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. 2020 Oct 13;26(7):2964–2976. doi: 10.1038/s41380-020-00910-4

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© The Author(s) 2020

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic of the brief timeline of the generation of small human cerebral organoids from human pluripotent stem cells. b Quantification of cortical cell populations in vitro at day 30 postdifferentiation (n = 30 organoids). c, g Representative immunostaining of human cerebral organoids for Ki67 (a proliferation marker) and PAX6 (a dorsal telencephalic progenitor marker) at 30 days postdifferentiation. d Double immunostaining for the ventricular zone (VZ) marker SOX2 and the mature neuronal marker NeuN. NeuN⁺ cells were found in the peripheral region of cerebral organoids. e, f Immunostaining for DCX (a newborn neuron marker), PKC-λ (an adherent junction marker), and Nestin (a neural stem cell marker). Few DCX⁺ and Nestin⁺ cells were found. h–l Sample images of immunostaining for the early stage forebrain marker FOXG1, the oRGC marker HOPX, the GABAergic interneuron marker GAD67, the cortical-layer neuron markers TBR1, CTIP2, and FOXP2, and neuronal markers TUJ1 and MAP2 in cerebral organoids. m Human cerebral organoids derived from human pluripotent stem cells expressed glutamate (a glutamatergic neuronal marker) in our culture system. n Glial fibrillary acidic protein (GFAP, human astrocytic marker) was expressed at a low level in cerebral organoids. Scale bars: 100 μm.