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

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.

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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.

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).

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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.

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Authors

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 or Valeria V. Orlova or Christine L. Mummery.

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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.

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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

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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

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