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. 2020 Dec 14;10:21900. doi: 10.1038/s41598-020-79082-8

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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 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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ECO lack hepatocyte-fate differentiation potential. Culturing organoids in hepatocyte differentiation medium (DM-hep) for 14 days result in morphological changes (A). Organoids became more dense (DM-hep) and stopped proliferating (bar = 1000 um). H&E staining of 5 µm organoid Sections (40×) show multiple cell layers and dense cellular structure in DM-hep. Bulk RNA sequencing analysis was performed and 35 selected genes associated with heptatocyte, cholangiocyte and liver progenitor cells¹⁵ provided an expression heatmap showing the relative expression of ECO and ICO organoids that were differentiated towards hepatocyte-fate (B). Relative gene expression levels are indicated in color, with blue for down-regulated genes and red for upregulated genes. None of the ECO-hep organoids clearly expressed hepatocyte-specific genes, whereas 2 of the 3 ICO-hep organoids showed expression towards a hepatocyte-like phenotype. Primary hepatocytes (PHH, adapted from Schneeberger et al.²³), served as positive controls. Assessing general differences in ICO and ECO gene expression showed that in DM-hep condition the difference between ICO and ECO is biggest whereas small differences in gene expression was observed in DM-chol (C). Gene expression levels of hepatocyte-specific genes albumin (D) and Cyp3A4 (E) were further confirmed by qRT-PCR analyses in independent experiments. Both albumin (D) and Cyp3A4 (E) were again found increased in ICO and not in ECO. (C). KRT19 (F) expression levels did not differ significantly between these organoid types. Significance is indicated as ***p < 0.0005. As a functional measure of hepatocyte metabolic activity, Cyp3A4 activity was measured using a commercially available kit (G). The relative Cyp3A4 activity was significantly higher in DM-hep conditions for ICO but not for ECO. Significance is indicated as *p < 0.01.