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. 2023 Mar 6;28(12):5077–5089. doi: 10.1038/s41380-023-01997-1

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© The Author(s) 2023

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. 3 — a Experimental timeline and sample harvesting scheme. b Hyper-IL-6 treatment does not lead to changes in size of the DFOs during the treatment (days 45–55, grey area) and at least up to day 90 of differentiation. Vehicle (n = 5–32) and Hyper-IL-6 (n = 5–32) DFOs from one batch per iPSC line, two iPSC lines. c Hyper-IL-6 treatment does not lead to changes in the proportion of the SOX2-positive areas and the number of the VZ-like regions in the DFOs. Ki-67 (cyan), SOX2 (magenta), nuclei stained by DRAQ5 (blue). Four sections of each DFO were analyzed. Each dot represents the mean value of four sections in a single organoid. Color represents cell line of origin, HMGU1 (green), BIONi010-C (orange). Vehicle (n = 6) and Hyper-IL-6 (n = 5) DFOs from one batch per iPSC line. d Hyper-IL-6 treatment leads to an increase in SOX2-positive ventricular RGs (vRG). Hyper-IL-6 treatment does not change the number of TBR2-positive IPCs. Hyper-IL-6 treatment leads to decreased proportion of TBR2-positive IPCs over SOX2-positive vRGs. Each dot represents individual VZ/SVZ-like region, 2–6 regions imaged per organoid. Color represents cell line of origin, HMGU1 (green), BIONi010-C (orange). Shape corresponds to individual DFO. Vehicle (n = 8) and Hyper-IL-6 (n = 6) DFOs from one batch per iPSC line. e Hyper-IL-6 treatment does not lead to changes in proportion of Ki-67-positive proliferative cells over SOX2-positive vRGs. Each dot represents individual VZ-like region, 2–3 regions imaged. Color represents cell line of origin, HMGU1 (green), BIONi010-C (orange). Shape corresponds to individual DFO. Vehicle (n = 6) and Hyper-IL-6 (n = 5) DFOs from one batch per iPSC line. f Hyper-IL-6 treatment does not change the number of CTIP2-positive deep-layer excitatory neurons (dExN) at day 90 of differentiation while increasing the number of SATB2-positive upper-layer excitatory neurons (uExN). Each dot represents an individual region-of-interest representing a putative cortical plate (CP)-like region, 2-3 regions per DFO imaged. Color represents cell line of origin, HMGU1 (green), BIONi010-C (orange). Shape corresponds to individual DFO. Vehicle (n = 12) and Hyper-IL-6 (n = 14) DFOs from three batches. g Hyper-IL-6 treatment leads to incorrect laminar positioning of both CTIP2-positive dExNs and SATB2-positive uExNs at day 90 of differentiation. The cortical structure from the surface of the organoid until the end of dense CTIP2-positive area was evenly divided into bins 1–10. Shown are curves representing the normalized abundance within each bin, calculated as [#CTIP2/SATB2+ cells in a bin/#total CTIP2/SATB2+ cells]. Same samples as in f are shown. In all panels, bars (for c–f) and points (for b and g) represent mean, error bars represent ± SEM. For panels b–f, comparisons were analyzed using Aligned Rank Transform (ART) ANOVA: n.s., non-significant p-value >0.05, *p-value < 0.05. For panel g, comparisons were analyzed using t-test: *p-value < 0.05.