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OPEN Evolution from adherent

to suspension: systems biology
of HEK293 cell line development
Magdalena Malm1,8, Rasool Saghaleyni2,8, Magnus Lundqvist1, Marco Giudici1,
Veronique Chotteau1, Ray Field3,4, Paul G. Varley3,5, Diane Hatton3, Luigi Grassi3,
Thomas Svensson2,6, Jens Nielsen2,7* & Johan Rockberg1*
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 h ­ umans1–4. 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 antibodies and antibody–drug conjugates, alternative or engineered
expression hosts are being explored. Extensive omics profiling of CHO cells has been carried out during recent
­years5–12, which has paved the way for cell line engineering efforts aiming to improve bioproduction efficiency
and product q ­ uality13–15. Moreover, human production cell lines, such as HEK293, have served as convenient
expression hosts for proteins with specific requirement for human post-translational ­modifications16,17.
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 ­1918,19. The expression of E1A and E1B enable
continuous culturing of HEK293 cells by inhibiting apoptosis and interfering with transcription and cell cycle
control ­pathways20. In addition, E1A and E1B are essential helper factors for adeno associated virus (AAV) pro-
duction, which makes HEK293 cells attractive production hosts for recombinant AAV ­particles21. HEK293 cell
lines have been reported to have a pseudotriploid genome with the adenoviral DNA inserted on chromosome
­1919,22,23. 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 d ­ rift22,24. Such abnormalities and genomic instability is, however, characteristic for immortalized
cells and have also been reported for CHO ­cells25–28.

1
KTH ‑ School of Engineering Sciences in Chemistry, Biotechnology, and Health, Dept. of Protein Science, Royal
Institute of Technology, 106 91 Stockholm, Sweden. 2Department of Biology and Biological Engineering, Chalmers
University of Technology, 412 96  Gothenburg, Sweden. 3Biopharmaceutical Development, BioPharmaceuticals
R&D, AstraZeneca, Milstein Building, Granta Park, Cambridge  CB21 6GH, UK. 4GammaDelta Therapeutics
Ltd, White City Place, London  W12 7FQ, UK. 5Kymab, Babraham Research Campus, Cambridge  CB22 3AT,
UK. 6NBIS ‑ Bioinformatics Systems Biology Support, Chalmers University of Technology, 412 96  Gothenburg,
Sweden. 7Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens
Lyngby, Denmark. 8These authors contibuted equally: Magdalena Malm and Rasool Saghaleyni. *email: nielsenj@
chalmers.se; [email protected]

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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 ­proteins16,17. Two
examples are 2­ 93T29 and ­293E30,31 cell lines, constitutively expressing the temperature sensitive allele of the large
T antigen of Simian virus 4­ 029, or the Epstein-Barr virus nuclear antigen EBNA1, ­respectively30,31. In addition,
several HEK293 cell lines have been adapted to high-density suspension growth in serum-free ­medium32–34,
enabling large-scale cultivation and bioproduction in ­bioreactors24. Two industrially relevant suspension cell
lines are 293-F and 293-H (Gibco, Thermo Fisher Scientific), which both enable fast growth and high transfectiv-
ity 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 adher-
ent 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 dif-
ferences 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.

Results
Genomic and transcriptomic profiling indicate clonal divergence between parental HEK293
and its progeny.  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. The parental cell line HEK293 showed a notable difference in transcriptome profile
compared to all the other cell lines along the first principal component (PC1). These results indicate a genomic
divergence of the clonal lineages compared to the parental HEK293 and suggest the presence of similar tran-
scriptomic traits between HEK293 progeny cell lines individually selected for during the isolation of each clone.
Hierarchical clustering of the cell lines based on SNVs gave a slightly different trend compared to the transcrip-
tomic comparison. A different pattern of overall clustering was observed, with the original HEK293 and 293E
cell lines separated from the rest on a separate branch and the three suspension cell lines grouped on a second
branch together with 293T. However, 293-F and Freestyle 293-F were, as expected, the most similar cell lines also
in this comparison (Supplementary Fig. S1). The overall number of genomic variations was similar between the
cell lines and variations located to similar genomic regions (Supplementary Fig. S1). Moreover, the ratio between
missense and synonomous SNVs in all cell lines ranged between 0.866 and 0.879, where the original HEK293
strain had the lowest ratio (Supplementary Fig. S1). Pairwise comparisons of SNVs and indels between HEK293
and each progeny cell line showed that the highest number of high (variants expected to have a disruptive impact
on the protein, for example protein truncation or loss of start/stop codon) and moderate (non-disruptive vari-
ant that might change protein effectiveness, for example a missense variant) SNVs and indels were seen for the
two adherent cell lines 293E and 293T (Supplementary Fig. S1). Genes with high impact SNVs in progeny cell
lines compared to the parental HEK293 can be found in Supplementary Table S2. Amongst these, five genes
had acquired high impact SNVs (PPP2R4, C9orf43, CTB-47B11.3, CYFIP2 and SGCD) in all progeny cell lines
compared to the parental strain.
The HEK293 cell line was originally immortalized by the random integration of viral genomic DNA of adeno-
virus ­518, which includes the E1A and E1B genes. In this study, overall high mRNA levels of E1A and E1B were
observed in all HEK293 cell lines (Supplementary Fig. S2). A comparison of mRNA levels of the viral element
E1A showed significantly (p < 0.05) higher expression in HEK293 compared to both 293T and Freestyle 293-F.
In addition, both 293E and 293-H had significantly higher expression than 293T, 293-F and Freestyle 293-F.
Further, 293-F had significantly higher expression than Freestyle 293-F. (Supplementary Fig. S1) The analysis of
the viral element E1B showed that 293-F had significantly higher expression (p < 0.05) than 293-H and Freestyle
293-F (Supplementary Fig. S1). As expected, the gene expression of Large T and EBNA-1 was detected in 293T

