic brain organoids from stem cell-derived single neural rosettes. Procedures for studying the organization of the different neural cells, as well as generating models of neurodevelopmental disorders are included.

  • Compared with other organoids that develop form multiple rosettes, single rosette-derived organoids have a predictable organization with a reproducible cellular composition and functional neural networks.

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Fig. 1: A schematic of the protocol for generating human telencephalic brain organoids from stem cell-derived SNRs.
Fig. 2: Representative images of cells during early steps of a successful differentiation.
Fig. 3: Representative images of SNR-derived organoids at different timepoints post isolation.
Fig. 4: Representative images of unsuccessful differentiation into SNR-derived organoids.
Fig. 5: MG-embedded SNR-derived organoids.
Fig. 6: CRISPR–Cas9 strategy for introducing complete hemizygous deletion of SHANK3 in human PS cells.
Fig. 7: Characterization of cell composition in 1- and 5-month-old SNR-derived organoids using scRNA-seq.
Fig. 8: Characterization of cell composition and organization in 1- and 5-month-old SNR-derived organoids using immunohistochemistry.
Fig. 9: Slice patch-clamp electrophysiology on 5-month-old SNR-derived organoids.

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Data availability

The data related to this protocol are included in the original paper43. Interactive visualization of our transcriptomic and electrophysiological data is provided in our online browser: http://organoid.chpc.utah.edu.

Code availability

We developed and open- sourced the Shiny Single Cell Browser software in R to build the single-cell RNA-seq browser, code available: https://github.com/yueqiw/shiny_cell_browser. The electrophysiology browser was developed in Python, code available at https://github.com/yueqiw/ephys_analysis.

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Acknowledgements

The authors are thankful to current and former members of the Shcheglovitov Lab for their contribution to the protocol development and to J. Cui for help with gRNA design and validation. Cartoons used in Figs. 1, 4a and 6d were created with BioRender.com. The study was supported by the National Institute of Mental Health (R01MH113670), NINDS (R01NS123849 and R21NS104963), Utah Neuroscience Initiative, and Utah Genome Project grants (to A.S.) and the NHGRI T32 Genomic Medicine Training Grant (T32HG008962) to H.M.A.U.

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Authors and Affiliations

  1. Department of Neurobiology, University of Utah, Salt Lake City, UT, USA

    H. M. Arif Ullah, Qiju Huang, Simone Chiola, Yueqi Wang & Alex Shcheglovitov

  2. Neuroscience Graduate Program, University of Utah, Salt Lake City, UT, USA

    Yueqi Wang & Alex Shcheglovitov

  3. Department of Biomedical Engineering, University of Utah, Salt Lake City, UT, USA

    Alex Shcheglovitov

  4. Department of Psychiatry, University of Utah, Salt Lake City, UT, USA

    Alex Shcheglovitov

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  1. H. M. Arif Ullah
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  2. Qiju Huang
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  3. Simone Chiola
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Contributions

H.M.A.U., Q.H., S.C. and Y.W. performed data acquisition and analyses. A.S. conceived the protocol idea and wrote the manuscript. All authors commented on the manuscript.

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Correspondence to Alex Shcheglovitov.

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Nature Protocols thanks Alysson Muotri, In-Hyun Park, Clive Svendsen, Zhexing Wen, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Wang, Y. et al. Nat Commun. 13, 5688 (2022): https://doi.org/10.1038/s41467-022-33364-z

Yang, G. et al. Mol. Psychiatry 28, 2525–2539 (2023): https://doi.org/10.1038/s41380-023-02035-w

Kubanek, J. et al. Heliyon 9 (8), e18482 (2023): https://doi.org/10.1016/j.heliyon.2023.e18482

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Supplementary Fig. 1.

Supplementary Video 1 (download MOV )

Preparation of glass hook from Pasteur pipette.

Supplementary Video 2 (download MOV )

Isolation of SNR using glass hook.

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Ullah, H.M.A., Huang, Q., Chiola, S. et al. Generating and characterizing human telencephalic brain organoids from stem cell-derived single neural rosettes. Nat Protoc 21, 718–748 (2026). https://doi.org/10.1038/s41596-025-01197-x

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