Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice.
The gene expression datasets generated and analyzed during the current study are available in the Gene Expression Omnibus repository: microarray data have been deposited under accession number GSE150051 and single-cell RNA sequencing data under accession number GSE150202. For the latter, to interrogate gene expression in the t-SNE plot, the .cloupe.gz file should be downloaded, unzipped and opened with the 10x Genomics Loupe Browser. All additional data supporting the findings of this study are available within the article and its Supplementary Information.
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We thank S. C. Den Hartogh and R. Passier (Department of Anatomy and Embryology, Leiden University Medical Centre) for providing the HES3 NKX2.5–eGFP cell line, E. G. Stanley and A. G. Elefanty (Monash Immunology and Stem Cell Laboratories, Monash University) for the HES3 MIXL1–GFP cell line, D. J. Anderson and D. A. Elliott (Murdoch Children’s Research Institute, Royal Children’s Hospital) for the HES3 NKX2.5–eGFP/eGFP cell line and A. Haase (LEBAO, Hannover Medical School, MHH) for the HSC_ADCF_SeV-iPS2 cell line. We also thank the Research Core Unit for Laser Microscopy at MHH for providing the opportunity to take confocal images. Microarray and scRNA-seq data used or referred to in this publication were generated by the Research Core Unit Genomics at MHH. We thank B. Andrée and M. Fischer for providing critical and helpful comments for this manuscript. R.Z. received funding from: the German Research Foundation (DFG; grants: Cluster of Excellence REBIRTH EXC 62/2, ZW64/4-1 and KFO311/ZW64/7-1), the German Ministry for Education and Science (BMBF, grants: 13N14086, 01EK1601A, 01EK1602A, 13XP5092B and 031L0249), ‘Förderung aus Mitteln des Niedersächsischen Vorab’ (grant: ZN3340), ‘Cortiss Stiftung’ and the European Union H2020 project TECHNOBEAT (grant: 66724). C.W.-S. received DFG grants: WA 2597/3-1 and SFB/TRR152. U.M. received BMBF grants: 82DZL00201 and 82DZL00401.
The authors declare no competing interests.
Peer review information Nature Biotechnology thanks Thomas Brand, Thomas Eschenhagen, Gordana Vunjak-Novakovic and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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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.
a, Paraffin sections from different parts of the HFO stained for cardiac troponin T (cTnT) showing that the cTnT-positive myocardial layer encloses the cTnT-negative inner core on its back side. b, Proportion of NKX2.5-eGFP/ ISL1-double-positive (second heart field-like) cells among all NKX2.5-eGFP-positive cells in HFOs from d7 – d13 of differentiation. n = 3 (d7, d13) or 4 (d10) derived from 3 independent experiments; data are presented as mean ± SEM. (c–j) Patch clamp analysis of HFO-derived NKX2.5-eGFP-positive cardiomyocytes in whole-cell current clamp mode. c–e, Representative traces for three different action potential (AP) phenotypes. Left panels show spontaneous electrical activity of the cells; right panels depict subsequently evoked APs of the same cell. Here, the cell membrane was hyperpolarized by application of negative holding currents (from −2 to −50 pA) to achieve comparable conditions for each cell mimicking physiological resting membrane potentials of around −80 mV. Short current pulses (1 ms, 400 – 1500 pA) were applied to evoke APs. The classification of the phenotypes was based on the AP duration of these traces: Cells were classified as ventricular-like when they showed APs with a plateau phase longer than 200 ms as measured at 50% repolarization level (APD50) (c). Cells were classified as atrial-like when they displayed a triangular shape with APD50 values of 20 – 200 ms (d). Cells showing APs without overshoot were termed atypical (e). f, Distribution of cardiac subtypes in HFOs based on the classification in c–e. (g–j) Additional AP parameters for ventricular-like cells isolated from HFOs (g: MDP/RMP, maximal diastolic potential/ resting membrane potential; h: APD50; i: AP amplitude; j: upstroke velocity). Spontaneous AP: n = 36 cells, evoked AP: n = 35 cells isolated from 3 HFOs from one experiment; data are presented as mean ± SEM. Scale bars: a: 200 µm.
a, Representative flow cytometry plot of an HFO stained for the endothelial cell (EC) marker CD31 (left) and EC amount in HFOs assessed by flow cytometry (right). Data are presented as mean ± SEM; n = 6 HFOs from 2 independent experiments. b, Paraffin section stained for CD31 and enlargements showing EC-lined cavities (dotted arrows) and a non-EC-lined cavity (arrowhead) in the inner core (IC) and ECs (solid arrow) between the IC and the myocardial layer (ML). c, Assessment of the average number of vessel-like structures (VL) present on paraffin sections of HFOs (front view). Left: Representative image of an HFO paraffin section stained for CD31, in which the VLs are marked with asterisks. Right: Number of VLs per 1 mm² IC area. Data are presented as mean ± SEM; n = 10 HFOs from 3 independent experiments. d, Cryosection stained for CD31 and NKX2.5 and enlargements showing CD31/ NKX2.5-double-positive endocardial-like cells (arrows) between the ML and the IC. (e) Multiphoton microscopy of an HFO. The figure shows still images of Supplementary Video 4 at different time points. Scale bars: b–d: 100 µm.