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Figure 1.  Comparisons of genomic and transcriptomic profiles of HEK293 cells showed taxonomic divergence
between parental HEK293 and progeny cell lines. (a) A schematic overview of the lineage relationship of the six
HEK293 cell lines used in this study. Blue dots represent adherent cells whereas grey dots represent suspension
cell lines. (b) Genomic comparison between HEK293 cell lines based on Spearman correlation coefficients
of read counts. Darker blue color indicates higher correlation. (c) Sample-to-sample comparison between
transcriptomes illustrated by a heatmap and hierarchical clustering of taxonomical divergence between samples.
Darker blue color indicates shorter Euclidean distance between samples and more similarity. (d) PCA plot
showing the separation in expression pattern between samples. (e) RNA expression levels (in DESeq2 median
of ratios) and standard deviations (n = 3) of stably integrated viral genes (EBNA-1, Large T, E1A and E1B) in
HEK293 cell lineages determined by RNA sequencing.

and 293E, respectively (Fig. 1e). Interestingly, expression of the Large T antigen was also observed in 293E, which
is not reported by the supplier (ATCC). The presence of a truncated version of Large T in the 293E genome was

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Figure 2.  Copy number variation analysis of HEK293 progeny cells compared to the parental HEK293
revealed conserved patterns of copy number gain and loss. (a) Genomic copy number gain (red) or loss (blue)
of chromosomes 1, 8, 9, 13 and 18 of progeny HEK293 cell lines compared to parental HEK293 cells. The black
line indicates the centromere position of each chromosome. (b) Genomic copy number gain or loss (log2 fold
change) compared to HEK293 for each cell line of the FH, KMO, TLE4 and ADAM3A genes.

confirmed by de novo assembly of all reads not mapping to the human reference genome (Supplementary Fig. S2).
Tracing the origin of the 293E cell l­ine31, the Large T expression of 293E may be derived from the pRSVneo
plasmid that was used to co-transfect HEK293 cells along with the pCMV-EBNA plasmid for the generation of
the stable EBNA-1 expressing clone (293c18) by geneticin (G418) selection. The pRSVneo plasmid contains a
truncated version of the Large T gene (according to the AddGene vector Database), which aligns perfectly with
the truncated Large T sequence found in the 293E genome (Supplementary Fig. S2).

Progeny cell lines displayed common patterns of copy number gain/loss at several genomic
loci compared to parental HEK293.  In order to evaluate the genomic variation between HEK293 and
its derivatives further, overall genomic copy number variation of all progeny cell lines compared to the parental
HEK293 was performed. A comparison of gained and lost regions on all chromosomes between all cell lines
can be found in Supplementary Fig. S3 and Table S3. Interestingly, a conserved pattern of copy number gain or
loss of large regions has occurred on several chromosomes of all HEK293 progeny cells compared to HEK293,
whereas other changes are more local or cell line specific. For instance, on chromosome 13, a region of > 15 Mb
has been amplified in all cell lines compared to the parental HEK293 strain (Fig. 2a). All elements with copy
number gain of > 1 log2 fold-change common to all progenitor cells are located in this region (Supplementary
Table S3). Amongst these, four out of seven protein-coding genes (BORA, MZT1, PIBF1 and KLHL1) belong to

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the cytoskeleton gene set (GO: 0005856). On chromosome 18, there is a conserved pattern of copy number loss
of most of the chromosome sequence for all progeny cell lines compared to the parental HEK293 strain, with the
exception of a high degree of copy number gain (> 0.8 log2 fold-change) of a region close to the centromere for
all cell lines except 293E (Fig. 2a). Within the region of conserved gain are several genes encoding cell adhesion
molecules within the desmocollin (DSC) and desmoglein (DSG) subfamilies, belonging to the cell–cell adhesion
gene set (GO: 0098609). When analyzing more local copy number variations between progeny cell lines and
the parental strain, some interesting loss or gain of full or partial elements compared to the parental HEK293
were identified. For instance, copy number loss was observed for the fumarate hydratase (FH) gene, which has
previously been reported to have lost several gene copies in HEK293 and hence been hypothesized to play a
role in the phenotypic transformation of ­HEK29322. Interestingly, the fumarate hydratase gene along with the
neighboring kynurenine 3-monooxygenase (KMO) gene, had a log2-fold copy ratio of < -1 in 293E, 293-F and
Freestyle 293-F cell lines compared to the parental HEK293 (Fig. 2b and Supplementary Fig. S4), suggesting that
these cells have half the number of copies compared to the parental cell line. Moreover, the 293T and 293-H cell
lines have a gain of the genomic loci surrounding the FH gene, while maintaining the copy number of the FH
gene compared to HEK293. Interestingly, the resulting FH expression levels of the cell lines only partly reflected
the gene copy number changes (Fig. 2b and Supplementary Fig. S4). Even though the gene copy number of the
parental HEK293 strain is the same as for 293T and 293-H lineages, the FH mRNA levels of HEK293 was as low
as the expression levels of the lineages with only half the number of FH gene copies. Moreover, the expression
levels of KMO was comparably low in all cell lines but did not correlate with gene copy number. Besides the
changes in gene copy number of the FH locus, a locus around the transducin-like enhancer protein 4 (TLE4)
gene, encoding a transcriptional co-repressor of Wnt signaling pathway members was found to have a log2-fold
copy number gain of > 1.5 in all progeny cell lines except for 293E (Fig. 2b). This gain in the TLE4 locus was
accordingly reflected in the transcription level of the gene with a higher level of expression in 293T, 293-H, 293-F
and Freestyle 293-F compared to 293E and HEK293 (Supplementary Fig. S4). In addition, a major loss of copy
number of the ADAM3A pseudogene was observed for all cell lines except 293-H with a maintained low or no
expression of the pseudogene observed in the cell lines (Fig. 2a,b and Supplementary Fig. S4).
Due to the observed pattern of common genomic changes to progeny cell lines compared to the parental
HEK293, an evaluation of common SNPs amongst all progeny cell lines but not HEK293 was performed. GO
enrichment analysis of common genes with high or moderate impact SNPs different in all progeny cell lines
compared to the original HEK293 (Supplementary Table S4), showed significant (adjusted p-value < 0.05) enrich-
ment of homophilic cell adhesion via plasma membrane adhesion molecules (GO:0007156; adjusted p-value
0.025; fold enrichment 10.26; data not shown) and cell–cell adhesion via plasma-membrane adhesion molecules
(GO:0098742; adjusted p-value 0.032; fold enrichment 7.53; data not shown). All genes with moderate or high
impact SNPs in progeny cell lines compared to HEK293 found amongst both these GO-terms were protocad-
herins (PCDH12, PCDHB10, PCDHB13, PCDHB15, PCDHB16 PCDHGA2, PCDHGA3 and PCDHGB2). In
addition, the Teneurin-2 gene (TENM2) (within GO:0098742) had an altered SNP allele in all progeny cell lines
compared to HEK293. These SNPs all result in missense mutations with unknown biological impact on the
gene products. However, the enrichment of common SNPs within this group of genes in all HEK293 progeny
cell lines may suggest an impact on the protein function and a selective advantage of such phenotypic changes
during continuous cell line cultivation.

