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. 2021 Feb 8;39(6):737–746. doi: 10.1038/s41587-021-00815-9

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© The Author(s), under exclusive licence to Springer Nature America, Inc. 2021, corrected publication 2021

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, Typical outcome of one experiment. b, Typical examples of successfully formed versus failed HES3 NKX2.5-eGFP-derived HFOs. Only successfully formed HFOs were used for subsequent experiments. c, HFO formation efficiency determined by the proportion of successfully formed HFOs from n = 10 independent experiments (418 HFOs in total). Data are presented as mean ± SEM. d, Whole mount immunofluorescence (IF) staining for NKX2.5 on an HFO derived from the hiPSC line HSC_ADCF_SeV-iPS2 showing a ring-like NKX2.5 pattern equivalent to HES3 NKX2.5-eGFP-derived HFOs. e, Representative images of HES3 NKX2.5-eGFP-derived HFOs differentiated in five different Matrigel lots. f, Comparison of HES3 NKX2.5-eGFP-derived HFOs differentiated in Matrigel, Geltrex or collagen I on d0 and d10 of differentiation. g, Representative images of HES3 NKX2.5-eGFP-derived HFOs from d7 – d10 of differentiation. h, Total area of HFOs from d7 – d10 of differentiation. n = 4 HFOs from one experiment; one-way ANOVA (Tukey’s multiple comparison test); data are presented as mean ± SEM; d7/d10: *P = 0.0115; d8/d10: *P = 0.0268. (i) Area of myocardial layer (ML) plus inner core (IC) from d7 – d10 of differentiation. n = 4 HFOs from one experiment; one-way ANOVA (Tukey’s multiple comparison test); data are presented as mean ± SEM; ns = not significant (P > 0.05). (j) Scheme of an HFO with defined axes. OL=outer layer. Scale bars: a–g: 500 µm.