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. 2024 Sep 23;56(10):2158–2173. doi: 10.1038/s41588-024-01875-8

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© The Author(s) 2024

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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Extended Data Fig. 1 — a, Epithelioid generation from a single esophageal explant. b, Live microscopy ‘Incucyte’ images 5, 11 and 15 days after plating Rosa26^mTmG esophageal explants. Color scale shows fluorescence intensity. Scale bar, 1000 μm. Images representative of 20 independent explants. c, Area of covered by cells from a single esophageal explant. Each dot represents an epithelioid. n = 6 epithelioids. d-e, Expansion velocity at Day 6 after plating and area of cellular outgrowth 8 days after plating. Each dot represents a cellular outgrowth and each colour a different mouse (n = 29 epithelioids from 6 mice). Red bar, mean. f, Number of cells in epithelioids 22 days after plating an explant of 1/32th of esophagus (Epithelioid) compared to the basal cell number in original explant (Original tissue). Each dot represents a mouse or an epithelioid. Dots with the same color correspond to same mouse. n = 9 epithelioids from 3 mice. Red bar, mean. g Protocol: 5 explants from Rosa26^mTmG mouse esophagus were plated in 75 mm diameter inserts and cultured for 20 days basal cell numbers quantified at the start and end of experiment. h optical sections of basal layer (top-left), suprabasal layer (top-right) with lateral views (bottom) of a representative area of 20 day culture from g. Grey, TP63, green, KRT4, and blue, DAPI. Scale bars, 20 μm. i and j, Immunostaining against PDGFRA (red, fibroblasts), KRT4 (green), CD45 (grey, immune cells) and DAPI (blue), of explant and surrounding cellular outgrowth. Optical section with orthogonal views of explant submucosa (I, scale bars=44 μm) and newly formed epithelioid basal layer membrane (j, scale bars=41 μm). Images representative of 3 biological replicates. k-n, Mouse esophageal epithelioids cultured in mFAD immunostained against (k) KRT13 (green) and DAPI (blue); (l) FABP5 (green), F-actin (red), ITGA6 (grey) and DAPI (blue); (m) LOR (green), ITGA6 (grey) and DAPI (blue); (n) KLF4 (green) and DAPI (blue). Scale bars correspond to 40μm (k, top panel), 30μm (k, bottom panel and l), 50μm (m) and 20μm (n). Source data in Supplementary Table 1. Images representative of 3 biological replicates.

Source data