Consensus differential expression analysis suggested a role of integral membrane proteins

in HEK293 progeny cell line development.  Based on the overall genomic and transcriptomic profiles
of the different HEK293 cell lines, the parental HEK293 strain stood out as different compared to all other cell
lines. In order to evaluate common changes between all progeny cell lines and the parental HEK293, differen-
tial expression analysis was performed. Results showed a significant consensus of down-regulation of genes
involved in extracellular matrix organization, locomotion and cell adhesion in progeny cells compared to the
parental HEK293 strain (Fig. 3a). Moreover, amino acid metabolism and metabolic process of small molecules
were found up-regulated in all progeny cell-lines. Along with changes in extracellular matrix genes, there is also
a consensus amongst progeny cell lines compared to HEK293 of differential expression of genes involved in
other types of cellular component organization such as cell morphogenesis, cytoskeleton-, membrane- and cell
junction organization. A comparison between gene expression fold changes and copy number variation of the
differentially expressed genes (log2-fold change >  ± 1) for each progeny cell line compared to HEK293 showed
a trend of gained gene copies amongst the majority of genes with up-regulated mRNA levels (Supplementary
Fig. S4). However, there was not a clear trend of loss in gene copy number amongst transcriptionally down-
regulated genes for any cell line.
For further evaluation of the transcriptomic similarities and changes between HEK293 cell lines, pairwise
differential expression comparisons between all cell lines were performed. As expected, the parental cell line had
the highest number of differentially expressed genes when compared to all other cell lines (Fig. 3b, Supplementary
Fig. S5 and Supplementary Table S5). In addition, when looking at differentially expressed genes unique to cer-
tain comparisons, the largest group of genes were found common to all pairwise comparisons between HEK293
and each of the progeny cell lines (green bar in Fig. 3b), again emphasizing a relatively high degree of common
transcriptomic changes amongst progeny cell lines differentiated from the parental HEK293. As the progeny
cell lines had an enrichment of differentially expressed genes associated with cellular component organization
compared to HEK293, we sought to evaluate to what cellular compartments the 329 genes common to all pairwise
comparisons between HEK293 and progeny cell lines localize. In line with the overall differential expression
evaluation (Fig. 3a), which emphasized changes in for instance cell adhesion and extracellular matrix organiza-
tion, there was a significant (padj < 0.05) enrichment of genes relating to the integral compartment of plasma

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Figure 3.  Differential expression analysis emphasized processes and genes with common changes in all progeny
cell lines compared to the parental HEK293. (a) Consensus heatmap of GO biological processes with a different
expression pattern between progeny cell lines compared to the parental HEK293. Low consensus scores,
represented by a dark blue color, indicate more significant differences. (b) Common differentially expressed
(DE) genes in pairwise comparisons of all HEK293 cells. Blue bars show number of DE genes in each pairwise
comparison. Green bar shows 329 common DE genes in pairwise comparisons of progeny with HEK293
parental cells. Red bar shows common 38 DE genes in the comparison of suspension cells against adherent cells.
(c) Top ten significant GO cellular components of the 329 common DE genes in pairwise comparisons between
progeny cells and HEK293.

membrane (GO:0005887) amongst the common differentially expressed (DE) genes unique to the comparisons
between HEK293 and all progeny cell lines (Fig. 3c). Moreover, gene set analysis of these 329 genes showed,
although non-significant in this limited set of genes, alterations in processes related to cell surface, cell adhesion
and epithelial to mesenchymal transition (Supplementary Fig. S6).

