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. 2023 Apr 3;41(12):1787–1800. doi: 10.1038/s41587-023-01718-7

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© The Author(s) 2023

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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Extended Data Fig. 1 — (a) Cross-sectional area of spheroids during 3D cardiac induction with retinoic acid (RA) or without (noRA). Box plots indicate the median, 25^th and 75^th percentile, with whiskers extending to the 5^th and 95^th percentiles. N per day provided in the Source Data; 3 independent differentiations/group; two-way ANOVA with Sidak’s multiple comparisons test, ***p < 0.0001. (b) Representative bright field images of spheroids at days 4 and 7 of differentiation. Yellow arrowheads indicate rounded protrusions typical of RA spheroids. Scale bars = 500 µm. (c) Percentage of beating spheroids at the indicated days of differentiation; mean ± SEM. noRA: N = 55 spheroids, RA: N = 96 spheroids; 3 independent differentiations/group. Two-way ANOVA with Sidak’s multiple comparisons test. (d) Immunostaining for cTnT, TJP1 and VIM in epicardioids (days 15 and 30). Arrowheads = mesothelial epicardium; arrows = epicardium-derived cells. Scale bars = 50 µm. (e) Immunostaining for cTnT and VIM in epicardioids (day 15). Arrowheads indicate examples of cTnT⁻ VIM⁺ mesenchymal cells in the subepicardial space (left) and within the myocardium (right). Scale bars = 50 µm. (f) Immunostaining for cTnT and CD31 or CDH5 in VEGF-treated epicardioids (day 15). Scale bars = 50 µm. Images in (d-f) are representative of 3 independent differentiations. (g,h) Percentage of spheroids with an absent, partial or complete epicardial layer at day 15 of epicardioid differentiation of different human induced pluripotent (hiPSC) or embryonic stem cell (hESC) lines. The starting cell number was adapted to the cell lines’ proliferation rate to obtain a consistent spheroid size at day 0. Mean ± SEM; hiPSC1: N = 6 independent differentiations, hiPSC2: N = 2, hiPSC3: N = 1, hESC: N = 2; N per differentiation provided in the Source Data. (g) Representative images of cTnT and cytokeratin 18 (KRT18) (top) or TBX18 (bottom) immunostaining of day-15 epicardioids from the different hPSC lines. The arrow indicates an example of a partial epicardial layer, arrowheads show examples of TBX18⁺ epicardial cells. Scale bars top = 200 µm, bottom = 50 µm. (h).