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Generation of iPSC-derived human forebrain organoids assembling bilateral eye primordia

Nature Protocols volume 18, pages 1893–1929 (2023)Cite this article

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Abstract

Induced pluripotent stem cell-derived brain organoids enable the developmental complexities of the human brain to be deconstructed. During embryogenesis, optic vesicles (OVs), the eye primordium attached to the forebrain, develop from diencephalon. However, most 3D culturing methods generate either brain or retinal organoids individually. Here we describe a protocol to generate organoids with both forebrain entities, which we call OV-containing brain organoids (OVB organoids). In this protocol, we first induce neural differentiation (days 0–5) and collect neurospheres, which we culture in a neurosphere medium to initiate their patterning and further self-assembly (days 5–10). Then, upon transfer to spinner flasks containing OVB medium (days 10–30), neurospheres develop into forebrain organoids with one or two pigmented dots restricted to one pole, displaying forebrain entities of ventral and dorsal cortical progenitors and preoptic areas. Further long-term culture results in photosensitive OVB organoids constituting complementary cell types of OVs, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections and electrically active neuronal networks. OVB organoids provide a system to help dissect interorgan interactions between the OVs as sensory organs and the brain as a processing unit, and can help model early eye patterning defects, including congenital retinal dystrophy. To conduct the protocol, experience in sterile cell culture and maintenance of human induced pluripotent stem cells is essential; theoretical knowledge of brain development is advantageous. Furthermore, specialized expertise in 3D organoid culture and imaging for the analysis is needed.

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Fig. 1: Overview of the four-stage OVB organoid differentiation from human iPSCs.
Fig. 2: Overview of preparing iPSCs for a successful differentiation into OVB organoids.
Fig. 3: Overview of OVB organoid generation stages and characterizations.
Fig. 4: Initial characterization of OVB organoids.
Fig. 5: Tracing experiment using labeled CTB.
Fig. 6: OVB organoids reveal visual pathway cell populations.

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Data availability

Source data are provided with the original paper5. Any other data are available from the corresponding author. The data presented here have not been published previously.

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Acknowledgements

This study was supported by the SPP2127-GO 2301/5-2 ‘Gene and Cell-Based Therapies to Counteract Neuroretinal Degeneration’ (J.G. and E.G.) and by the Fritz Thyssen Stiftung (E.G). V.B. acknowledges support by the Volkswagen Foundation (Freigeist—A110720) and the Deutsche Forschungsgemeinschaft SPP2127-BU 2974/4-1, EXC-2151-390873048-Cluster of Excellence—ImmunoSensation2 at the University of Bonn.

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Authors and Affiliations

  1. Institute of Human Genetics, University Hospital, Heinrich-Heine-University, Düsseldorf, Germany

    Elke Gabriel, Anand Ramani, Aruljothi Mariappan & Jay Gopalakrishnan

  2. Institute for Neurophysiology, University of Cologne, Cologne, Germany

    Walid Albanna & Toni Schneider

  3. Department of Neurosurgery, RWTH Aachen University, Aachen, Germany

    Walid Albanna

  4. Department of Ophthalmology, Medical Faculty, University of Bonn, Bonn, Germany

    Giovanni Pasquini & Volker Busskamp

  5. Institute of Pathology, University Medicine Göttingen, Georg-August University Göttingen, Göttingen, Germany

    Natasa Josipovic & Argyris Papantonis

  6. Center of Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

    Natasa Josipovic & Argyris Papantonis

  7. Department of Life Sciences and Medical Biotechnology University of Siena, Siena, Italy

    Maria Giovanna Riparbelli & Giuliano Callaini

  8. Department of Psychiatry, Washington University in St. Louis, St. Louis, MO, USA

    Celeste M. Karch

  9. Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France

    Olivier Goureau

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  1. Elke Gabriel
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Contributions

J.G. and E.G.: conception and protocol design. The rest of the authors have contributed their expertise in the respective methods of the protocol. E.G.: organoid generation and characterization; G.P. and V.B.: scRNA-seq analysis; W.A. and T.S.: ERGs; A.P. and N.J.: RNA-seq analyses; A.R. and A.M.: 3D imaging; M.G.R. and G.C.: TEM and analysis; O.G.: iPSCs, organoids and data analysis; C.M.K.: iPSC lines and data analysis.

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Correspondence to Jay Gopalakrishnan.

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Key references using the protocol

Gabriel, E. et al. EMBO J. 35, 803–819 (2016): https://doi.org/10.15252/embj.201593679

Gabriel, E. et al. Cell Stem Cell 20, 397–406.e5 (2017): https://doi.org/10.1016/j.stem.2016.12.005

Gabriel, E. et al. Cell Stem Cell 28, 1740–1757.e8 (2021): https://doi.org/10.1016/j.stem.2021.07.010

Supplementary information

Supplementary Video 1

Microinjection of Cholera toxin B (CTB-488 and CTB-647) to label axon-like projections. Representative movie of volume-rendered optic vesicles from specimen shown in Fig. 5c microinjected with CTB-488 and CTB-647 at two distinct sites. The injection sites are marked with arrows (00:03 min). Individual channels (GFP and Cy5) have been shown to highlight the distribution of injected toxins. The converging nature of optic tracts is demonstrated by the strong co-localization of axons labeled by both CTB-488 and CTB-647. Scale bar, 200 μm. The representative movie, cell line IMR-90.

Supplementary Table 1

Visual pathway genes

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Gabriel, E., Albanna, W., Pasquini, G. et al. Generation of iPSC-derived human forebrain organoids assembling bilateral eye primordia. Nat Protoc 18, 1893–1929 (2023). https://doi.org/10.1038/s41596-023-00814-x

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