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Reproducibility Issues – The Ghosts of HeLa

Listeria monocytogenes is an intracellular pathogen and the causative agent of the food-borne disease listeriosis. This disease primarily affects pregnant women, newborns, older adults, and people with an impaired or weakened immune system. L. monocytogenes is able to not only invade macrophages but also non-phagocytic cells. In such cases, adhesion and internalization are mediated mainly through a group of proteins called internalins.
In 2014, Radosław Stachowiak and colleagues at the University of Warsaw set out to find a general mechanism to explain how invasion of human cells would work in different tissues – an important aspect for finding new and better treatment options. The scientists compared how the bacterium invaded three different cell lines: INT 407 cells (as a model of the pathogen’s intestinal target), HEp-2 cells (squamous cell laryngeal cancer), and HeLa cells. They found that a specific internalin protein is involved in listerial invasion in all three different cell lines tested and concluded that a general mechanism has been developed by the pathogen to enter different cell types and tissues.

But it turned out that INT 407 and HEp-2 cells were impostors!

In reality, the study tested a single cervical cancer cell line, HeLa, three times. Therefore, the Biological Unit of observation was not three but one and the statement made in the original publication was incorrect.
A year after the original study was published, the authors took the right steps and issued a correction of the paper: “The discussion section contains false conclusion drawn on the basis that the three cell lines used in the study are ostensibly different. Since it was tested only on HeLa cells, it is currently not clear whether the observed mechanism of invasion is restricted to HeLa cells or not. To assess the specificity and the more detailed role of this internalin-like protein future studies are needed.”

Cell line contamination is not a new problem but researchers have mostly ignored or denied the issue. In the 1970s, biologist Walter Nelson-Rees was determined to expose impostor cell lines and pushed for regular authentication. However, for his efforts Nelson-Rees was confronted with so much vilification and dispraise that he left science altogether in 1981. He then specialized in buying, selling and cataloguing historical paintings by early California artists and became a well-known contributor to art endeavors in the Bay Area and California.

In vitro cell culture systems are crucial research tools for analyzing complex mechanisms regulating cell biology. However, cell lines can be misidentified, meaning that they no longer correspond to the donor from whom the cells were first obtained. This problem may arise due to cross-contamination: the accidental introduction of cells from another culture. The contaminant, which is often a rapidly dividing cell line, will overgrow and replace the original culture. The end result is a misidentified cell line, also known as a false or imposter cell line. Today, over 480 misidentified cell lines routinely used in published studies were identified as contaminated, according to the International Cell Line Authentication Committee(ICLAC).
This list also includes the two above mentioned HEp-2 and Intestine 407, which are false cell lines widely used in the scientific literature. They were shown to be cross-contaminated in 1967, and have no known authentic stock. A geneticist from the University of Colorado, Christopher Korch, has studied this issue at length. He has identified nearly 5,800 articles in 1,182 journals that may have confused HeLa cells for HEp-2, and another 1,336 articles in 271 journals that may have mixed up HeLa with INT 407 cells. Together, more than 7,000 papers that have been cited some 214,000 times were likely using the wrong cells for their research.

This analysis clearly shows the need for action on false cell lines.
The first line of defense is Good Research Practice which ensures rigorous sterile and strict cell line management. Original cell lines must be derived from a reputed cell bank and “gift” lines from other colleagues need to be carefully checked. Cell line authentication can be achieved using e.g. short tandem repeat (STR) profiling, a verification step that should be conducted regularly.
In the past, the technology to check cell lines did not exist and it was very difficult for researchers to control for cross-contamination and authentication. But today, STR profiling is cheap and easy and the technology is widely available. There is no excuse for conducting experiments on cells without first making sure that these cells are indeed what they are supposed to be. To be fair, some journals, such as Nature and Cell (STAR Methods), now require authors to attest that they have identity-checked their cell lines. But, crucially, editors do not insist to see a proof. In any case, this step will only guard against future mix-ups; it does not help to purge the literature of past studies built on misidentified or contaminated cell lines.
One step in the right direction could be to label all papers on PubMed using cell lines that were identified by Korch and/or ICLAC to make sure that scientists pay extra attention to this issue when planning to build their own research projects on the results published in these articles.

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