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Cited by 536Pluripotent Stem Cells ResearchCongenital heart defects researchTissue Engineering and Regenerative Medicine

Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease

Open access
Lewis-Israeli, Yonatan R., Wasserman, Aaron H., Gabalski, Mitchell A., Volmert, Brett D., Ming, Yixuan, Ball, Kristen A., Yang, Weiyang, Zou, Jinyun +6 more·2021·Nature Communications
DOI 10.1038/s41467-021-25329-5PMC PMC8390749

Published version of a preprint

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Abstract

Congenital heart defects constitute the most common human birth defect, however understanding of how these disorders originate is limited by our ability to model the human heart accurately in vitro. Here we report a method to generate developmentally relevant human heart organoids by self-assembly using human pluripotent stem cells. Our procedure is fully defined, efficient, reproducible, and compatible with high-content approaches. Organoids are generated through a three-step Wnt signaling modulation strategy using chemical inhibitors and growth factors. Heart organoids are comparable to age-matched human fetal cardiac tissues at the transcriptomic, structural, and cellular level. They develop sophisticated internal chambers with well-organized multi-lineage cardiac cell types, recapitulate heart field formation and atrioventricular specification, develop a complex vasculature, and exhibit robust functional activity. We also show that our organoid platform can recreate complex metabolic disorders associated with congenital heart defects, as demonstrated by an in vitro model of pregestational diabetes-induced congenital heart defects.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper reports a fully defined, reproducible method to generate self-assembling human heart organoids (hHOs) from human pluripotent stem cells via three-step Wnt signaling modulation. The hHOs recapitulate fetal-like cardiac structure, multi-lineage composition, chamber formation, vascularization, and electrophysiology, and can model pregestational diabetes-induced congenital heart defects in vitro.

Key findings

  • hHOs were generated by sequential CHIR99021/Wnt-C59/CHIR99021 treatment; beating appeared as early as day 6, was robust by day 10, and lasted at least 8 weeks. Optimal first Wnt activation was 1–4 µM CHIR99021, with 4 µM yielding 64.9 ± 5.3% TNNT2+ cells at day 15.
  • hHOs contained multiple cardiac lineages: approximately 59% cardiomyocytes, 16% epicardial cells, 14% endocardial cells, 12% cardiac fibroblasts, and 1.6% endothelial cells; at day 15, atrial cardiomyocytes were ~48% and ventricular cardiomyocytes ~18% of total cells.
  • hHOs formed 4–6 internal chambers ranging from 5.5e−4 mm3 to 1.3e−2 mm3, with organoid size up to 1 mm in diameter (~0.45 mm3), and showed vascular networks, action potentials, and calcium transients.
  • In a pregestational diabetes model using 11.1 mM glucose and 1.14 nM insulin versus 3.5 mM glucose and 170 pM insulin, diabetic hHOs were larger, showed arrhythmic action potentials, decreased oxygen consumption, increased glycolysis, fewer mitochondria, more lipid droplets, decreased MYL2+ ventricular cells, enlarged MYL7+ atrial regions, and disrupted epicardial/myocardial organization.

Methods

  • hPSCs were seeded at 10,000 cells/well in ultra-low attachment 96-well plates to form embryoid bodies, then differentiated with a three-step Wnt modulation protocol using CHIR99021, Wnt-C59, and a second CHIR99021 exposure; BMP4 and Activin A were tested as additives.
  • Characterization included RNA sequencing across days 0–19, immunofluorescence/confocal imaging, optical coherence tomography, TEM, microelectrode array electrophysiology, GCaMP6f calcium imaging, and Seahorse metabolic assays.
  • Statistical analysis used unpaired two-tailed Student’s t-tests and 1-way or 2-way ANOVA with multiple-comparison corrections; data are mean ± s.d. from at least 3 independent experiments unless otherwise stated.

Limitations

  • Stated: organoids tend to deviate from normal developmental pathways over time and become less relevant the longer they are cultured; their recapitulation of heart development remains limited compared with models such as mice.
  • Stated: the technology needs improvement in morphological/anatomical fidelity and in forming effective vascular networks that can provide nutrients; further studies are needed to assess vascular maturity, connectivity, and resemblance to coronary vasculature.
  • Stated and evident: immunofluorescence and bulk RNA sequencing may not fully capture organoid heterogeneity, and single-cell/single-organoid omics are proposed for future work; the study is in vitro only and does not report in vivo validation or transplantation.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41467-021-25329-5

Cites

95 of 95 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 68 cited sources checked · 0 supported · 0 flagged

  • Findingnone given

    The hHO protocol relies mainly on three sequential Wnt modulation steps (activation/inhibition/activation) at specific time points on suspension embryoid bodies.