a, 3D reconstruction of the inner core. The endodermal cavities are depicted in different colors. The figure shows still images of Supplementary Video 5 at different time points. b, Comparison of HFOs with human embryonic hearts. Heat map generated from the microarray data of five HFOs versus four 5–7 weeks old human embryonic hearts and three hPSC samples (q≤0.01; variance filtering = 0.5; fold change ≥ 10 to view the 100 most significantly up-/ downregulated genes). c, Additional t-SNE plots showing the expression of the indicated genes in two combined d13 HFOs.
Extended Data Fig. 5 Comparison to conventional cardiac differentiation and long-term culture of HFOs.
a, Selected genes, which were up- or downregulated in HFOs compared to cardiomyocytes generated by a conventional 2D differentiation protocol and respective gene functions according to the GeneCards database38. FC= fold change. b, Cryosections of aggregates generated by a conventional 3D cardiac differentiation protocol stained for the cardiac markers NKX2.5, cTnT and sarcomeric actinin (SA). c, Long-term culture of HFOs. HES3 NKX2.5-eGFP-derived HFOs were cultured up to 146 days in suspension after dissolving the surrounding Matrigel. HFOs were stained with DiI-Ac-LDL to visualize endothelial cells. Scale bars: b: 100 µm; c: 500 µm.
a, Paraffin section of an NKX2.5-KO organoid stained for CD31 to visualize the vascular structures. Dotted arrows point at endodermal cavities in the inner core, solid arrows point at endodermal islands in the outer layer. b, Hematoxylin-eosin staining of HFOs and enlargements highlighting the reduced myocardial layer (ML) compactness in NKX2.5-KO organoids compared to control HFOs. c, ML compactness of control 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; ns = not significant (P>0.05). d, Exemplary images of an NKX2.5-KO organoid on d10 and d20 of differentiation. e, ML compactness of NKX2.5-KO organoids over time compared to the ML compactness of d10 HFOs. Control: n = 14 HFOs, NKX2.5-KO: n = 3 (d10 – d13) or 4 (d14 – d20) HFOs from 3 (control) or 1 (NKX2.5-KO) experiments; one-way ANOVA (Tukey’s multiple comparison test); data are presented as mean ± SEM; Control d10/ KO d12: *P = 0.0196; KO d10/d12: *P = 0.0269; KO d10/d13: **P = 0.0016; ****P ≤ 0.0001; ns = P > 0.05. f, Cardiomyocyte (eGFP-positive cell) content in NKX2.5-KO versus control HFOs determined by flow cytometry. Control: n = 6 HFOs, NKX2.5-KO: n = 4 HFOs from 2 independent experiments; two-tailed t-test; data are presented as mean ± SEM; ns = not significant (P > 0.05). g, Light microscopy (LM; overview in left column; toluidine blue staining) and transmission electron microscopy (TEM) pictures of sarcomeres in control and NKX2.5-KO organoids. h, Microarray analysis of control versus NKX2.5-KO organoids: Selected genes, which were up- or downregulated in NKX2.5-KO organoids and respective gene functions according to the GeneCards database38. FC=fold change. Scale bars: a, b: 100 µm; d = 500 µm; g = 200 µm (LM) or 1 µm (TEM).
The myocardial layer (ML) compactness was defined as the eGFP-positive area in proportion to the ML area. The figure shows stepwise how the compactness was determined using ImageJ software. Scale bars: 500 µm.
Schematic comparison of an HFO with the early embryonic heart/ foregut region (sagittal plane).
SLOT of an HFO. The analysis via SLOT shows the typical layered 3D structure of HFOs.
Calcium imaging of an HFO with a circular beating pattern. The HFO was stained with the calcium-sensitive dye Rhod-4 to highlight the contractions.
Calcium imaging of an HFO with a contraction initiation point. The HFO was stained with the calcium-sensitive dye Rhod-4 to highlight the contractions.
MPM of an HFO. The HFO was stained with DAPI (white) and CD31 (purple) to visualize the endothelial structures. The video starts at the front of the HFO showing the IC and progresses through the ML to the OL.
A 3D reconstruction of the IC of an HFO. The large, branched endodermal cavities, which are distributed all over the IC of the HFO, are depicted in different colors.
Calcium imaging of an NKX2.5-KO organoid. The HFO was stained with the calcium-sensitive dye Rhod-4 to highlight the contractions.
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Drakhlis, L., Biswanath, S., Farr, CM. et al. Human heart-forming organoids recapitulate early heart and foregut development.
Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-00815-9