Differential expression between suspension and adherent HEK293 cell lines identified key
changes related to cholesterol metabolism.  The growth morphology of bioproduction cell lines is
of great importance for culture maintenance and efficiency of industrial bioprocessing. In order to look into
gene expression variations correlating with adherent and suspension HEK 293 cell lines, differential expres-
sion analysis between adherent and suspension HEK293 progeny cell lines was performed. As results from the
overall comparison of transcriptomic profiles of the HEK293 cell lines showed that the parental HEK293 cell
line is highly differentiated from all of the progeny cell lines and moreover, that the Freestyle 293-F cell line is
very similar to the 293-F cell line, HEK293 and Freestyle 293-F were excluded from this analysis, so as not to
skew the data. Enrichment analysis of the differentially expressed genes between adherent (293T and 293E) and
suspension (293-H and 293-F) progeny cell lines showed significant expression differences of similar gene sets as
in the comparison between progeny cell lines and the parental HEK293 (Figs. 3a, 4a, Supplementary Table S6).
For instance, the suspension progeny cell lines had a significant up-regulation of gene sets involved in cellular
compartment organization such as cell morphogenesis, cell junction-, cell membrane- and cytoskeleton organi-
zation. Interestingly, there is no significant change in the expression of the extracellular matrix organization gene
set between suspension and adherent HEK293 progeny cell lines. Perhaps as expected, there was significant dif-
ferential expression observed for the cell adhesion, cell differentiation, cell morphogenesis and cell motility gene
sets. All of the above-mentioned gene sets, including cell adhesion, were up-regulated in suspension HEK293
cells as compared to adherent. When looking at the most significantly differentially expressed genes (adjusted
p-value < 0.01) amongst the cell adhesion gene set, many genes of the cadherin superfamily of cell adhesion

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Figure 4.  Gene set analysis identified biological process and metabolic changes between suspension and
adherent HEK293 progeny cell lineages. (a) Heatmap of gene set analysis using GO biological processes and
Piano consensus scores showed a different expression pattern between suspension cells (293-H and 293-F)
and adherent cells (293E and 293T). Low consensus scores, represented by a dark blue color, indicate more
significant differences. (b) Metabolic genes set analysis for comparing metabolic differences between suspension
and adherent HEK293 progeny cell lines. The size of each node corresponds to the number of genes in each
of these pathways, thickness of connections between nodes corresponds to the number shared genes between
pathways and the colors of the nodes shows the p-value for the given metabolic process.

molecules were found up-regulated in suspension cell lines compared to adherent HEK293 progeny cells (Sup-
plementary Table S7).
In order to evaluate what metabolic impact the differentially expressed genes may have on cells in suspen-
sion compared to the adherent state, a generic human metabolic model, H ­ MR235, was used to generate a set
of metabolic genes and their assigned pathways to find metabolic pathways with altered expression between
adherent and suspension HEK293 progeny cell lines. As shown in Fig. 4b, pathways related to aromatic amino
acids and oxidative phosphorylation were significantly down-regulated in suspension cells compared to adher-
ent and had amongst the highest number of differentially expressed genes. Pathways related to retinol, linoleate
and nucleotide metabolism were also significantly down-regulated in suspension cell-lines. On the other hand,
biosynthesis and metabolism of cholesterol were found to be most significantly up-regulated amongst metabolic
pathways in suspension compared to adherent cells. In addition, pathways related to protein modification and
fatty acid metabolism (omega-3/6 fatty acid metabolism, fatty acid desaturation, fatty acid biosynthesis and fatty
acid elongation) were up-regulated in suspension compared to adherent HEK293 progeny cell lines. All results
from the metabolic gene set analysis are provided in Supplementary Table S8.
Focusing on the pairwise comparisons between HEK293 cell lines, 38 differentially expressed genes were iden-
tified common to all adherent to suspension pairwise comparisons (red column in Figs. 3b, 5a, Supplementary
Table S5). Three of the genes (ARRDC3, HMGCS1 and PCYOX1L) had the same directional gene copy number
variation (gain or loss) compared to gene expression fold-changes (up or down) of all suspension compared to
adherent cells, which may at least partly explain the differential expression of these genes between the groups.
Gene enrichment analysis of this set of 38 differentially expressed genes between adherent and suspension cells,
performed using ­Enrichr36, predicted the cholesterol biosynthetic process pathway as the cellular pathway most
affected by this expression variation (Fig. 5b). This result further emphasizes the differential expression between

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Figure 5.  Evaluation of common DE genes between adherent and suspension HEK293 cells identified
cholesterol biosynthesis as the main enriched pathway. (a) Heat map with TPM values for each DE gene
common to all adherent to suspension comparisons. (b) The top ten most enriched biological GO terms of the
38 common DE genes between adherent and suspension cells based on gene enrichment analysis. Length and
color of bars both show significance of adjusted p-value for the hypergeometric test. Also, genes mentioned in
each bar are the genes that belong to enriched GO term and present in the list of 38 DE genes.

adherent and suspension cells of genes involved in the cholesterol pathway, mentioned above. Among the 38
common differentially expressed genes MSMO1, IDI1, NPC1L1, INSIG1 and HMGCS1 are directly related to
cholesterol biosynthesis (GO:0006695 and/or GO:0008203, Fig. 5b). Each of these genes had at least a two-fold
increase in expression in the suspension cells compared with the adherent cell-lines. Based on these findings, we
sought to predict the effect of the differentially expressed genes between adherent and suspension HEK293 cell
lines on the cholesterol biosynthesis of the cells using Ingenuity pathway analysis (IPA). Although the MSMO1,
IDI1 and HMGCS1 genes were all up-regulated in 293-F and 293-H compared to HEK293, the down-regulation
of the lathosterol oxidase gene (SC5D), which gene product is in downstream steps of the pathway, resulted in a
predicted reduction in cholesterol production in 293-F and 293-H cells compared to HEK293 (Supplementary
Fig. S7). Comparisons between suspension cell lines (293-F and 293-H) and adherent progeny cell lines (293E
and 293T) did not result in any predicted changes in cholesterol biosynthesis (data not shown).
As four out of the 38 differentially expressed genes (LOX, SMAD7, ID1 & TXNIP) have previously been
shown to have a role in the epithelial to mesenchymal transition (EMT) ­pathway37, we evaluated the role of this
pathway in the transition from adherent to suspension cell growth of these HEK293 cell lines. The normalized
expression of a set of EMT markers showed that the parental HEK293 actually had the highest level of expression
of various mesenchymal markers (N-cadherin, vimentin and fibronectin) of all the six cell lines (Supplementary
Fig. S8). Moreover, when predicting EMT pathway outcomes for suspension cells (293F and 293H) compared
to the parental HEK293 strain using IPA, the results predicted reduced EMT in the suspension progeny cells
compared to HEK293 (Supplementary Fig. S8). However, suspension cells were predicted to have increased
disruption of adherence junctions, which is consistent with the suspension cell phenotype. Taken together the
comparison between adherent and suspension HEK293 progeny cell lines suggest that the transition between
adherent to suspension cell growth is not equivalent to the epithelial to mesenchymal transition even though
several EMT-associated genes may be key to the difference between cell lines. Instead key changes were found
associated with cholesterol biosynthesis and fatty acid metabolism.