    “Our protocol relies mainly on three sequential Wnt modulation steps (activation/inhibition/activation) at specific time points on suspension embryoid bodies (EBs)”
    Quote in the paperSubstantial claim
  • Findingnone given

    hHOs are comparable to age-matched human fetal cardiac tissues at the transcriptomic, structural, and cellular level.

    “Heart organoids are comparable to age-matched human fetal cardiac tissues at the transcriptomic, structural, and cellular level.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The developmental stage of hHOs at day 19 is age-matched to human fetal hearts at gestational days 57–67.
  • Findingown data

    hHOs develop sophisticated internal chambers with well-organized multi-lineage cardiac cell types.

    “They develop sophisticated internal chambers with well-organized multi-lineage cardiac cell types”
    Quote in the paperSubstantial claim
  • Findingown data

    The paper reports a small molecule-based methodology to create highly complex and physiologically relevant self-assembling human heart organoids using hPSCs by manipulating cardiac developmental programs.

    “Here we report a method to generate developmentally relevant human heart organoids by self-assembly using human pluripotent stem cells.”
    • Also stated in: § Introduction · plain
      “Here, we report a small molecule-based methodology to create highly complex and physiologically relevant self-assembling human heart organoids (hHOs) using hPSCs by manipulating cardiac developmental programs.”
    Quote in the paperSubstantial claim
  • Findingnone given

    The hHO protocol produces significant heart-like structures in terms of structure, organization, functionality, cardiac cell type complexity, ECM composition, and vascularization.

    “produces significant heart-like structures in terms of structure, organization, functionality, cardiac cell type complexity, ECM composition, and vascularization.”
    Quote in the paperSubstantial claim
  • May combine two claims. 48
  • Classified as probably not a substantial claim. 22

  • May combine two claims.

    Findingown data

    hHOs developed sarcomeres as early as day 7, with clearer sarcomere banding and fiber assembly by day 15.

    “Confocal microscopy for the cardiomyocyte-specific marker TNNT2 showed that organoids developed sarcomeres as early as day 7 (Fig. 1b), with much clearer sarcomere banding and fiber assembly by day 15”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Beating hHOs appeared as early as day 6, with robust and regular beating by day 10 in all organoids and lasting for at least 8 weeks in culture.

    “Beating hHOs appeared as early as day 6 of the differentiation protocol, with robust and regular beating by day 10 in all organoids and lasting for at least 8 weeks in culture”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Optimal conditions for the first Wnt activation were critical for successful heart organoid formation and differed from those reported for cardiac lineage monolayer differentiation.

    “We found out that optimal conditions for the first Wnt activation were critical for successful heart organoid formation and differed from those reported for cardiac lineage monolayer differentiation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    High concentrations or long exposure times of CHIR99021 at the second Wnt activation inhibited cardiac cell type formation and provoked extensive epicardial expansion.

    “however, high concentrations or long exposure times inhibited cardiac cell type formation and provoked an undesired extensive epicardial expansion”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The hHO cell composition is 12.49 ± 1.01% cardiac fibroblasts, 13.82 ± 1.54% endocardial cells, and 1.63 ± 0.21% endothelial cells.

    “Figure 3f shows a quantification of the contribution of the different cardiac cell populations to the organoids, with a composition of 12.49 ± 1.01% cardiac fibroblasts, 13.82 ± 1.54% endocardial cells, and 1.63 ± 0.21% endothelial cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    hHOs recapitulate heart field formation and atrioventricular specification.

    “recapitulate heart field formation and atrioventricular specification”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Imaging revealed a robust interconnected network of endothelial cells (PECAM1+) and vessel-like tube formation throughout the organoid assembling between day 11 and 13.

