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Conditional immortalization of human atrial myocytes for the generation of in vitro models of atrial fibrillation

Nature Biomedical Engineering volume 6, pages 389–402 (2022)Cite this article

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Abstract

The lack of a scalable and robust source of well-differentiated human atrial myocytes constrains the development of in vitro models of atrial fibrillation (AF). Here we show that fully functional atrial myocytes can be generated and expanded one-quadrillion-fold via a conditional cell-immortalization method relying on lentiviral vectors and the doxycycline-controlled expression of a recombinant viral oncogene in human foetal atrial myocytes, and that the immortalized cells can be used to generate in vitro models of AF. The method generated 15 monoclonal cell lines with molecular, cellular and electrophysiological properties resembling those of primary atrial myocytes. Multicellular in vitro models of AF generated using the immortalized atrial myocytes displayed fibrillatory activity (with activation frequencies of 6–8 Hz, consistent with the clinical manifestation of AF), which could be terminated by the administration of clinically approved antiarrhythmic drugs. The conditional cell-immortalization method could be used to generate functional cell lines from other human parenchymal cells, for the development of in vitro models of human disease.

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Fig. 1: Generation and selection of hiAM monoclones.
Fig. 2: Characterization of the hiAM phenotype during proliferation and after 12 days of differentiation.
Fig. 3: Characterization of differentiated hiAM and hESC-AM transcriptomes.
Fig. 4: Electrophysiological characteristics of differentiated hiAMs.
Fig. 5: Robustness and effect of cryopreservation on hiAM differentiation capacity.
Fig. 6: hESC-AM-based and hiAM-based atrial arrhythmia models.
Fig. 7: Effects of antiarrhythmic drugs in the hiAM-based atrial arrhythmia model.

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Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. Certain raw and analysed datasets generated during the study are too large to be publicly shared, but they are available for research purposes from the corresponding author on reasonable request. The RNA-sequencing data are available at the NCBI’s Gene Expression Omnibus (GEO) under GEO accession numbers GSE156824 and GSE178473. The whole-genome-sequencing data are available under BioProject accession number PRJNA760786.

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Acknowledgements

We thank J. Liu (LUMC, Leiden, the Netherlands) for constructing plasmid pLV.iMHCK7.SV40-LT-WT and producing LV.iMHCK7.SV40-LT-WT particles, T. v. Herwaarden (LUMC, Leiden, the Netherlands) for collecting human foetal atrial tissue, B. Klein (LUMC, Leiden, the Netherlands) for donating the SV40 LT-encoding plasmid pAT153.SV40ori, S. Hauschka (University of Washington, Seattle, WA) for providing the construct +aMHCKChCAT encoding the MHCK7 promoter, D. Trono (Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland) for making available the LV shuttle plasmid pLVET-tTR-KRAB, C. Jost (LUMC, Leiden, the Netherlands) for assistance with interpretation and annotation of transmission electron microscopy data and U. Ravens (University of Freiburg, Freiburg, Germany) for useful discussions.This publication received financial support from the LUMC Executive Board (PhD fellowship to N.H.) and from the Leiden Regenerative Medicine Platform Holding (LRMPH project 8212/41235 to A.A.F.d.V.). Additional support was provided by the research programme ‘More Knowledge with Fewer Animals’ (MKMD, project 114022503, to A.A.F.d.V.), which is (partly) financed by the Netherlands Organisation for Health Research and Development (ZonMw) and by the Dutch Society for the Replacement of Animal Testing (dsRAT).

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Author notes

  1. These authors contributed equally: Daniël A. Pijnappels, Antoine A.F. de Vries.

Authors and Affiliations

  1. Department of Cardiology, Leiden University Medical Center, Leiden, the Netherlands

    Niels Harlaar, Sven O. Dekker, Juan Zhang, Lente J. S. Lerink, Martin J. Schalij, Daniël A. Pijnappels & Antoine A. F. de Vries

  2. Department of Cardiothoracic Surgery, Leiden University Medical Center, Leiden, the Netherlands

    Niels Harlaar, Robert J. M. Klautz & Thomas J. van Brakel

  3. Department of Molecular Developmental Biology, Radboud University, Nijmegen, the Netherlands

    Rebecca R. Snabel & Gert Jan C. Veenstra

  4. Department of Clinical and Experimental Cardiology, Amsterdam UMC, Amsterdam, the Netherlands

    Marieke W. Veldkamp, Arie O. Verkerk & Mathilde R. Rivaud

  5. Department of Medical Biology, Amsterdam UMC, Amsterdam, the Netherlands

    Arie O. Verkerk

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

    Carla Cofiño Fabres, Verena Schwach & Robert Passier

  7. Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands

    Aat A. Mulder & Marie José T. H. Goumans

  8. Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands

    Willem E. Corver

  9. Institute of Pharmacology, University Duisburg-Essen, Essen, Germany

    Dobromir Dobrev

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  1. Niels Harlaar
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Contributions

N.H., D.A.P. and A.A.F.d.V. conceived the study, interpreted results and wrote the manuscript. N.H. generated and characterized the hiAM lines with the assistance of S.O.D, J.Z. and L.J.S.L. M.W.V., A.O.V. and M.R.R. performed and analysed patch-clamping experiments. V.S., C.C.F. and R.P. generated hESC-AM layers and assisted with the associated analyses. R.R.S. and G.J.C.V. performed the comparative transcriptome analyses of the different cell types and tissues. A.A.M. generated and interpreted transmission electron microscopy data. W.E.C. performed the flow cytometric ploidy analysis. M.J.T.H.G. provided human foetal atrial material. D.D. assisted in the design and interpretation of the arrhythmia studies. T.J.v.B., R.J.M.K. and M.J.S. provided clinical input to the study. All authors refined the manuscript.

Corresponding author

Correspondence to Antoine A. F. de Vries.

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Competing interests

M.J.S., D.A.P. and A.A.F.d.V. are inventors of a patent application (US16/480,280, ‘Conditionally immortalized cells and methods for their preparation’) related to this work. R.P. is a cofounder of Pluriomics (Ncardia) and River Biomedics. D.D. is a member of the scientific advisory boards of OMEICOS Therapeutics and Acesion Pharma. All other authors declare no competing interests.

Additional information

Peer review information Nature Biomedical Engineering thanks Lior Gepstein and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Supplementary information

Main Supplementary Information

Supplementary methods, results, discussion, references, figures, tables and video captions.

Reporting Summary

Video 1

Contractions of hfAMs and hiAMs.

Video 2

Optical voltage mapping of hESC-AM and hiAM layers.

Video 3

Induction of reentrant activity in hESC-AM and hiAM layers.

Video 4

hiAM arrhythmic activity with varying degrees of complexity.

Video 5

Effects of antiarrhythmic drugs on reentrant activity in hiAM layers.

Data 1

Differentially expressed genes: hiAM D0 vs D12.

Data 2

Differentially expressed genes: hiAM vs hESC-AM.

Data 3

Individual (raw) data points and P values.

Data 4

Uncropped western blots.

Peer Review File

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Harlaar, N., Dekker, S.O., Zhang, J. et al. Conditional immortalization of human atrial myocytes for the generation of in vitro models of atrial fibrillation. Nat. Biomed. Eng 6, 389–402 (2022). https://doi.org/10.1038/s41551-021-00827-5

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