Identification of five genes with potential key roles in differences between human adherent
and suspension cell lines.  For identification of key genes involved in the transition from adherent to
suspension morphology, expression data from an additional set of 63 different human cell lines deposited in
the Human Protein Atlas ­database38 were analyzed. Principal component analysis of these cell lines resulted in
clustering of suspension cell lines in a distinct group separated from adherent cell lines (Fig. 6a). However, since
most of the suspension cell lines are lymphoid or myeloid-derived this clustering may be a result of the similar
origin of the suspension cell lines. Transcription data of the 38 previously identified differentially expressed
genes from 47 adherent and 16 suspension cell lines (Supplementary Table S9) was compared between the two
groups using a Mann–Whitney U-test39,40. Within this set, nine genes (LOX, ID1, ADAMTS1, ZIC1, KCNMA1,
DHRS3, RARG, COL4A6 and ARRDC4) had significant different expression levels between adherent and sus-
pension cell lines with p-values < 0.01 (Fig.  6b,c). Four of these genes (ADAMTS1, KCNMA1, COL4A6 and
ARRDC4) had the opposite directional change in the extended data set compared to the differential expression
between only HEK293 strains. Based on these findings, the remaining five genes (LOX, ID1, ZIC1, DHRS3 and
RARG), which showed a consistent down-regulation in suspension cell lines compared to adherent cells, may
play important roles in the morphological differences between the adherent and suspension cell lines.

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Figure 6.  Gene expression validation of the 38 previously identified differentially expressed genes in 63

human cell lines, identified nine significantly differentially expressed genes between suspension and adherent
cell lines. (a) PCA transcriptomic data of 63 human cell-lines from the Human Protein Atlas shows a clear
separation of suspension and adherent cell lines from different tissues. (b) Range of normalized counts in
HPA cell lines for each of the previously identified 38 genes, differentially expressed between all adherent and
suspension HEK293 cell lines. The black line in each box shows median of normalized counts for the gene. (c)
Genes that are differentially expressed between adherent and suspension cells using a Mann–Whitney U-test,
with p-values < 0.01, are highlighted in purple, where length of bars shows logarithmic fold change of median
between two groups and the color of bars denotes degree of significance of p-value. Non-significant genes have
gray bars.

Discussion
Due to the extensive usage of HEK293 cells as a bioproduction platform for pharmaceutical proteins and AAV
vectors, characterization of the HEK293 genome and transcriptome is relevant for bioprocess development. A
deeper knowledge of the HEK293 genomic and transcriptomic traits can for instance pave the way for more
rational cell line engineering approaches, aiming to improve bioproduction efficiency and quality of protein
products. As different HEK293 lineages are propagated under different conditions and the observation that
immortalized continuously cultured cell lines, such as HEK293, have a high degree of genomic instability with
frequent chromosome r­ earrangements22,25 it can be expected that different HEK293 lineages are differentiated at
the genomic and transcriptomic level compared to the parental cell line. Here, the genomes and transcriptomes
of the original HEK293 along with five progeny cell lineages were analyzed (Fig. 1a). The overall comparison of
genomic and transcriptomic profiles confirmed the picture of clonally diverged progeny cells as compared to the
parental HEK293 (Fig. 1b,c). As expected, there was a high degree of genomic and transcriptomic similarities of
the Freestyle 293-F and 293-F cell lines (Fig. 1b–d). The results presented here indeed show that they are highly
similar both on a genomic and transcriptomic level and confirm the previously reported findings that standard
propagation of HEK293 cell lines does not alter the genomic profile to a large e­ xtent22. Furthermore, based on
the hierarchical clustering, the adherent progeny cell lines showed a higher degree of divergence from each other
compared to suspension cells. This may be a result of the independent transformation and isolation of the 293T
and 293E lineages by the stable integration of different viral genes in different labs. The relatively low expression
level of EBNA1 observed in 293E cells may be an effect of cultivating cells in the absence of geneticin in this study,
in order to minimize differences in cultivation conditions between cell lines. Interestingly, a truncated version of
the Large T antigen was also found to be expressed in 293E cells, which has to our knowledge not been reported

