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. 2023 Sep 1;55(9):2005–2024. doi: 10.1038/s12276-023-01074-1

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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. 1 — A Schematic overview of organoid differentiation from PSCs. B Representative images of Day 0 (D0) pluripotent spheroids, left panel brightfield. The remaining panels show whole-mount immunostaining of the pluripotency markers OCT4, SOX2 and NANOG; the scale bar is 200 µm. The highlighted area is magnified in Supplementary Fig. 9. C Whole-mount immunostaining of the definitive endoderm (DE) marker FOXA2 at D2 of differentiation. D RT‒qPCR analysis of pluripotency- and DE-associated genes at D1 and D2 of differentiation relative to D0 spheroids on a log10 scale. The results from three independent experiments are presented as the mean ± SD. E Graph showing the shift in size of organoids from D0 to D2. The size is expressed as the mean diameter (µm) with error bars showing the standard deviation, representing a total of 570 organoids. Day 0 vs. Day 1 p = 0.001, Day 0 vs. Day 2 p < 0.0001, Day 1 vs. Day 2 p < 0.0001 from Kruskal‒Wallis test with Dunn`s multiple comparisons test. F RT‒qPCR analysis of DE and early liver development genes at D7 of differentiation relative to D2 on a log10 scale. The results from three independent experiments are presented as the mean ± SD. G Whole-mount immunostaining of D7 organoids showing epithelial (ECAD) and early hepatocyte markers (FOXA2, CK8, HNF4α, AFP) on the outer surface of the organoids. The highlighted area is magnified in Supplementary Fig. 9. H Whole-mount immunostaining of D7 organoids showing heterogeneous expression of mesoderm (MESP1)- and mesenchymal (ALCAM, WT1)-associated markers. The highlighted area is magnified in Supplementary Fig. 9. I Brightfield image of D20 organoids. Scale bar is 500 µm. J Violin plot of D20-D27 organoid diameter from three representative experiments. The solid line represents the mean, while the dotted lines represent the quartiles. K RT‒qPCR analysis of both early and later (developmentally) hepatocyte genes at D20 of differentiation relative to D2 on a log10 scale. The results from three independent experiments are presented as the mean ± SD. L Whole-mount immunostaining of D20 organoids showing expression of the hepatocyte markers HNF4α and albumin on the outer surface of the organoids. M Demonstration of increasing secretion of HGF into the culture medium by the organoids throughout differentiation as measured by ELISAs. The results from three independent experiments are presented as the mean ± SD. All of the above experiments were performed with the hiPSC line AG27. All scale bars are 100 µm unless stated otherwise.