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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. 6 — (a) Number of cell-cell interactions inferred between all scRNA-seq clusters in epicardioids (day 30). (b) Selected interactions between mesothelial epicardium and the indicated clusters (day 30). Circle size = two-tailed permutation-based p-values, color = mean of the expression level of the interacting molecules. (c) Immunostaining for cTnT and IGF2 (left) or IGF1R (right) in day-15 epicardioids (representative of 2 independent differentiations). Scale bar left = 50 µm, right = 100 µm. (d) Spheroids differentiated without retinoic acid (noRA) were treated with DMSO or IGF2 from days 11 to 15. (e) Representative images of immunostaining for cTnT and Ki67 in treated and control noRA spheroids (day 15). Hatched lines = outer edge of the myocardium; dotted lines = separation between the OM and IM. Scale bars = 50 µm. (f) Percentage of Ki67⁺ CMs in the OM and IM of treated and control noRA spheroids (day 15). All data points; lines connect the values for OM and IM within the same sample. Two-way ANOVA with Tukey’s multiple comparisons test; *p = 0.01; **p = 0.001; ***p < 0.0001; asterisks indicate the p-values obtained from comparing the OM or IM of treated samples with the corresponding layer of controls. N = 7 spheroids, 3 independent differentiations/group. (g) Ratio of CM density (CMs/mm²) OM/IM in treated and control noRA spheroids (day 15). Mean ± SEM; one-way ANOVA with Tukey’s multiple comparisons test. N = 7 spheroids, 3 independent differentiations/group. (h) Immunostaining for cTnT and NRP2 in day-15 epicardioids (representative of 2 independent differentiations). Scale bar = 50 µm. (i) Representative images of immunostaining for cTnT and Ki67 in day-15 epicardioids treated with DMSO or 500 µg/mL NRP2 blocking antibody. Scale bars = 100 µm. (j) Percentage of Ki67⁺ and Ki67⁻ epicardial cells in treated or control epicardioids (d15). Mean ± SD. N = 9 epicardioids, 3 independent differentiations/group. (k) NRP2 blocking antibody targeted with a secondary antibody alongside immunostaining for cTnT and CDH1 in day-15 epicardioids treated with 500 µg/mL NRP2 blocking antibody (representative of 2 independent differentiations). Scale bar = 100 µm.