    “Further imaging revealed a robust interconnected network of endothelial cells (PECAM1 + ), and vessel-like tube formation throughout the organoid assembling between day 11 and 13”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Immunofluorescence of day 7 hHO cryosections showed well-differentiated regions of HAND1 and HAND2 cells in the same organoid, suggesting both FHF and SHF progenitors are present and segregated into their respective heart fields.

    “Immunofluorescence of day 7 hHO cryosections showed well-differentiated regions of HAND1 and HAND2 (Fig. 4a) cells in the same organoid, suggesting that both FHF and SHF progenitors are present and segregated into their respective heart fields.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    NHOs were slower to grow and exhibited patterning and elongation between days 4 and 8.

    “NHOs were slower to grow and exhibited patterning and elongation between days 4 and 8”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Atrial cardiomyocytes made up most of the cell population (~48%) while ventricular cardiomyocytes made up about one fifth (~18%) of the total cells in the organoids at day 15.

    “Atrial cardiomyocytes made up most of the cell population (~48%) while ventricular cardiomyocytes made up about one fifth (~18%) of the total cells in the organoids at day 15”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    BMP4/ActA treatment did not significantly change the percentage of cardiomyocyte (TNNT2+) or epicardial (WT1+TJP1+) positive cells relative to control.

    “a Percent of cardiomyocyte and epicardial positive cells as a percentage of total DAPI + nuclei (n = 7 organoids per condition, value = mean ± s.d., 2-way ANOVA Sidak’s multiple comparisons test, ns: p = 0.76 for TNNT2 and p = 0.97 for WT1 + TJP1)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    BMP4 and Activin A treatment caused a 160% increase in PECAM1+ cells, indicating a significant effect on organoid vascularization.

    “Notably, immunofluorescence analysis of organoids treated with BMP4 and Activin A showed a 160% increase in PECAM1+ cells too, indicating a significant effect on organoid vascularization”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    hHOs contain 4-6 chambers near the center, with volumes ranging from 5.5e−4 mm3 to 1.3e−2 mm3.

    “revealing 4–6 chambers near the center of the organoid ranging from 5.5e−4 mm3 to 1.3e−2 mm3 (Supplementary Fig. 6d, Supplementary Movie 10).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    hHOs exhibit robust beating and normal electrophysiological activity with action potential waves reminiscent of QRS, T, and P waves and regular action potentials.

    “we could detect robust beating and normal electrophysiological activity with well-defined action potential waves reminiscent of QRS complexes, T and P waves, and regular action potentials across multiple organoids (Fig. 6d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The two different populations localized to different regions of the organoid and were in close proximity, mirroring the expression pattern seen in human hearts.

    “The two different populations localized to different regions of the organoid and were in close proximity, which mirrors the expression pattern seen in human hearts”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SHF markers appeared later, only becoming prominent around day 7, and expressing throughout the organoid up to day 9.

    “SHF markers appeared later in the process, only becoming prominent around day 7, and expressing throughout the organoid up to day 9”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PGDHOs contained epicardial tissue surrounded by myocardial tissue, whereas NHOs contained epicardial tissue on top of or beside myocardial tissue.

    “PGDHOs contained epicardial tissue surrounded by myocardial tissue, whereas NHOs contained epicardial tissue on top of or beside myocardial tissue as physiologically expected (Fig. 7h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Diabetic hHOs showed decreased MYL2+ ventricular cardiomyocytes and enlarged MYL7+ atrial cardiomyocyte regions compared with normal glycemia.

    “Compared with normal glycemia conditions, diabetic hHOs showed decreased MYL2 + ventricular cardiomyocytes and enlarged MYL7 + atrial cardiomyocyte regions”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treatment with BMP4 and Activin A in addition to CHIR increased PECAM1+ cells by 160% relative to control, indicating a significant effect on organoid vascularization.

    “with BMP4 and Activin A showed a 160% increase in PECAM1+ cells too, indicating a significant effect on organoid vascularization (Fig. 5f, g).”
    • Also stated in: § BMP4 and Activin A improve heart organoid chamber formation and vascularization · plain
      “f PECAM1 + cells as a percentage of total DAPI + nuclei, (n = 7 organoids per condition, value = mean ± s.d., two-tailed, unpaired t-test, **p = 0.0023).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    FHF markers were observed as early as day 3 (prior to TNNT2 detection), confined to specific regions on the organoid, and reducing in expression around day 9.

