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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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Both ECO and ICO have cholangiocyte-fate differentiation potential. Culturing organoids in cholangiocyte differentiation medium (DM-chol) for 14 days result in morphological changes, Organoids became more dense, had thicker outer walls as compared to EM condition and stopped proliferating (A). Shown are representative bright field microscopic images (bar = 1000 um). At gene expression level, stem cell and proliferation markers LGR5 and Ki76 were significantly downregulated in DM-chol both for ECO and ICO (B). Gene expression clustering is based on 20 genes selected for their high expression in cholangiocytes by single cell RNA sequencing. For benchmarking the cholangiocyte-differentiated ICO and ECO bile duct tissue gene expression profiles were used (Rimland et al.¹⁷). Both ICO and ECO showed clear upregulation of cholangiocyte genes in DM-chol conditions (C). Fluorescent tracer dye was used to determine transporter channel activity in DM-chol ECO (D) As shown in left panel, Rhodamine123 is actively transported into the lumen of ECO-chol. This luminal transport of rhodamine was completely inhibited by MDR1 inhibitor, verapamil (10 nM, right panel). Blocking the Notch-pathway by DAPT, inhibit cholangiocyte-fate differentiation of ECO (E). This confirms in vitro that Notch signals are important drives of cholangiocyte lineage development.