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previously (Fig. 1e and Supplementary Fig. S2). This sequence was likely derived from the pRSVneo plasmid that
was co-transfected with pCMV-EBNA1 during the isolation of the 293E c18 ­clone31.
The overall comparison of the genomic and transcriptomics profiles of HEK293 cell lines suggests that the
parental HEK293 strain has the highest divergence amongst the cell lines. Common changes in gene copy number
gain or loss (Fig. 2 and Supplementary Fig. S3) and consensus differential expression alterations (Fig. 3) were
observed amongst all progeny cell lines when compared to HEK293. For instance, a common dense pattern of
copy number gain and loss for progeny cell lines was observed on chromosome 13 and 18 (Fig. 2a). Such patterns,
found across all or several lineages of HEK293 isolated independently by different methods, suggests a selective
advantage for altered copy numbers of such loci in regard to the phenotypes of the cell lines. On chromosome 13,
several genes associated with the cytoskeleton (BORA, MZT1, PIBF1, DACH1 and KLHL1) had a copy number
gain in all progeny cell lines (Supplementary Table S3). Indeed, consensus gene expression changes associated
with cytoskeleton organization were observed between all progeny HEK293 cell lines and HEK293 (Fig. 3a).
In addition, BORA, MZT1 and DACH1 were found amongst the 329 genes commonly differentially expressed
between all adherent and suspension cell lines (Supplementary Table S3). Moreover, amongst the five genes with
high impact SNVs found in all progeny cell lines compared to HEK293, one gene (SGCD) encodes the cytoskel-
etal protein delta-sarcoglycan. Within the gained region of chromosome 18 common to all progeny cell lines
except 293E, there are several cell adhesion molecules (DSC1, DSC2, DSC3, DSG1, DSG2, DSG3 and DSG4),
which may render this region prone to gene copy number variation during cell line development. The observed
enrichment of cell adhesion GO-terms (GO:0,007,156 and GO:0,098,742) amongst genes with common high/
moderate impact SNPs unique to progeny cell lines compared to HEK293 cells also supported common genomic
alterations in progeny cell lines involved in cell adhesion. Combined with the observed down-regulation of the
entire cell adhesion gene set in progeny cell lines compared to HEK293 (Fig. 3a), results highlight changes in
cytoskeleton and cell adhesion during continuous cultivation and cell line development of HEK293 cells. Such
traits associated with cell adhesion, cell motility and extracellular matrix organization may result from selective
pressure, through inefficiency of detaching the most adherent cells, during single cell cloning. Indeed, the adher-
ent progeny cell lines 293E and 293T are easier to detach by trypsinization compared to the parental HEK293,
potentially through altered expression of cell adhesion, cytoskeletal and cell membrane proteins in accordance
with the observed consensus changes between progeny cell lines and the parental HEK293.
Moreover, specific genomic regions of more local gain or loss of specific genes were observed, including a loss
of fumarate hydratase (FH) gene copies. The loss of FH copies was previously observed for HEK293 by Lin and
coworkers and was suggested to play an important part in the transformed phenotype of the cell l­ine22. In line
with this, our results showed that several of the HEK293 progeny cell lines (293E, 293-F and Freestyle 293-F) were
found to only maintain half the number of FH gene copies compared to the original HEK293 (Supplementary
Fig. S4), supporting an advantageous loss of the FH gene in the HEK293 cell lineages. Furthermore, a conserved
pattern of substantial gain (> 1.5 log2-fold change) of the TLE4 gene and surrounding loci, was observed for all
progeny cells except 293E (Supplementary Fig. S4). Interestingly, TLE4 has previously been reported to have
both a tumor suppressor function and to be associated with promoting tumor growth in different studies of
different ­cancers41,42. Moreover, a significant loss of the ADAM3A pseudogene (< -1 log2-fold change), which
has previously been associated with different c­ ancers43,44, was observed in all progeny cell lines except 293-H
compared to HEK293 (Supplementary Fig. S4). The specific gain of TLE4 and/or loss of ADAM3A loci and their
association with tumor development, suggest important functions of these genes in the evolution of HEK293 cell
lines, potentially through effects on proliferation and/or evasion of normal cell senescence of progeny cell lines.
In bioproduction processes for pharmaceutical proteins, suspension cell lines enable large-scale cultivation
in bioreactors, which is required in order to meet the demands for marketed drugs. However, the adaptation
of cells from adherent to suspension growth and the differential cultivation procedures between adherent and
suspension cells induces phenotypic changes to the cell lines. In order to develop a deeper understanding of
such changes, we evaluated differences in gene expression levels between adherent and suspension progeny
HEK293 cells. Consensus differential expression results were found related to up-regulation of genes associated
with cell component organization such as membrane, cytoskeleton and cell junction in suspension compared to
adherent cells (Fig. 5a). Noteworthy, cell adhesion was found up-regulated in suspension compared to adherent
cells. Amongst the most significant differentially expressed genes (adjusted p-value < 0.01) in the cell adhesion
gene set (Supplementary Table S7), several members of the cadherin superfamily, including many different pro-
tocadherins (PCDH), desmoglein 2 (DSG2) and desmocollin 2 (DSC2) were found significantly up-regulated
in suspension cells compared to adherent cells. This family of genes are involved in the formation of adherence
junctions between c­ ells45. Notably, DSG2 and DSC2 are located in the region on chromosome 18 that had gained
genomic copies in all progeny cell lines except 293E compared to the parental strain (Fig. 2a). Moreover, four pro-
tocadherin members showed the highest fold-change of up-regulated genes in suspension cells (Supplementary
Table S7). The higher expression of such cell adhesion molecules in suspension cell lines compared to adherent
progeny HEK293 cells may be explained by the loss of culture dish support to grow on in case of suspension cells.
Upon disruption of adhesion interactions with other cells and extracellular matrix, a natural cellular response
may be to increase or maintain the expression of adhesion molecules in an attempt to restore such connections.
The ability of the suspension cell lines to form cell aggregates during suspension cultivation and the ease of the
cells to attach to culture dish surfaces upon cultivation without shaking, can be speculated to support these
findings. Such cell adhesion molecules found up-regulated in suspension cell lines may thus be appropriate cell
line engineering targets for improved bioprocess performance of suspension cell lines.
Further evaluation of the differentially expressed genes between adherent and suspension HEK293 progeny
cell lines, based on metabolic gene set analysis, highlighted changes in biosynthesis of aromatic amino acids and
pathways related to lipids and/or cholesterol metabolic processes (Fig. 4b). These metabolic changes could be a
result of different growth media compositions used for the cultivation of either adherent or suspension cells that

