Reproducibility in research can be compromised by both biological and technical variation, but most of the focus is on removing the latter. Here we investigate the effects of biological variation in HeLa cell lines using a systems-wide approach. We determine the degree of molecular and phenotypic variability across 14 stock HeLa samples from 13 international laboratories. We cultured cells in uniform conditions and profiled genome-wide copy numbers, mRNAs, proteins and protein turnover rates in each cell line. We discovered substantial heterogeneity between HeLa variants, especially between lines of the CCL2 and Kyoto varieties, and observed progressive divergence within a specific cell line over 50 successive passages. Genomic variability has a complex, nonlinear effect on transcriptome, proteome and protein turnover profiles, and proteotype patterns explain the varying phenotypic response of different cell lines to Salmonella infection. These findings have implications for the interpretation and reproducibility of research results obtained from human cultured cells.
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We thank G. Rosenberger, A. Beyer, B. Collins and S. Nikolaev for discussions. We thank L. Reiter, R. Bruderer and O. Rinner from Biognosys AG for sharing their thoughts about cell line proteome analysis from a commercial perspective. We thank H. Zhang and J. Chen from Johns Hopkins University, D. Pflieger and O. Filhol-Cochet from CEA Grenoble, M. Riwanto from University Hospital Zurich, U. Greber and M. Suomalainen from the University of Zurich, C. Arrieumerlou from the University of Basel (through InfectX), M. Beck and M.-T. Mackmull from the European Molecular Biology Laboratory, C. Jorgensen and J. Worboys from the Cancer Research UK Manchester Institute, M. Peter and C. Barnes from ETH Zurich, and A. Venkitaraman and C. Williams from the University of Cambridge for providing us their HeLa cells.
The work was supported by the SystemsX.ch project PhosphoNetX PPM (to R.A.), TargetInfectX (to C.D.), the Swiss National Science Foundation (grant 3100A0-688 107679 to R.A.), the European Research Council (ERC-20140AdG 670821 to R.A.), the JRC for Computational Biomedicine (which was partially funded by Bayer AG, to J.S.-R.), the Swiss National Science Foundation (grant 163180 to S.E.A.), the European Research Council (grants AdG 249968 to S.E.A. and 616441-DISEASEAVATARS to G.T.), the Umberto Veronesi Foundation (fellowship to P.-L.G.), the ERA-NET Neuron Program (P.-L.G.), Regione Lombardia (Ricerca Indipendente 2012 to G.T.) and the Italian Ministry of Health (Ricerca Corrente to G.T.) E.G.W. was supported by an NIH F32 Ruth Kirchstein Fellowship (F32GM119190).
R.A. holds shares of Biognosys AG, which operates in the field covered by the article.
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Liu, Y., Mi, Y., Mueller, T. et al. Multi-omic measurements of heterogeneity in HeLa cells across laboratories. Nat Biotechnol 37, 314–322 (2019). https://doi.org/10.1038/s41587-019-0037-y
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