    “FHF markers were observed as early as day 3 (prior to TNNT2 detection), confined to specific regions on the organoid, and reducing in expression around day 9”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Compared with existing cardiomyocyte monolayer differentiation methods, hHOs showed higher expression of genes associated with conduction, contractile function, calcium handling, and various cardiac cell populations, which better resemble gene expression data from human fetal hearts.

    “When compared with existing cardiomyocyte monolayer differentiation methods, hHOs showed higher expression of genes associated with conduction, contractile function, calcium handling, and various cardiac cell populations, which better resemble gene expression data retrieved from human fetal hearts.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    hHOs form multiple chambers lined with NFATC1+ endocardial cells that are interconnected.

    “The hHOs reported here form multiple chambers lined with NFATC1+ endocardial cells which are interconnected as seen in the OCT cross-sectional imaging (Supplementary Movies 7–10).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Hybrid cardiac-foregut organoids from human PSCs have been reported with external heart layers and an internal endodermal core.

    “Hybrid cardiac-foregut organoids from human PSCs have also been reported very recently, with external heart layers and an internal endodermal core19”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Drakhlis, L. et al. Human heart-forming organoids recapitulate early heart and foregut development. Nat. Biotechnol. 39, 737–746 (2021). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    These cardioids relied heavily on complex growth factor mixtures, co-culture approaches to include epicardial clusters, and independent differentiation protocols.

    “While demonstrating a large leap in self-assembling capabilities, these cardioids still relied heavily on complex growth factor mixtures, co-culture approaches to include epicardial clusters, and independent differentiation protocols to achieve their final results.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [19] Drakhlis, L. et al. Human heart-forming organoids recapitulate early heart and foregut development. Nat. Biotechnol. 39, 737–746 (2021). source

      Not checked in the free run.

    • cites [20] Hofbauer, P. et al. Cardioids reveal self-organizing principles of human cardiogenesis. Cell. 184, 3299–3317 (2021). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    The hHO method is automatable, scalable, and amenable to high-content/high-throughput pharmacological screenings.

    “It is also automatable, scalable, and amenable to high-content/high-throughput pharmacological screenings.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Mouse ESCs have been used to generate precardiac organoids showing distinct heart field specifications, cardiac crescent-like structures juxtaposed with primitive gut tube, and atrial and ventricular cardiomyocyte lineages.

    “Recently, mouse embryonic stem cells (ESCs) were used to generate precardiac organoids showing distinct heart field specifications16, cardiac crescent-like structures juxtaposed with primitive gut tube17, and atrial and ventricular cardiomyocyte lineages18.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [16] Andersen, P. et al. Precardiac organoids form two heart fields via Bmp/Wnt signaling. Nat. Commun. 9, 1–13 (2018). source

      Not checked in the free run.

    • cites [17] Rossi, G. et al. Capturing cardiogenesis in gastruloids. Cell Stem Cell 28, 230–240 (2020). source

      Not checked in the free run.

    • cites [18] Lee, J. et al. In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix. Nat. Commun. 11, 1–18 (2020). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Cardioids have been reported with a large internal chamber, relevant cardiac cell lineages and single cell transcriptomic analysis.

    “cardioids (an alternative nomenclature for heart or cardiac organoids) have been reported with a large internal chamber, relevant cardiac cell lineages and single cell transcriptomic analysis20.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Hofbauer, P. et al. Cardioids reveal self-organizing principles of human cardiogenesis. Cell. 184, 3299–3317 (2021). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    The hHO method is low-cost compared to growth factor-based approaches and involves less steps and manipulation than currently existing methods.

    “Our method is low-cost when compared to growth factor-based approaches and involves less steps and manipulation than currently existing methods.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Tissue engineering approaches for heart-on-a-chip or heart organoid models allow high control of the end construct but tend to be expensive, work-intensive, and not readily scalable.

    “While these approaches allow for high control of the end construct, they tend to be expensive, work-intensive, and not readily scalable.”
    Quote in the paperSubstantial claim0 of 8 sources checked
    • cites [8] Richards, D. J. et al. Inspiration from heart development: biomimetic development of functional human cardiac organoids. Biomaterials 142, 112–123 (2017). source

      Not checked in the free run.

