Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Generation, functional analysis and applications of isogenic three-dimensional self-aggregating cardiac microtissues from human pluripotent stem cells

Nature Protocols volume 16, pages 2213–2256 (2021)Cite this article

Subjects

Abstract

Tissue-like structures from human pluripotent stem cells containing multiple cell types are transforming our ability to model and understand human development and disease. Here we describe a protocol to generate cardiomyocytes (CMs), cardiac fibroblasts (CFs) and cardiac endothelial cells (ECs), the three principal cell types in the heart, from human induced pluripotent stem cells (hiPSCs) and combine them in three-dimensional (3D) cardiac microtissues (MTs). We include details of how to differentiate, isolate, cryopreserve and thaw the component cells and how to construct and analyze the MTs. The protocol supports hiPSC-CM maturation and allows replacement of one or more of the three heart cell types in the MTs with isogenic variants bearing disease mutations. Differentiation of each cell type takes ~30 d, while MT formation and maturation requires another 20 d. No specialist equipment is needed and the method is inexpensive, requiring just 5,000 cells per MT.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Outline of in vitro differentiation protocol from hiPSCs to ECs, CFs and CMs.
Fig. 2: Outline of in vitro 3D cardiac MT formation and analysis.
Fig. 3: Morphology of undifferentiated hiPSCs.
Fig. 4: hiPSC-EC characterization.
Fig. 5: hiPSC-EPI and hiPSC-CF characterization.
Fig. 6: hiPSC-CM characterization.
Fig. 7: Examples of optimal and sub-optimal cardiac MTs a24h after formation.
Fig. 8: Overview of correct Seahorse plating conditions.
Fig. 9: Characterization of 3D MTs after 21 d in culture.

Similar content being viewed by others

Data availability

The datasets generated and/or analyzed during the current study are available in Giacomelli et al. 202011 and from the corresponding authors upon request.

Code availability

MUSCLEMOTION is freely accessible on the web at https://gitlab.com/bjvanmeer/MUSCLEMOTION or https://github.com/l-sala/MUSCLEMOTION/. The license is a GNU GENERAL PUBLIC LICENSE version 3 (GPL v3), which can be found at https://github.com/l-sala/MUSCLEMOTION/blob/master/LICENSE. After downloading the macro, users can access the code through a text editor or directly through FIJI. When using MUSCLEMOTION, users are encouraged to read Sala, L. et al.61 (https://www.ahajournals.org/doi/suppl/10.1161/CIRCRESAHA.117.312067).

References

  1. Broutier, L. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat. Protoc. 11, 1724–1743 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. Koike, H. et al. Modelling human hepato-biliary-pancreatic organogenesis from the foregut-midgut boundary. Nature 574, 112–116 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).

    Article  CAS  PubMed  Google Scholar 

  4. Tirziu, D., Giordano, F. J. & Simons, M. Cell communications in the heart. Circulation 122, 928–937 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Bergmann, O. et al. Dynamics of cell generation and turnover in the human heart. Cell 161, 1566–1575 (2015).

    Article  CAS  PubMed  Google Scholar 

  6. Zhou, P. & Pu, W. T. Recounting cardiac cellular composition. Circ. Res. 118, 368–370 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pinto, A. R. et al. Revisiting cardiac cellular composition. Circ. Res. 118, 400–409 (2016).

    Article  CAS  PubMed  Google Scholar 

  8. Banerjee, I., Fuseler, J. W., Price, R. L., Borg, T. K. & Baudino, T. A. Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse. Am. J. Physiol. Heart Circ. Physiol. 293, H1883–H1891 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Furtado, M. B., Nim, H. T., Boyd, S. E. & Rosenthal, N. A. View from the heart: cardiac fibroblasts in development, scarring and regeneration. Development 143, 387–3971 (2016).

    Article  CAS  PubMed  Google Scholar 

  10. Brutsaert, D. L. Cardiac endothelial-myocardial signaling: its role in cardiac growth, contractile performance, and rhythmicity. Physiol. Rev. 83, 59–1151 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Giacomelli, E. et al. Human-iPSC-derived cardiac stromal cells enhance maturation in 3D cardiac microtissues and reveal non-cardiomyocyte contributions to heart disease. Cell Stem Cell 26, 862–879 e811 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Garry, D. J. & Olson, E. N. A common progenitor at the heart of development. Cell 127, 1101–1104 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Moretti, A. et al. Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification. Cell 127, 1151–1165 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. van Wijk, B. & van den Hoff, M. Epicardium and myocardium originate from a common cardiogenic precursor pool. Trends Cardiovasc. Med. 20, 1–7 (2010).

