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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. 9 — (a) Schematic representation of the lentiviral vector used for lineage tracing of mesothelial epicardial cells in epicardioids derived from AAVS1-CAG-FRT-flanked STOP-mKate2-HA reporter hiPSCs, consisting of the sequence encoding flippase (FLP) driven by the CDH1 promoter. (b) (Left) Immunostaining for the HA-tag and CDH1 in epicardioids at day 18, 72 h after infection with the CDH1-FLP lentivirus. The arrowhead shows an exemplary HA-tag⁺ CDH1⁺ epicardial cell at the outer layer. (Right) Corresponding uninfected negative control. Images are representative of 3 independent differentiations. Scale bars = 50 µm. (c) Immunostaining for the HA-tag and cardiac troponin T (cTnT) in infected organoids at day 24, showing HA-labeled cardiomyocytes (CMs) close to the epicardium (representative of 5 independent differentiations). Scale bars = 50 µm. Insets show exemplary labeled CMs at higher magnification; scale bars = 10 µm. (d) Violin plots showing the expression levels of markers of mesothelial epicardium (KRT19), EMT (TWIST1), and cycling cells (TOP2A) in epicardial metacell subclusters obtained from analysis of epicardioids by paired scRNA-seq and scATAC-seq. (e) Heatmap showing the relative gene activity of FB (VIM, TNC, FN1, COL1A2), CM (TNNT2, TTN, ACTN2, MYH7) and mural (RGS5, KCNJ8, ACTA2, CSPG4) lineage markers (orange) and the relative gene expression of EMT markers (TWIST1, SNAI2) (blue) along an alternative mural differentiation trajectory as the one presented in Fig. 5g. CM: cardiomyocyte; FB: fibroblast; EPDC: epicardium-derived cell; EMT: epithelial-to-mesenchymal transition.