    • cites [9] Richards, D. J. et al. Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity. Nat. Biomed. Eng. 4, 446–462 (2020). source

      Not checked in the free run.

    • cites [10] Fischer, B. et al. A complete workflow for the differentiation and the dissociation of hiPSC-derived cardiospheres. Stem Cell Res. 32, 65–72 (2018). source

      Not checked in the free run.

    • cites [11] Davis, D. R. et al. Validation of the cardiosphere method to culture cardiac progenitor cells from myocardial tissue. PLoS ONE 4, e7195 (2009). source

      Not checked in the free run.

    • cites [12] Ronaldson-Bouchard, K. et al. Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature 556, 239–243 (2018). source

      Not checked in the free run.

    • cites [13] Li, Q. et al. Cyborg organoids: implantation of nanoelectronics via organogenesis for tissue-wide electrophysiology. Nano Lett. 19, 5781–5789 (2019). source

      Not checked in the free run.

    • cites [14] Mills, R. J. et al. Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest. Proc. Natl Acad. Sci. USA 114, E8372–E8381 (2017). source

      Not checked in the free run.

    • cites [15] Ma, Z. et al. Self-organizing human cardiac microchambers mediated by geometric confinement. Nat. Commun. 6, 1–10 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The hHO model has endocardial lined chambers within myocardial tissue, interspersed with cardiac fibroblasts and a network of endothelial cells, and complete with external epicardial tissue, features highly recapitulative of the developing human heart.

    “Together, these observations depict a complex and sophisticated human heart organoid model with endocardial lined chambers within myocardial tissue, interspersed with cardiac fibroblasts and a network of endothelial cells, and complete with external epicardial tissue, features that are highly recapitulative of the developing human heart”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    hHOs have significant enrichment in structural and cellular complexity, bringing them in line with fetal hearts and further away from monolayer-based differentiation.

    “These data suggest a significant enrichment in the structural and cellular complexity of our hHOs, thus bringing them in line with fetal hearts and further away from monolayer-based differentiation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    FHF and SHF are two cell populations found in the developing heart.

    “The first and second heart fields (FHF, SHF) are two cell populations found in the developing heart.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [36] Brade, T., Pane, L. S., Moretti, A., Chien, K. R. & Laugwitz, K. L. Embryonic heart progenitors and cardiogenesis. Cold Spring Harb. Perspect. Med. 3, 1–18 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    hHO expression profiles are similar to those of fetal hearts, and their global transcriptomes are closer to those of fetal hearts than monolayer protocols.

    “Taken together, these data suggest hHO expression profiles are similar to those of fetal hearts, and their global transcriptomes are closer to those of fetal hearts than monolayer protocols, as determined by hierarchical clustering”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The organoid platform can recreate complex metabolic disorders associated with congenital heart defects, as demonstrated by an in vitro model of pregestational diabetes-induced congenital heart defects.

    “We also show that our organoid platform can recreate complex metabolic disorders associated with congenital heart defects, as demonstrated by an in vitro model of pregestational diabetes-induced congenital heart defects.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The resulting hHOs contained significant myocardial tissue with epicardial tissue clustered near the outer surface of the organoids, mimicking the anatomical structure of the developing embryonic heart.

    “Overall, the resulting hHOs contained significant myocardial tissue with epicardial tissue clustered near the outer surface of the organoids, mimicking the anatomical structure of the developing embryonic heart”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    The hHO differentiation protocol was reproducible across multiple hPSC lines (iPSC-L1, AICS-37-TNNI1-mEGFP, iPSCORE_16_3, H9), all of which exhibited similar differentiation efficiencies, beat metrics, growth rates, and sizes.

    “Our hHO differentiation protocol was reproducible across multiple hPSC lines (iPSC-L1, AICS-37-TNNI1-mEGFP, iPSCORE_16_3, H9), all of which exhibited similar differentiation efficiencies, beat metrics, growth rates, and sizes”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    BMP4/ActA treatment improves morphological and structural organization in developing hHOs, including increased chamber interconnectivity and vascularization.

