High variability of genomic instability and gene expression profiling in different HeLa clones

Frattini, Annalisa; Fabbri, Marco; Valli, Roberto; Paoli, Elena De; Montalbano, Giuseppe; Gribaldo, Laura; Pasquali, Francesco; Maserati, Emanuela

doi:10.1038/srep15377

Cited by 79 publications

(58 citation statements)

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“…HeLa alone were used in more than 70,000 articles and 11,000 patents. However, tumor cells differ dramatically in their genetic repertoire and thus physiology: In 2015, Frattini and co-workers 15 reported the tremendous genetic differences in HeLa cells from different laboratories. The four samples showed gain and loss of genomic material varying widely between laboratories, drastically affecting basal gene expression.…”

Section: Shortcomings Of Traditional Cell Culturesmentioning

confidence: 99%

21st Century Cell Culture for 21st Century Toxicology

Pamies

Hartung

2016

Chem. Res. Toxicol.

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There is no good science in bad models. Cell culture is especially prone to artifacts. A number of novel cell culture technologies have become more broadly available in the 21st century, which allow overcoming limitations of traditional culture and are more physiologically relevant. These include the use of stem-cell derived human cells, cocultures of different cell types, scaffolds and extracellular matrices, perfusion platforms (such as microfluidics), 3D culture, organ-on-chip technologies, tissue architecture, and organ functionality. The physiological relevance of such models is further enhanced by the measurement of biomarkers (e.g., key events of pathways), organ specific functionality, and more comprehensive assessment cell responses by high-content methods. These approaches are still rarely combined to create microphysiological systems. The complexity of the combination of these technologies can generate results closer to the in vivo situation but increases the number of parameters to control, bringing some new challenges. In fact, we do not argue that all cell culture needs to be that sophisticated. The efforts taken are determined by the purpose of our experiments and tests. If only a very specific molecular target to cell response is of interest, a very simple model, which reflects this, might be much more suited to allow standardization and high-throughput. However, the less defined the end point of interest and cellular response are, the better we should approximate organ- or tissue-like culture conditions to make physiological responses more probable. Besides these technologic advances, important progress in the quality assurance and reporting on cell cultures as well as the validation of cellular test systems brings the utility of cell cultures to a new level. The advancement and broader implementation of Good Cell Culture Practice (GCCP) is key here. In toxicology, this is a major prerequisite for meaningful and reliable results, ultimately supporting risk assessment and product development decisions.

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Section: Shortcomings Of Traditional Cell Culturesmentioning

confidence: 99%

21st Century Cell Culture for 21st Century Toxicology

Pamies

Hartung

2016

Chem. Res. Toxicol.

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“…To date it has not been thoroughly explored to what extent 59 cultured cell lines remain genetically stable over time and to what extent potential genotypic 60 variability induces proteotype and phenotype variations in a given cell line with the "same name" 61 across laboratories. This question deserves critical consideration because anecdotal, non-systematic 62 observations suggest that many cultured cell lines might be genomically unstable 17,18 . Therefore, 63 even careful experimentation cannot fully alleviate the problem.…”

Section: Introductionmentioning

confidence: 99%

“…Second, HeLa cells have been reported for decades to 74 contaminate and dominate other cell lines 23,24 and conversely, they are less likely to be 75 contaminated themselves, minimizing the possibility of cell misidentification for HeLa-focused 76 studies 15,16 . Third, due to extensive genome instability during passaging and between labs HeLa 77 cells were reported to contain an exceptional number of genomic variants 18,[25][26][27][28] . For example, the 78 currently widely used HeLa variants include HeLa CCL2, the "original" HeLa cell line; HeLa S3 79 (also called CCL2.2), the third clone isolated from an early HeLa culture; and HeLa Kyoto, the 80 popular Kyoto version.…”

Section: Introductionmentioning

confidence: 99%

“…were reported between the two 30 . Furthermore, different HeLa Kyoto clones between labs were 83 reported to vary in terms of mRNA expression 18 . However, little is known about the manner by 84 which such genomic variability affects the proteomes or phenotypes of the different HeLa strains 85 between individual labs, the effect of successive passages within a stock-derived line on its 86 molecular make up, and how these would affect a biological research outcome 31,32 .…”

Section: Introductionmentioning

confidence: 99%

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Genomic, Proteomic and Phenotypic Heterogeneity in HeLa Cells across Laboratories: Implications for Reproducibility of Research Results