    Article  PubMed  Google Scholar 

  15. Giacomelli, E. et al. Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells. Development 144, 1008–1017 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Iwamiya, T., Matsuura, K., Masuda, S., Shimizu, T. & Okano, T. Cardiac fibroblast-derived VCAM-1 enhances cardiomyocyte proliferation for fabrication of bioengineered cardiac tissue. Regen. Ther. 4, 92–102 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Guadix, J. A. et al. Human pluripotent stem cell differentiation into functional epicardial progenitor cells. Stem Cell Reports 9, 1754–1764 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tallquist, M. D. & Molkentin, J. D. Redefining the identity of cardiac fibroblasts. Nat. Rev. Cardiol 14, 484–491 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Zhao, J. et al. Efficient differentiation of TBX18(+)/WT1(+) epicardial-like cells from human pluripotent stem cells using small molecular compounds. Stem Cells Dev. 26, 528–540 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bich, L., Pradeu, T. & Moreau, J. F. Understanding multicellularity: the functional organization of the intercellular space. Front. Physiol. 10, 1170 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Wan, A. C. A. Recapitulating cell-cell interactions for organoid construction—are biomaterials dispensable? Trends Biotechnol. 34, 711–721 (2016).

    Article  CAS  PubMed  Google Scholar 

  22. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Nunes, S. S. et al. Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes. Nat. Methods 10, 781–787 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gao, L. et al. Myocardial tissue engineering with cells derived from human-induced pluripotent stem cells and a native-like, high-resolution, 3-dimensionally printed scaffold. Circ. Res. 120, 1318–1325 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ong, C. S. et al. Biomaterial-free three-dimensional bioprinting of cardiac tissue using human induced pluripotent stem cell derived cardiomyocytes. Sci. Rep. 7, 4566 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Goldfracht, I. et al. Engineered heart tissue models from hiPSC-derived cardiomyocytes and cardiac ECM for disease modeling and drug testing applications. Acta Biomater. 92, 145–159 (2019).

    Article  CAS  PubMed  Google Scholar 

  27. Wang, G. et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat. Med. 20, 616–623 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ma, Z. et al. Contractile deficits in engineered cardiac microtissues as a result of MYBPC3 deficiency and mechanical overload. Nat. Biomed. Eng. 2, 955–967 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wanjare, M. et al. Vascularization of engineered spatially patterned myocardial tissue derived from human pluripotent stem cells in vivo. Front. Bioeng. Biotechnol. 7, 208 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Zhao, Y. et al. A platform for generation of chamber-specific cardiac tissues and disease modeling. Cell 176, 913–927 e918 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Tulloch, N. L. et al. Growth of engineered human myocardium with mechanical loading and vascular coculture. Circ. Res. 109, 47–59 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Schaaf, S. et al. Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. PLoS ONE 6, e26397 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Burridge, P. W. et al. Multi-cellular interactions sustain long-term contractility of human pluripotent stem cell-derived cardiomyocytes. Am. J. Transl. Res. 6, 724–735 (2014).

    PubMed  PubMed Central  Google Scholar 

  34. Hinson, J. T. et al. Heart disease. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy. Science 349, 982–986 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Mannhardt, I. et al. Human engineered heart tissue: analysis of contractile force. Stem Cell Reports 7, 29–42 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Keung, W. et al. Non-cell autonomous cues for enhanced functionality of human embryonic stem cell-derived cardiomyocytes via maturation of sarcolemmal and mitochondrial KATP channels. Sci. Rep. 6, 34154 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Tiburcy, M. et al. Defined engineered human myocardium with advanced maturation for applications in heart failure modeling and repair. Circulation 135, 1832–1847 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Lee, E. K. et al. Machine learning of human pluripotent stem cell-derived engineered cardiac tissue contractility for automated drug classification. Stem Cell Reports 9, 1560–1572 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Shadrin, I. Y. et al. Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues. Nat. Commun. 8, 1825 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Ronaldson-Bouchard, K. et al. Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature 556, 239–243 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Tsuruyama, S., Matsuura, K., Sakaguchi, K. & Shimizu, T. Pulsatile tubular cardiac tissues fabricated by wrapping human iPS cells-derived cardiomyocyte sheets. Regen. Ther. 11, 297–305 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Mills, R. J. et al. Drug screening in human PSC-cardiac organoids identifies pro-proliferative compounds acting via the mevalonate pathway. Cell Stem Cell 24, 895–907 e896 (2019).