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may imply different concentrations of for instance amino acids, glucose or serum. When reducing the number
of differentially expressed genes to those that consistently showed differential expression between adherent and
suspension cells in pairwise comparisons of all cell lines, the cholesterol and sterol biosynthesis and metabolism
pathway were found to be most significantly different between the cell types (Fig. 5b). Moreover, five of the con-
sistently up-regulated genes in suspension HEK293 compared to adherent encode enzymes that have either direct
roles in the cholesterol biosynthesis pathway (MSMO1, HMGCS1 and IDI1)46, or proteins that are associated with
cholesterol metabolism by various processes (NPC1L1 and INSIG1)47,48. As suspension cell lines are cultivated
under serum free conditions, the increased expression of genes associated with for instance cholesterol in suspen-
sion cell lines may be a result of a lower cholesterol content in the medium. However, as cholesterol is a major
component of the cell membrane and has an important function for membrane structure and cell ­signaling49, the
differential expression of genes associated with cholesterol synthesis and metabolism may also be of importance
for the different morphologies between adherent and suspension HEK293 cells. Indeed, previous studies have
shown that cholesterol plays a critical role in regulating the formation of cell-to-cell interactions in endothelial
­cells50 and that depletion of cholesterol reduces cell adhesion and increases endothelial cell ­stiffness51,52. Increased
cell surface stiffness has been reported for HEK293 cells in suspension compared to adherent state as a result of
up-regulation and re-organization of the actin c­ ytoskeleton53. This may partly be a result of altered cholesterol
levels in the cell membrane since cholesterol is a regulator of the actin cytoskeleton and cholesterol depletion
has been shown to induce actin ­polymerization54. Interestingly, the Insulin-induced gene 1 protein (INSIG1),
which was up-regulated in suspension compared to adherent HEK293, is a negative regulator of cholesterol
synthesis and important for cholesterol h ­ omeostasis48 and knockout of INSIG1 has previously been shown to
55
result in cholesterol ­accumulation . Notably, a lower cholesterol biosynthesis in suspension cell lines compared
to the original HEK293 strain was indeed predicted using IPA (Supplementary Fig. S7). It should however be
noted that this prediction does not take into consideration the effect of INSIG1, instead the predicted reduction
in cholesterol biosynthesis in suspension cells compared to the HEK293 cell line is a result of down-regulation
of SC5D (lathosterol oxidase). From a bioprocess perspective, differences in intracellular cholesterol synthesis
and metabolism may also be of interest with regards to the secretory capacity of a cell line since previous find-
ings has shown that cholesterol is essential for ER to Golgi transport within the secretory p ­ athway56 and that
secreted productivity of CHO cells increases upon elevated intracellular cholesterol levels, through silencing of
INSIG1, possibly due to increasing the volume of the Golgi c­ ompartment57. It would therefore be of interest to
gain further knowledge about the cholesterol content and distribution within HEK293 cell lines and potentially
evaluate if this pathway can be targeted for enhanced bioproductivity without having a deleterious impact on
suspension growth or cell morphology.
Four of the 38 genes (ID1, SMAD7, TXNIP and LOX) that were consistently differentially expressed between
adherent and suspension HEK293 have previously been annotated to play a role in epithelial to mesenchymal
transition (EMT)37, the event where stationary epithelial cells lose their cell–cell adhesion and change into
motile and invasive mesenchymal ­cells58. However, when evaluating the expression of common markers for
mesenchymal and endothelial phenotypes as well as predicting the outcome of the EMT pathway using IPA,
the parental HEK293 strain showed the most mesenchymal-like phenotype whereas suspension cell lines were
predicted to have reduced transition from epithelial to mesenchymal phenotype compared with HEK293 (Sup-
plementary Fig. S8). These results indicate that the suspension adaptation of HEK293 lineages does not follow
the EMT pathway.
Altogether nine of the 38 identified genes (LOX, ID1, ADAMTS1, ZIC1, KCNMA1, DHRS3, RARG, COL4A6
and ARRDC4), with differential expression between all adherent and suspension comparisons of HEK293
(Fig. 5a), were shown to have significantly different expression between adherent and suspension cells also in an
extended validation of the genes in a set of 63 human cell lines from the HPA ­database38 (Fig. 6c). Five of these
genes (LOX, ID1, ZIC1, DHRS3 and RARG) showed a consistent expression profile (same direction of up- or
downregulation) between adherent and suspension cells compared to the results presented in Fig. 5b, suggesting
a key role of these genes in the morphologies of adherent and suspension human cell lines. In support of this
hypothesis, up-regulation of ID1, as found in adherent cells compared to suspension cell lines, has been associated
with the mesenchymal-to-epithelial ­transition59. Moreover, ID1 silencing has also been shown to significantly
reduce adhesion of neural stem c­ ells60 and conversely, increased ID1 expression in epithelial cells has been related
to increased a­ dhesion61. In addition, lysyl oxidase (LOX), an enzyme responsible for the covalent cross-linking
between elastin and collagen in the extracellular matrix, has been shown to be important for cell–matrix adhe-
sion formation, supporting the adherent phenotype of adherent cells but is also associated with cell invasion and
induction of E ­ MT62,63. Besides the EMT-related genes, the additional three genes (RARG, ZIC1 and DHRS3),
consistently up-regulated in adherent cells compared to suspension cell lines, have previously been associated
with increased cell adhesion through the retinoic acid signaling p ­ athway64–67. In line with this, retinol metabolism
was found to be down-regulated in suspension cells in the metabolic gene set analysis (Fig. 4b).

Conclusions
Our study has outlined the genomic and transcriptomic variations between six industrially relevant HEK293
cell lines, in an attempt to improve the understanding of their respective differences in phenotype. We report
a selective pressure to develop certain expression profiles during the evolution and continuous cultivation,
evidenced by the numerous genes and pathways detailed here. The key common changes between HEK293
and its progeny cell lines involve in particular cell membrane proteins and processes related to cell adhesion,
motility and the organization of various cellular components such as the cytoskeleton and extracellular matrix.
In addition, changes associated with differences between adherent and suspension cell growth in particularly
involve changes in cell adhesion protein expression, cholesterol metabolism and a set of six key genes (RARG,

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ID1, ZIC1, LOX and DHRS3) with potentially key roles in the differentiation between the two groups. These
results could be of importance when pursuing further cell line engineering or bioprocess optimization of these
and other human cell lines.