    “These results suggest improved structural organization in the developing organoids under BMP4/ActA, in agreement with previously reported differentiation methods using BMP4 and Activin A alone16.”
    • Also stated in: § Discussion · plain
      “The addition of these growth factors to the initial CHIR exposure in our hHO differentiation protocol led to improved morphological features, such as increased chamber interconnectivity and vascularization.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Andersen, P. et al. Precardiac organoids form two heart fields via Bmp/Wnt signaling. Nat. Commun. 9, 1–13 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    hHOs were successfully derived from four independent iPSC lines and one ESC line, demonstrating reproducibility.

    “Notably, hHOs were successfully derived from four independent iPSC lines and one ESC line, demonstrating reproducibility.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    PGDHOs show dysfunctional lipid metabolism and a more glycolytic profile.

    “suggesting dysfunctional lipid metabolism and a more glycolytic profile (Fig. 7f, Supplementary Fig. 8e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In human hearts, the left ventricle ultimately forms from FHF progenitors, and the atria form from SHF progenitors.

    “In human hearts, the left ventricle ultimately forms from FHF progenitors, and the atria form from SHF progenitors38.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [38] Kelly, R. G., Buckingham, M. E. & Moorman, A. F. Heart fields and cardiac morphogenesis. Cold Spring Harb. Perspect. Med. 4, 1–11 (2014). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Cells from the FHF contribute to linear heart tube formation, followed by migrating SHF cells that contribute to further expansion and chamber formation.

    “Cells from the FHF contribute to the linear heart tube formation, followed by migrating cells belonging to the SHF that contribute to further expansion and chamber formation36.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [36] Brade, T., Pane, L. S., Moretti, A., Chien, K. R. & Laugwitz, K. L. Embryonic heart progenitors and cardiogenesis. Cold Spring Harb. Perspect. Med. 3, 1–18 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    hHOs contain most cardiac lineages and have multicell type and morphological complexity reminiscent of the developing human fetal heart, including chamber formation, atrioventricular specification, electrophysiological activity, and vascularization.

    “These conditions lead to the formation of most cardiac lineages in a self-assembling heart organoid with similar properties to the fetal heart.”
    • Also stated in: § Discussion · plain
      “Heart organoids present multicell type and morphological complexity reminiscent of the developing human fetal heart, including chamber formation, atrioventricular specification, electrophysiological activity and vascularization.”
    • Also stated in: § Discussion · hedged
      “These data provide a strong indication of the increased complexity of our hHO system and its close resemblance to fetal heart tissue.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    HAND1 and HAND2 are members of the Twist family of bHLH transcription factors that play key roles in the developing heart.

    “HAND1 (FHF) and HAND2 (SHF) are members of the Twist family of basic helix-loop-helix (bHLH) transcription factors that play key roles in the developing heart37.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [37] George, R. M. & Firulli, A. B. Hand factors in cardiac development. Anat. Rec. 302, 101–107 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The differentiation of hHOs involves heart field formation, atrioventricular specification and chamber formation, further emphasizing their recapitulation of human cardiac development.

    “Taken together, these data suggest that the differentiation of our hHOs involves heart field formation, atrioventricular specification and chamber formation, all of which further emphasizes their recapitulation of human cardiac development.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    BMP4 and Activin A are endogenous morphogens that pattern the early embryonic cardiogenic mesoderm and determine heart field specification in vivo.

    “The growth factors bone morphogenetic protein 4 (BMP4) and Activin A have frequently been used as alternatives to small molecule-mediated Wnt signaling manipulation, since they are the endogenous morphogens that pattern the early embryonic cardiogenic mesoderm and determine heart field specification in vivo16,39–41.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [16] Andersen, P. et al. Precardiac organoids form two heart fields via Bmp/Wnt signaling. Nat. Commun. 9, 1–13 (2018). source

      Not checked in the free run.

    • cites [39] Sa, S. & McCloskey, K. E. Activin A and BMP4 signaling for efficient cardiac differentiation of H7 and H9 human embryonic stem cells. J. Stem Cells Regen. Med. 8, 198–202 (2012). source

      Not checked in the free run.

    • cites [40] Kattman, S. J. et al. Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240 (2011). source

      Not checked in the free run.

    • cites [41] Elliott, D. A. et al. NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat. Methods 8, 1037–1040 (2011). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    BMP4 and Activin A induce cardiac mesoderm in the embryo, and established differentiation protocols using them differentiate to various cardiac mesoderm progenitors.

