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. 2024 Jan 31;626(8000):881–890. doi: 10.1038/s41586-023-06984-8

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 8 — a, Schematic of experimental paradigm for transient inhibition of EZH2 in forebrain organoids. WA09 hPSC-derived organoids were transiently treated with GSK343 or DMSO after the specification of cortical identity (d17-d25 or d17-37 depending on the experiment as specified in the figure panels) and analysed for the generation of cortical cell identities using representative markers at d45 and d65. b, Representative images of FOXG1 and PAX6 staining in organoids at d45 showing the acquisition of cortical identity in EZH2i and DMSO control conditions. c, Representative images of organoids at d45 and d65 stained for the cortical layer markers TBR1, CTIP2 and SATB2 showing the reduction of SATB2-expressing neurons at d65 in EZH2i versus DMSO control conditions. Insets show the general morphology of organoids with the nuclei staining DAPI. d–e, Representative images of organoids at d45 and d65 stained for the NPC marker SOX2 and the astrocyte marker GFAP showing the precocious emergence of astrocytes surrounding SOX2⁺ neural rosettes in EZH2i versus DMSO control conditions (d-e). The appearance of GFAP+ cells is enhanced in longer treatment condition with EZH2 inhibitor GSK343 (e). f, Representative images of organoids at d65 stained with the intermediate progenitor cell marker TBR2 and the astrocyte marker AQP4 confirming the precocious emergence of astrocytes in EZH2i versus DMSO control conditions. Dotted boxes depict organoid regions showed at higher magnification in each panel. Arrows in (e) and (f) mark GFAP⁺ fibers and AQP4⁺ cells respectively. n = 5–6 organoids from 2 independent experiments per condition. Scale bars are 500 μm in whole organoid sections and 100 μm for the regions identified by dotted box and showed at higher magnification in all figure panels.