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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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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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.
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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.
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Supplementary information
Main Supplementary Information
Supplementary methods, results, discussion, references, figures, tables and video captions.
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.
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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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DOI: https://doi.org/10.1038/s41551-021-00827-5