    “These growth factors lead to the induction of cardiac mesoderm in the embryo70 and established differentiation protocols using them show effective differentiation to various cardiac mesoderm progenitors23,70,71.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [70] Später, D., Hansson, E. M., Zangi, L. & Chien, K. R. How to make a cardiomyocyte. Dev 141, 4418–4431 (2014). source

      Not checked in the free run.

    • cites [23] Breckwoldt, K. et al. Differentiation of cardiomyocytes and generation of human engineered heart tissue. Nat. Protoc. 12, 1177–1197 (2017). source

      Not checked in the free run.

    • cites [71] Kim, M. et al. Activin-A and Bmp4 levels modulate cell type specification during CHIR-induced cardiomyogenesis. PLoS ONE. 10, 1–16 (2015). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Only samples with over 90% viability were used in the Seahorse assay.

    “Only samples with over 90% viability were used in the assay.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Study design and conductnone given

    All PSC lines used were validated for pluripotency and genomic stability.

    “All PSC lines were validated for pluripotency and genomic stability.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Identical numbers of cells were plated for the Seahorse assay.

    “Identical numbers of cells were plated for the assay.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductnone given

    The paper describes a highly reproducible and high-throughput human heart organoid generation method relying on self-assembly triggered by developmental cues and provides proof-of-concept for modeling a congenital heart disorder.

    “In summary, we describe here a highly reproducible and high-throughput human heart organoid generation method relying on self-assembly triggered by developmental cues and provide proof-of-concept for the modeling of a congenital heart disorder.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Maternal diabetes is one of the most common causes of newborn CHD, with up to 12% of newborns from diabetic mothers having some form of CHD.

    “Maternal diabetes is one of the most common causes of newborn CHD (up to 12% of newborns from diabetic mothers have some form of CHD88).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [88] Narchi, H. & Kulaylat, N. Heart disease in infants of diabetic mothers. Images Paediatr. Cardiol. 2, 17–23 (2000).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    hHOs tend to deviate from their normal developmental pathway as a function of time, becoming less relevant the longer they are cultured.

    “First, organoids tend to deviate from their normal developmental pathway as a function of time, becoming less relevant the longer they are cultured.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The axial resolution of the SD-OCT system was measured to be ~7 mm in tissue.

    “The axial resolution of the SD-OCT system was measured to be ~7 mm in tissue.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    All data represent a minimum of 3 independent experiments with at least 3 technical replicates unless otherwise stated.

    “All data are presented as mean ± s.d. and represent a minimum of 3 independent experiments with at least 3 technical replicates unless otherwise stated.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Calcium transient graphs are representative of 6 independent experiments.

    “All micrograph images are representative of at least 6 independent experiments per condition/marker and calcium transient graphs are representative of 6 independent experiments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    A 5X objective lens was used to achieve a transverse image resolution of ~7 mm.

    “A 5X objective lens (5X Plan Apo NIR, Mitutoyo) was used to achieve a transverse image resolution of ~7 mm”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All micrograph images are representative of at least 6 independent experiments per condition/marker.

    “All micrograph images are representative of at least 6 independent experiments per condition/marker and calcium transient graphs are representative of 6 independent experiments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The sensitivity of the OCT system was measured as ~104 dB when operating at a 20 kHz A-scan rate.

    “The sensitivity of the OCT system was measured as ~104 dB when operating at a 20 kHz A-scan rate.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    BMP4 and Activin A have been used successfully for cardiomyocyte differentiation.

    “successful cardiomyocyte differentiation has been achieved in the past using morphogens such as BMP4 and Activin A21,26.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [21] Lian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat. Protoc. 8, 162–175 (2013). source

      Not checked in the free run.

    • cites [26] Lian, X. et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc. Natl. Acad. Sci. USA 109, 1–10 (2012). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Epicardial-myocardial interactions are important in mammalian heart development and function; epicardial cells increase cardiomyocyte growth in 3D engineered heart tissues, and co-transplantation of both cell types into rat hearts increases endothelial cell production.

    “These epicardial-myocardial interactions are important in mammalian heart development and function, as epicardial cells increase cardiomyocyte growth in 3D engineered heart tissues, and co-transplantation of both cell types into rat hearts increases endothelial cell production63.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [63] Bargehr, J. et al. Epicardial cells derived from human embryonic stem cells augment cardiomyocyte-driven heart regeneration. Nat. Biotechnol. 37, 895–906 (2019). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Gradient exposures to specific concentrations of BMP4 and Activin A have been studied in specification of first and second heart field formation.