    Article  CAS  PubMed  Google Scholar 

  43. Goldfracht, I. et al. Generating ring-shaped engineered heart tissues from ventricular and atrial human pluripotent stem cell-derived cardiomyocytes. Nat. Commun. 11, 75 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Jang, Y. et al. Modulating cardiomyocyte and fibroblast interaction using layer-by-layer deposition facilitates synchronisation of cardiac macro tissues. Soft Matter 16, 428–434 (2020).

    Article  CAS  PubMed  Google Scholar 

  45. Dostanic, M. et al. A miniaturized EHT platform for accurate measurements of tissue contractile properties. J. of Microelectromech. Syst. 29, 881–887 (2020).

    Article  CAS  Google Scholar 

  46. Masumoto, H. et al. The myocardial regenerative potential of three-dimensional engineered cardiac tissues composed of multiple human iPS cell-derived cardiovascular cell lineages. Sci. Rep. 6, 29933 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Huebsch, N. et al. Miniaturized iPS-cell-derived cardiac muscles for physiologically relevant drug response analyses. Sci. Rep. 6, 24726 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Zimmermann, W. H. et al. Tissue engineering of a differentiated cardiac muscle construct. Circ. Res. 90, 223–230 (2002).

    Article  CAS  PubMed  Google Scholar 

  49. Ravenscroft, S. M., Pointon, A., Williams, A. W., Cross, M. J. & Sidaway, J. E. Cardiac non-myocyte cells show enhanced pharmacological function suggestive of contractile maturity in stem cell derived cardiomyocyte microtissues. Toxicol. Sci. 152, 99–112 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Pointon, A. et al. From the cover: high-throughput imaging of cardiac microtissues for the assessment of cardiac contraction during drug discovery. Toxicol. Sci. 155, 444–457 (2017).

    Article  CAS  PubMed  Google Scholar 

  51. Forsythe, S. D. et al. Environmental toxin screening using human-derived 3D Bioengineered liver and cardiac organoids. Front. Public Health 6, 103 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Stevens, K. R., Pabon, L., Muskheli, V. & Murry, C. E. Scaffold-free human cardiac tissue patch created from embryonic stem cells. Tissue Eng. Part A 15, 1211–1222 (2009).

    Article  CAS  PubMed  Google Scholar 

  53. Polonchuk, L. et al. Cardiac spheroids as promising in vitro models to study the human heart microenvironment. Sci. Rep. 7, 7005 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Sebastiao, M. J. et al. Bioreactor-based 3D human myocardial ischemia/reperfusion in vitro model: a novel tool to unveil key paracrine factors upon acute myocardial infarction. Transl. Res. 215, 57–74 (2020).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Qiao, X. et al. Uncoupling DNA damage from chromatin damage to detoxify doxorubicin. Proc. Natl Acad. Sci. USA 117, 15182–15192 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Veerman, C. C. et al. Immaturity of human stem-cell-derived cardiomyocytes in culture: fatal flaw or soluble problem? Stem Cells Dev. 24, 1035–1052 (2015).

    Article  CAS  PubMed  Google Scholar 

  58. Gaudesius, G., Miragoli, M., Thomas, S. P. & Rohr, S. Coupling of cardiac electrical activity over extended distances by fibroblasts of cardiac origin. Circ. Res. 93, 421–428 (2003).

    Article  CAS  PubMed  Google Scholar 

  59. De Simone, S. A. et al. The role of membrane capacitance in cardiac impulse conduction: an optogenetic study with non-excitable cells coupled to cardiomyocytes. Front. Physiol. 11, 194 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Quaife-Ryan, G. A. et al. Multicellular transcriptional analysis of mammalian heart regeneration. Circulation 136, 1123–1139 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Sala, L. et al. MUSCLEMOTION: a versatile open software tool to quantify cardiomyocyte and cardiac muscle contraction in vitro and in vivo. Circ. Res. 122, e5–e16 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Mastikhina, O. et al. Human cardiac fibrosis-on-a-chip model recapitulates disease hallmarks and can serve as a platform for drug testing. Biomaterials 233, 119741 (2020).