Liu

Mueller

et al. 2018

Preprint

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1The independent reproduction of research results is a cornerstone of experimental research, yet it is 2 beset by numerous challenges, including the quality and veracity of reagents and materials. Much of 3 life science research depends on life materials, including human tissue culture cells. In this study we 4 aimed at determining the degree of variability in the molecular makeup and the ensuing phenotypic 5 consequences in commonly used human tissue culture cells. We collected 14 stock HeLa aliquots 6 from 13 different laboratories across the globe, cultured them in uniform conditions and profiled the 7 genome-wide copy numbers, mRNAs, proteins and protein turnover rates via genomic techniques 8 and SWATH mass spectrometry, respectively. We also phenotyped each cell line with respect to the 9 ability of transfected Let7 mimics to modulate Salmonella infection. 10 11We discovered significant heterogeneity between HeLa variants, especially between lines of the 12 CCL2 and Kyoto variety. We also observed progressive divergence within a specific cell line over 13 50 successive passages. From the aggregate multi-omic datasets we quantified the response of the 14 cells to genomic variability across the transcriptome and proteome. We discovered organelle-specific 15 proteome remodeling and buffering of protein abundance by protein complex stoichiometry, 16 mediated by the adaptation of protein turnover rates. By associating quantitative proteotype and 17 phenotype measurements we identified protein patterns that explained the varying response of the 18 different cell lines to Salmonella infection. 19 20Altogether the results indicate a striking degree of genomic variability, the rapid evolution of 21 genomic variability in culture and its complex translation into distinctive expressed molecular and 22 phenotypic patterns. The results have broad implications for the interpretation and reproducibility of 23 research results obtained from HeLa cells and provide important basis for a general discussion of the 24 value and requirements for communicating research results obtained from human tissue culture cells. 25 7 molecular analyses, we centrally cultured all 14 cell lines for an additional three passages in our 126 group. To minimize experimental variability, the same researcher cultured the cells using the same 127 culture media and protocol. 128 129As a starting point for the evaluation of HeLa cell heterogeneity, we measured gene copy number 130 variation (CNV) by array-CGH (aCGH). Previous studies reported genome instability within and 131 between different HeLa strains 18, 25-28 . To investigate and display the ploidy variation of the strains 132 studied we projected the CNV data onto the reference human genomic map (Figure 1b). We 133 discovered pervasive CNV differences organized by domains, large chromosomal segments, and 134 even whole chromosomes. Particularly notable were ploidy changes at Chromosomes (Chr) 1, 2, 6, 135 9, 10, 17, 19, 21, 22, and X. (Figure 1b). On average, the HeLa genome of all cells tested...

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“…Another limitation in using cell lines is their genetic variability. There is a longstanding and general awareness that genetic variation exists in cell lines [Frattini et al 2015;Kleensang et al 2016]; however, a recent study elucidated the astonishing magnitude of cell line genetic drift and variance [Ben-David et al 2018]. In this study, the authors conducted a comprehensive genomic characterization in a large number of cell lines and reported extensive genetic variation within the same cell lines across passage numbers and laboratories, even for single-cell derived subclones.…”

Section: Introductionmentioning

confidence: 99%

Genetic instability of in vitro cell lines: Implications for genetic toxicity testing

Settivari

LeBaron

2019

Environ and Mol Mutagen

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Cell line‐based in vitro testing has been widely used as an important component of the genotoxicity testing battery; however, the use of cell lines is constrained by several limitations, including the genetic drift and variability. A study recently reported in the literature comprehensively examined genomic changes in a large number of cell lines and reported extensive genetic variations within the same cell lines across passage numbers and laboratories, even for single‐cell derived subclones. The primary objective of this communication is to raise awareness and stimulate discussion within the genotoxicity testing community of the extent of genetic variability of cell lines in general and how these variables could potentially influence the results and reproducibility of genotoxicity testing. Meanwhile, some recommendations for good cell culture practices are highlighted to mitigate, at least to some extent, the concern about genetic variation. Environ. Mol. Mutagen. 60:559–562, 2019. © 2019 Wiley Periodicals, Inc.

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High variability of genomic instability and gene expression profiling in different HeLa clones

Cited by 79 publications

References 50 publications

21st Century Cell Culture for 21st Century Toxicology

21st Century Cell Culture for 21st Century Toxicology

Genomic, Proteomic and Phenotypic Heterogeneity in HeLa Cells across Laboratories: Implications for Reproducibility of Research Results

Genetic instability of in vitro cell lines: Implications for genetic toxicity testing

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