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Generation of heart-forming organoids from human pluripotent stem cells

Nature Protocols volume 16, pages 5652–5672 (2021)Cite this article

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Abstract

Heart-forming organoids (HFOs) derived from human pluripotent stem cells (hPSCs) are a complex, highly structured in vitro model of early heart, foregut and vasculature development. The model represents a potent tool for various applications, including teratogenicity studies, gene function analysis and drug discovery. Here, we provide a detailed protocol describing how to form HFOs within 14 d. In an initial 4 d preculture period, hPSC aggregates are individually formed in a 96-well format and then Matrigel-embedded. Subsequently, the chemical WNT pathway modulators CHIR99021 and IWP2 are applied, inducing directed differentiation. This highly robust protocol can be used on many different hPSC lines and be combined with manipulation technologies such as gene targeting and drug testing. HFO formation can be assessed by numerous complementary methods, ranging from various imaging approaches to gene expression studies. Here, we highlight the flow cytometry-based analysis of individual HFOs, enabling the quantitative monitoring of lineage formation.

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Fig. 1: Scheme of an HFO.
Fig. 2: Schematic illustration of the HFO differentiation from hPSCs.
Fig. 3: Morphology of hPSCs on MEFs and in monolayer culture.
Fig. 4: Preculture of HFOs.
Fig. 5: Differentiation of HFOs.
Fig. 6: Monitoring and collection of d10 HFOs.
Fig. 7: HFO differentiation efficiency and tissue characterization.
Fig. 8: Gating strategy for a flow cytometry analysis of an HFO.

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Data availability

The flow cytometry data are available in the FlowRepository under accession code FR-FCM-Z4C4. All remaining data generated or analyzed during this study are included in this published article and its supplementary files.

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Acknowledgements

We thank S. C. Den Hartogh and R. Passier (Department of Anatomy and Embryology, Leiden University Medical Centre; current affiliation: Faculty of Science and Technology, University of Twente) for providing the HES3 NKX2.5-eGFP cell line, C. Guibentif and N.-B. Woods (Lund Stem Cell Center, Lund University) for the iPSC-CB1RB9_WAS-GFP cell line, A. Haase and U. Martin (LEBAO, MHH) for the hHSC_Iso4_ADCF_SeV-iPS2 cell line, and E. G. Stanley and A. G. Elefanty (Monash Immunology and Stem Cell Laboratories, Monash University) for the HES3 MIXL1-GFP cell line. R.Z. received funding from: German Research Foundation (DFG; grants Cluster of Excellence REBIRTH EXC 62/2, ZW64/4-1, ZW64/4-2, KFO311/ZW64/7-1), German Ministry for Education and Science (BMBF, grants 13N14086, 01EK1601A, 01EK1602A, 13XP5092B, 031L0249), ‘Förderung aus Mitteln des Niedersächsischen Vorab’ (grant ZN3340), ‘Cortiss Stiftung’, and the European Union H2020 project TECHNOBEAT (grant 66724).

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Author notes

  1. These authors contributed equally: Lika Drakhlis, Santoshi Biswanath Devadas.

Authors and Affiliations

  1. Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), REBIRTH—Research Center for Translational Regenerative Medicine, Hannover Medical School (MHH), Hannover, Germany

    Lika Drakhlis, Santoshi Biswanath Devadas & Robert Zweigerdt

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  1. Lika Drakhlis
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  2. Santoshi Biswanath Devadas
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Contributions

L.D., S.B.D. and R.Z. designed the experiments. L.D. and S.B.D. performed the experiments and analyzed the data. R.Z. supervised the project. L.D., S.B.D. and R.Z. wrote the manuscript.

Corresponding author

Correspondence to Robert Zweigerdt.

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The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Chulan Kwon, Zhen Ma and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key reference using this protocol

Drakhlis, L. et al. Nat. Biotechnol. 39, 737–746 (2021): https://doi.org/10.1038/s41587-021-00815-9

Supplementary information

Supplementary Video 1

Movement of a T25 flask inside an incubator to ensure equal distribution of cells after seeding.

Supplementary Video 2

Embedding of an hPSC aggregate in a Matrigel droplet (d-2).

Supplementary Video 3

Removing the culture medium with 1000 µL and 200 µL tips.

Supplementary Video 4

Removing the culture medium with stacked tips (10 µL tip put on a 200 µL tip using a 20 – 200 µL pipet).

Supplementary Video 5

Transfer of a d10 HFO from the 96-U-well plate to a 12-well suspension plate using a 100 – 1000 µL pipet.

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Drakhlis, L., Devadas, S.B. & Zweigerdt, R. Generation of heart-forming organoids from human pluripotent stem cells. Nat Protoc 16, 5652–5672 (2021). https://doi.org/10.1038/s41596-021-00629-8

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