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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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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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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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
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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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DOI: https://doi.org/10.1038/s41596-023-00814-x
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