    “Recently, gradient exposures to specific concentrations of BMP4 and Activin A have been studied in the specification of first and second heart field formation16.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Andersen, P. et al. Precardiac organoids form two heart fields via Bmp/Wnt signaling. Nat. Commun. 9, 1–13 (2018). source

      Not checked in the free run.

61 of 61

  • The paper relies on this without support.

    Current cell models are still far from the structural and cellular complexity of the tissues and organs they intend to represent.

    “However, current cell models are still far away from the structural and cellular complexity of the tissues and organs they intend to represent (e.g., lack of 3D matrix, disorganized cells, and absence of multicell-type interactions).”

  • The paper relies on this without support.

    The developmental stage of hHOs at day 19 is age-matched to human fetal hearts at gestational days 57–67.

    “Heart organoids are comparable to age-matched human fetal cardiac tissues”
    “human fetal heart tissue (gestational age days 57–67)33”

  • The paper relies on this without support.

    Pregestational diabetes is associated with a substantially increased risk of congenital heart disease in the first trimester, and the underlying mechanisms are poorly understood in humans.

    “Diabetes affects a large sector of the female population in reproductive age and comes associated with significant epidemiological evidence linking it to CHD during the first trimester of pregnancy (up to 12-fold risk increase, 12% vs. 1% risk for healthy females), but little understanding of the underlying mechanisms exists, especially in humans.”

  • The paper relies on this without support.

    All data presented a normal distribution.

    “All data presented a normal distribution.”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • HAND1/HAND2 cryosections are dated day 7 and day 8

    Minor

    The results describe the cryosections as day 7, while the caption for the cited panel identifies them as day 8.

  • Diabetes-results text cites the epicardial panel for atrial and ventricular cells

    Minor

    The text cites Fig. 7g for MYL2-positive ventricular and MYL7-positive atrial regions, but the caption says panel g shows TNNT2 and WT1 cardiac and epicardial staining. The caption assigns MYL2 and MYL7 to panel h.

  • Diabetes-results text cites the atrial/ventricular panel for epicardial tissue

    Minor

    The text cites Fig. 7h for myocardial and epicardial organization, but the caption says panel h shows ventricular and atrial staining; it assigns cardiac and epicardial staining to panel g.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Scott F. & Gilbert, M. J. F. B. Lateral Plate Mesoderm: Heart and Circulatory System. In Developmental Biology 591–610 (Sinauer Associates is an imprint of Oxford University Press, 2019).
  2. Eilers, P. H., & Boelens, H. F. Baseline correction with asymmetric least squares smoothing. Leiden University Medical Centre Report. 1, 5 (2005).

Cited by

Sorted by citations

Related

Authors
Lewis-Israeli, Y. R., Wasserman, A. H., Gabalski, M. A., Volmert, B. D., Ming, Y., Ball, K. A., Yang, W., Zou, J., Ni, G., Pajares, N., Chatzistavrou, X., Li, W., Zhou, C., & Aguirre, A.
Affiliations
  • Michigan State University (US)
  • Washington University in St. Louis (US)
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2021-08-26
Volume
12
Issue
1
Pages
5142-5142
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-021-25329-5
PMC
PMC8390749
OpenAlex
W3113822795
Semantic Scholar
91a81864d4ccfc2cf918084c7bfec9df49ea139c
Cited by
536
References
95
Citations by year
2026: 128 · 2025: 126 · 2024: 106 · 2023: 86 · 2022: 81 · 2021: 8 · 2020: 1
FWCI
21.94
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
  • Health Research Alliance (cc-by)
Funders
  • American Heart Association (19IPLOI34660342)
  • U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (K01HL135464, R01EB025209)
  • National Science Foundation (ECCS-2024270)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Congenital heart defects research
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
Keywords
self-assembly, heart development, congenital heart disease, human pluripotent stem cells, cardiac vasculature, pregestational diabetes, in vitro disease model
MeSH terms
Cell Differentiation, Female, Heart, Heart Defects, Congenital, Humans, Male, Organoids, Pluripotent Stem Cells, Wnt Signaling Pathway

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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