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. 2021 Feb 9;128(2):225–241. doi: 10.1007/s00702-021-02303-5

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© The Author(s) 2021

Open AccessThis 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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Fig. 1 — Differentiation of hiPSCs into 5-HT specific progenitors. A Scheme of the differentiation protocol describing the generation of hiPSCs-derived 5-HT specific neurons. B Generation of 5-HT specific progenitors illustrated by epifluorescence microscopy. a, b Specification of rostral hindbrain progenitors. After 1 week of neural induction cells were stained for typical neural markers, such as Nestin, SOX2, and SOX1. High expression level of HOXA2, a marker for hindbrain progenitors, was additionally found during that time point in these cells. c, d Ventralization of cells was shown by positive IF staining of NKX2.2, NKX6.1, GATA2 and negative staining of PAX3/7, when treated with 1000 ng/ml SHH for 1 week. e Ventralized rostral progenitors were treated with FGF4 for one further week to generate 5-HT progenitors with immunoreactivity for GATA2 and FOXA2. Scale bar: 50 μm (a–c), 100 µm (d, e). Cell nuclei were counterstained with DAPI, and Alexa Fluor 488 (SOX2, Nestin, NKX2.2, NKX6.1, GATA2), and Alexa Fluor 555 (SOX1, HOXA2, GATA2, PAX3/7, FOXA2) were used to visualize target proteins. 5-HT specific progenitor differentiation was verified using the JMUi001-A iPS line (data not shown)