HEK? No!
Authors: Karoly Toth, William S.M. Wold
Journal: Molecular Therapy (2002)

For the last 25 years, HEK 293 (or, in short, 293) cells have been a staple both for virologists studying human adenoviruses (Ads) and for anyone who needed an easily transfectable cell line. With the advent of viral gene therapy, 293 cells were used to produce the E1A-deleted Ad vectors, and they became one of the few cell lines approved by the FDA for virus production for clinical trials. The 293 cell line was created by Frank Graham over 25 years ago by transfecting human embryonic kidney (hence the name HEK 293) cells with sheared Ad5 DNA. The cell line was later confirmed to have incorporated the Ad5 E1A and E1B genes into chromosome 19. After a quarter of a century of extensive use, a paper published in The FASEB Journal by Shaw et al. questions the "K" of the cell line's original name [1Shaw, G, Morse, S, Ararat, M, and, Graham, F. L. Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells, FASEB J. (in press).Google Scholar]. This question stems from a chance discovery. In one experiment using 293 cells, the researchers used a monoclonal antibody specific to the major subunit of the human neurofilament NF-M, a marker for cells of neural origin. NF-M is not supposed to be expressed in a cell line derived from kidney cells (at least primary embryonic kidney cells do not express it). To their surprise, the authors found that 293 cells expressed copious amounts of NF-M. And, if this were not surprising enough, the immunofluorescent staining showed characteristic filamentous localization. The authors confirmed their results by using antibodies specific to other components of neurofilament and observed that, with some variation in intensity, all components stain positively. Strangely, 293 cells also stain very strongly with antibodies specific to the epithelial markers keratin 8 and keratin 18. The researchers extended their search to other kidney-derived cell lines that were created using cloned Ad5 DNA or SV40, and found that three out of three Ad5-transformed cell lines, but not SV40-transformed lines, share this neurofilament-positive phenotype. Using microarray techniques, the authors found that expression of a group of neuron-specific mRNAs was upregulated. All these findings should not be as surprising, looking at past publications that describe expression of neural markers (for example, endogenous corticotrophin releasing factor type I receptor, endogenous β2-adrenergic receptor, muscarinic acetylcholine receptor, and voltage activated ion channels) by 293 cells. These findings were discounted because it seemed improbable that a cell line derived from human primary kidney culture would have a neuronal origin. However, it is known that at early developmental stages, cells expressing early neuronal markers can be found in the kidney. It is also known that Ad5 transforms human embryonic retinal (HER) cells much more readily than it does HEK cells. According to Shaw et al., these Ad5-transformed HER cells also express neuronal markers. The authors speculate that the approximately 100-fold difference in the success of obtaining transformed cell lines from HEK and HER cultures is due to the higher proportion of cells with neuronal origin in HER cultures. They cautiously provide an alternative hypothesis to the neuronal origin of 293 cells: the cell line may have just a generally unstable transcriptional profile. However, as pointed out by the authors, other, nonneuronal filaments are not expressed by 293 cells. We can draw two conclusions from this paper. First, with some luck and with the use of new powerful techniques such as microarray analysis, one can make discoveries that question even such fundamental beliefs as the origin of a cell line. Second, attention should be paid to facts pointing to a certain direction, even if this direction seems improbable. There is a third, more practical consideration: 293 can no longer be used as a kidney cell line.