after pre - stimulation, 2 × 10 cells per well were transduced with the lv - gfp or a3x3g ( dk ) lentiviral vector at an moi of 20 in the presence of 5 μg / ml polybrene for 24 h. after lentiviral vector transduction, hspcs were washed to remove vector, cultured for 6 days, and then analyzed by flow cytometry for the proportion of gfp or eyfp cells, as previously described. the authors declare no competing interests. the need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. the human cell line hek293 has bio - synthetic potential for human - like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used hek293 cell lines. changes in gene copy and expression between industrial progeny cell lines and the original hek293 were associated with cellular component organization, cell motility and cell adhesion. changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes ( rarg, id1, zic1, lox and dhrs3 ), a pattern validated in 63 human adherent or suspension cell lines of other origin. the production of protein therapeutics is a fast - growing field as it allows for the generation of sophisticated molecules with high specificity and activity in humans. even though the chinese hamster ovary ( cho ) cell line is a successfully used mammalian platform for the production of advanced recombinant proteins with the need for proper protein folding and post translational modifications, there is an increasing demand for improved and more efficient bioproduction platforms. with an increasing number of difficult - to - express proteins entering clinical development, including bispecific antibod ies and antibod y – drug conjugates, alternative or engineered expression hosts are being explored. extensive omics profiling of cho cells has been carried out during recent years, which has paved the way for cell line engineering efforts aiming to improve bioproduction efficiency and product quality. moreover, human production cell lines, such as hek293, have served as convenient expression hosts for proteins with specific requirement for human post - translational modifications. the human cell line hek293 is the most commonly utilized human cell line for expression of recombinant proteins for a multitude of research applications. this cell line originate from the kidney of an aborted human female embryo and was originally immortalized in 1973 by the integration of a 4 kbp adenoviral 5 ( ad5 ) genome fragment including the e1a and e1b genes, at chromosome 19. the expression of e1a and e1b enable continuous culturing of hek293 cells by inhibiting apoptosis and interfering with transcription and cell cycle control pathways. in addition, e1a and e1b are essential helper factors for adeno associated virus ( aav ) production, which makes hek293 cells attractive production hosts for recombinant aav particles. hek293 cell lines have been reported to have a pseudo tripl oid genome with the adenoviral dna inserted on chromosome 19. the organization of the hek293 genome is continuously evolving through the events of chromosomal translocations and copy number alterations, suggesting that long - term cultivation and subcloning of cells result in karyotypic drift. such abnormalities and genomic instability is, however, characteristic for immortalized cells and have also been reported for cho cells. several hek293 cell lineages have been established from the parental hek293 lineage with the objective to improve recombinant protein production and are used for the production of therapeutic proteins. two examples are 293t and 293e cell lines, constitutively expressing the temperature sensitive allele of the large t antigen of simian virus 40, or the epstein - barr virus nuclear antigen ebna1, respectively. in addition, several hek293 cell lines have been adapted to high - density suspension growth in serum - free medium, enabling large - scale cultivation and bioproduction in bioreactors. two industrially relevant suspension cell lines are 293 - f and 293 - h ( gibco, thermo fisher scientific ), which both enable fast growth and high transfectivity in serum - free medium. in addition, the 293 - h cell line, which was originally derived from a more adherent hek293 cell clone, shows strong adherence during plaque assays. despite extensive usage of cho and hek in both suspension and adherent mode and several empirical protocols for adaptation in either direction, molecular knowledge of the key genes involved in the transition between the two growth states are limited. while adherent cells have traditionally been widely used for the production of viruses, e. g. aav and lenti virus for clinical research, suspension growth is the platform of choice for bioproduction of therapeutic proteins. whereas certain experimental steps are more efficient in adherent mode, e. g. chemical transfection and viral infection, the ability to increase the volumetric cell density by growth in suspension without cell clump formation, which results in oxygen limitations, is a key step from a manufacturing perspective. even though different hek293 strains have all been derived from the same original cell line, significant genomic and transcriptomic changes between parental and progenitor cell lines can be expected due to the genomic instability of hek293 as discussed above. here, we present a genomic and transcriptomic analysis of the hek293 parental cell line along with five widely used hek293 derivatives. an overall analysis of the differences in genomic landscape and transcriptomic profiles was performed in order to provide novel molecular insights into the differences between cell lines that have occurred during the process of clonal isolation and expansion. furthermore, we focus on transcriptomic differences between adherent and suspension hek293 cells and the impact of the differentially expressed genes on metabolic pathways and the phenotype of the cells from a bioprocess perspective. in this study, six industrially relevant hek293 cell lines ( fig. 1a ) were subjected to omics profiling. this set of cell lines includes the parental hek293 as well as five additional cell lines that have all been clonally derived from parental hek293 cells. the cell lines can be divided into either adherent ( hek293, 293e and 293t ) or suspension ( 293 - h, 293 - f and freestyle 293 - f ) cells. the genomes and the transcriptomes of these six cell lines were sequenced using illumina hiseq. supplementary table s1 provides full results of transcript levels ( tpm ) for all cell lines. comparisons of the genomes and transcription profiles between the cell lines show overall similar results ( fig. 1b, c ). hierarchical clustering divided the progeny cell lines into two different taxonomic groups, of either adherent ( 293t, 293e ) or suspension cell lines ( 293 - h, 293 - f and freestyle 293 - f ), diverged from the parental hek293. interestingly, the original hek293 cell line was the most distant from all other cell lines. as expected, the two 293 - f lineages ( 293 - f and freestyle 293 - f ) showed very similar profiles. the same pattern of gene expression clustering was visualized by principal component analysis ( fig. 1d ), where the suspension cell - lines grouped together in the plot, with a very close clustering of 293 - f and freestyle 293 - f cells. on the other hand, the adherent cell - lines 293e and 293t showed larger variations in gene expression patterns between cell lines.