    Article  PubMed  Google Scholar 

  63. van den Berg, C. W., Elliott, D. A., Braam, S. R., Mummery, C. L. & Davis, R. P. Differentiation of human pluripotent stem cells to cardiomyocytes under defined conditions. Methods Mol. Biol. 1353, 163–180 (2016).

    Article  PubMed  Google Scholar 

  64. Sala, L., Ward-van Oostwaard, D., Tertoolen, L. G. J., Mummery, C. L. & Bellin, M. Electrophysiological analysis of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) using multi-electrode arrays (MEAs). J. Vis. Exp. https://doi.org/10.3791/55587 (2017).

  65. Giacomelli, E., Bellin, M., Orlova, V. V. & Mummery, C. L. Co-differentiation of human pluripotent stem cells-derived cardiomyocytes and endothelial cells from cardiac mesoderm provides a three-dimensional model of cardiac microtissue. Curr. Protoc. Hum. Genet. 95, 21.9.1–21.9.22 (2017).

    Google Scholar 

  66. van den Brink, L. et al. Cryopreservation of human pluripotent stem cell-derived cardiomyocytes is not detrimental to their molecular and functional properties. Stem Cell Res. 43, 101698 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  67. Burridge, P. W. et al. Chemically defined generation of human cardiomyocytes. Nat. Methods 11, 855–860 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Lian, X. et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions. Nat. Protoc. 8, 162–175 (2013).

    Article  CAS  PubMed  Google Scholar 

  69. Tohyama, S. et al. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 12, 127–137 (2013).

    Article  CAS  PubMed  Google Scholar 

  70. Moretti, A. et al. Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N. Engl. J. Med. 363, 1397–1409 (2010).

    Article  CAS  PubMed  Google Scholar 

  71. Zhang, M. et al. Recessive cardiac phenotypes in induced pluripotent stem cell models of Jervell and Lange-Nielsen syndrome: disease mechanisms and pharmacological rescue. Proc. Natl Acad. Sci. USA 111, E5383–E5392 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Meraviglia, V. et al. Generation of human induced pluripotent stem cell line LUMCi027-A and its isogenic gene-corrected line from a patient affected by arrhythmogenic cardiomyopathy and carrying the c.2013delC PKP2 mutation. Stem Cell Res. 46, 101835 (2020).

    Article  CAS  PubMed  Google Scholar 

  73. Assou, S., Bouckenheimer, J. & De Vos, J. Concise review: assessing the genome integrity of human induced pluripotent stem cells: what quality control metrics? Stem Cells 36, 814–821 (2018).

    Article  CAS  PubMed  Google Scholar 

  74. Stacey, G. N. et al. Stem cell culture conditions and stability: a joint workshop of the PluriMes Consortium and Pluripotent Stem Cell Platform. Regen. Med. 14, 243–255 (2019).

    Article  CAS  PubMed  Google Scholar 

  75. Miller, L. R. et al. Considering sex as a biological variable in preclinical research. FASEB J. 31, 29–34 (2017).

    Article  CAS  PubMed  Google Scholar 

  76. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  77. Pioner, J. M. et al. Isolation and mechanical measurements of myofibrils from human induced pluripotent stem cell-derived cardiomyocytes. Stem Cell Reports 6, 885–896 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Ferrantini, C. et al. Impact of detubulation on force and kinetics of cardiac muscle contraction. J. Gen. Physiol. 143, 783–797 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. van Meer, B. J. et al. Quantification of muscle contraction in vitro and in vivo using MUSCLEMOTION software: from stem cell-derived cardiomyocytes to zebrafish and human hearts. Curr. Protoc. Hum. Genet. 99, e67 (2018).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. Ward-van Oostwaard, M. P. H. Mol, L. G. J. Tertoolen, A. Krotenberg Garcia and A. Cochrane for technical assistance. This project was supported by European Research Council (ERCAdG 323182 STEMCARDIOVASC; ERCStG 638030 STEMCARDIORISK); European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 602423; European Union’s Horizon 2020 research and innovation Programme (H2020 Technobeat) under grant agreement No. 668724; The Netherlands Organ-on-Chip Initiative is an NWO Gravitation project (024.003.001) funded by the Ministry of Education, Culture and Science of the government of the Netherlands; Transnational Research Project on Cardiovascular Diseases (JTC2016_FP-40-021 ACM-HF); The Netherlands Organisation for Health Research and Development (ZonMW) (MKMD project No. 114022504; VIDI project No. 91715303; TAS project No. 446002501); Health~Holland TKI-LSH PPP-allowance (LSHM17013-H007); Individual Fellowship under the Marie Sklodowska Curie Grant Agreement No. 838985.

