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. 2018 Oct 2;9:4027. doi: 10.1038/s41467-018-06334-7

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© The Author(s) 2018

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. 4 — Organoid generation and characterization. a Human fetal cochlea at W10 (sample E1201) immunostained for EPCAM and FACS plot showing the gating of EPCAM-positive cochlear duct cells. Also shown is a schematic overview of the procedure for organoid generation. b Representative examples of EPCAM+ and EPCAM− derived cultures at day 7 (DIV7) and day 20 (DIV20) in vitro (sample E1220). EPCAM negative cells immunostained at day 13 for nestin (NES), peripherin (PRPH), and βIII Tubulin (TUBB3). EPCAM-positive organoids immunostained at day 20 in vitro for MYO7A. F-actin was labeled with phalloidin. Scale bar = 100 μm. c–d Immunostaining of 1st generation organoids (E1220 and E1253) at DIV14 and DIV20 for the proteins indicated. Colored boxes indicate marker classes: epithelial (green), cell cycle (red), cochlear duct (light blue), and PSD markers (orange) are shown. Scale bar = 100 μm. e–f Immunostaining of 2nd generation organoids (E1224) at DIV16 + DIV20 (days 1st generation + days 2nd generation) for the proteins indicated; marker classes are indicated by the colored boxes. Scale bar = 100 μm