Author information

Author notes

  1. Elisa Giacomelli

    Present address: The Center for Stem Cell Biology, Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY, USA

  2. These authors contributed equally: Giulia Campostrini, Viviana Meraviglia.

Authors and Affiliations

  1. Department of Anatomy and Embryology, Leiden University Medical Centre, Leiden, the Netherlands

    Giulia Campostrini, Viviana Meraviglia, Elisa Giacomelli, Ruben W. J. van Helden, Loukia Yiangou, Richard P. Davis, Milena Bellin, Valeria V. Orlova & Christine L. Mummery

  2. Department of Biology, University of Padua, Padua, Italy

    Milena Bellin

  3. Veneto Institute of Molecular Medicine, Padua, Italy

    Milena Bellin

  4. Department of Applied Stem Cell Technologies, University of Twente, Enschede, the Netherlands

    Christine L. Mummery

Authors
  1. Giulia Campostrini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Viviana Meraviglia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Elisa Giacomelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ruben W. J. van Helden
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Loukia Yiangou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Richard P. Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Milena Bellin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Valeria V. Orlova
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Christine L. Mummery
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.C. and V.M. designed and performed the experiments, analyzed the data and wrote the manuscript. E.G developed and optimized the cardiac MT protocol, acquired and analyzed the data and wrote the manuscript. R.v.H. and L.Y acquired and analyzed the data and wrote the manuscript. R.P.D. contributed to drafting the manuscript and revising it for important intellectual content. M.B., V.O. and C.L.M. conceived, designed and supervised the protocol, acquired the funding and wrote and approved the final manuscript.

Corresponding authors

Correspondence to Milena Bellin, Valeria V. Orlova or Christine L. Mummery.

Ethics declarations

Competing interests

C.L.M. is co-founder of Pluriomics (now Ncardia) bv.

Additional information

Peer review information Nature Protocols thanks Sara Nunes de Vasconcelos, Wolfram H Zimmermann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Giacomelli, E. et al. Cell Stem Cell 26, 862–879 e811 (2020): https://doi.org/10.1016/j.stem.2020.05.004

Giacomelli E. et al. Curr. Protoc. Hum. Genet. 95, 21.9.1–21.9.22 (2017): https://doi.org/10.1002/cphg.46

Giacomelli, E. et al. Development 144, 1008–1017 (2017): https://doi.org/10.1242/dev.143438

Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting Summary

Supplementary Table 1

Raw data for results summarized in Table 3. Table contains morphological, contraction and single-cell electrophysiological parameters measured in LUMC0099iCTRL04 and LUMC0020iCTRL06 hiPSC lines.

Supplementary Video 1

Beating monolayer hiPSC-CMs after 21 d of differentiation using LI-BPEL protocol with lactate purification

Supplementary Video 2

Beating monolayer hiPSC-CMs after 21 d of differentiation using mBPEL protocol

Supplementary Video 3

Time-lapse movie of the first 72 h of MT formation (time frame: 15 min)

Supplementary Video 4

Movie of representative MT showing CD31 positive ECs organized in a vessel-like structure (in red) and CMs stained with ACTN2 (in green) (15 f.p.s.)

Supplementary Video 5

Contracting MT paced at 1 Hz at after 21 d in culture

Supplementary Video 6

Contracting MT loaded with Fluo-4, paced at 1.5 Hz after 21 d in culture

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Campostrini, G., Meraviglia, V., Giacomelli, E. et al. Generation, functional analysis and applications of isogenic three-dimensional self-aggregating cardiac microtissues from human pluripotent stem cells. Nat Protoc 16, 2213–2256 (2021). https://doi.org/10.1038/s41596-021-00497-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-021-00497-2

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research