Methods
Cell cultivation for DNA and RNA preparation.  The adherent cell lines HEK293 (ATCC-CRL-1573),
HEK293T (ATCC-CRL-3216) and 293E (ATCC-CRL-10852) were obtained from ATCC and propagated in
DMEM (D6429) supplemented with 10% FBS at 37 °C in a humidified incubator with 5% ­CO2 in air. Suspension
cell lines 293-F, 293-H and Freestyle 293-F (Gibco) were obtained from Thermo Fisher Scientific and cultivated
in 293 SFM II medium (Gibco) supplemented with Glutamax at a final concentration of 4 mM (Gibco). Suspen-
sion cells were cultivated in 125-ml Erlenmeyer shake flasks (Corning) at 37 °C and 120 rpm in a humidified
incubator with 8% ­CO2 in air. All cells were propagated from frozen stocks for no longer than 20 passages.

RNA and DNA preparation and sequencing.  Adherent cells were detached by trypsinization and both
adherent and suspension cells were harvested by centrifugation. Genomic DNA was extracted using the Blood
and Cell Culture DNA Mini Kit (Qiagen) according to the manufacturer’s guidelines and concentrations were
determined by using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Genome sequencing was
performed at the National Genomics Infrastructure (Scilifelab, Solna, Sweden) using the Illumina HiSeq X plat-
form. For RNA extraction, cells grown in log phase from three biological replicates were collected (derived
from successive propagations). Cell pellets were resuspended in RNAlater Stabilization Solution (Invitrogen)
according to the manufacturer’s recommendations until RNA extraction. Total RNA was extracted from three
replicates of each cell line using Qiagen’s RNeasy plus Mini Kit according to the manufacturer’s instructions.
Concentrations were determined with a NanoDrop ND-1000 spectrophotometer and RNA quality was assessed
on a 2100 Bioanalyzer (Agilent Technologies) using RNA 6000 Nano chips (Agilent Technologies). All samples
had an RNA integrity number of at least 9.9. RNA sequencing was performed at GATC (Konstanz, Germany)
using the Inview Transcriptome Advance service and an Illumina HiSeq instrument.

DNA‑sequencing analysis.  Genome sequencing reads were aligned to the reference (human_g1k_v37.
fasta) using bwa (0.7.12)68. The raw alignments were then deduplicated, recalibrated and cleaned using GATK
(version 3.3–0-geee94ec, gatk-bundle/2.8)69. Quality control information was gathered using Qualimap (v2.2)70.
SNVs and indels have been called using the GATK ­HaplotypeCaller69,71. These variants were then functionally
annotated using snpEff (4.1) and snpEff reference GRCh37.7572. The Piper pipeline from the National Genom-
ics Infrastructure was u­ sed73. The correlation between BAM files was assessed using multibamsummary and its
plotCorrelation function from ­deepTools274. Spearman was used to calculate correlation coefficients between
samples, and the clusters are joined with the nearest neighbor. The R package seqCAT​75 was used to compare
SNVs between samples, its compare_profiles function mode parameter was set to the default value “intersec-
tion”. The heatmap in Supplementary Fig. S1 was based on the similarity scores between the cell lines and Euclid-
ean ­distances76. To compare the Large T antigen sequences of 293T and 293E, unmapped reads were extracted to
new bam files using S­ AMtools77, converted to fastq with B
­ EDTools78, and de novo assembled with M­ EGAHIT79.
NCBI BLAST was used to identify the Large T antigen in the assembled contigs. To evaluate and visualize copy
number variations, ­CNVkit80 was used with its whole-genome sequencing method, cbs ­segmentation81,82 and
the HEK293 alignment as reference. GO enrichment analysis of genes with high or moderate impact SNPs was
performed using PANTHER classification ­system83.

RNA‑sequencing data.  Kallisto84 was used to quantify transcripts by pseudo-alignment based on human
genome assembly version GrCh37. Log transformed normalized data by DESeq2 was used for cell line clustering
and calculation of Euclidean distances of samples. The expression comparison of the viral elements was based
on normalized counts from DESeq2. Significant testing of differential mRNA expression of E1A/B elements
was done by Welch two sample t-test85. For differential expression analysis, raw count data from Kallisto was
imported using the tximport p ­ ackage86 and analyzed with D
­ ESeq287. Wald tests were used to calculate p-values,
and the BH method was used for multiple testing correction. Throughout the article a gene is considered dif-
ferentially expressed if log2- fold change >  ± 1 and FDR < 0.05. In the differential expression analysis between
adherent and suspension cells, all suspension cell-lines were compared to all adherent cell-lines, and addition-
ally, all pairwise combinations between suspension and adherent cell-lines were evaluated. For evaluating dif-
ferential expression of 38 common differentially expressed genes between adherent and suspension HEK293
cell lines in a set of 63 human cell lines, RNA-seq data from each cell line deposited in the HPA database was
used. Based on the growth characteristics, cells were divided into two groups of adherent and suspension cells.
A Mann–Whitney U-test was used to compare normalized counts based on library size between the two groups
for each of the 38 differentially expressed ­genes39,40.

Gene set analysis.  To discover significant alterations of gene sets and metabolic pathways between HEK293
cell lines, the Piano package in R was ­used88. The adjusted p-values and fold changes from the differential expres-
sion was used in combination with a gene set collection based on “goslim_generic Biological Process”. The heat-
map for the progeny cells lines vs. HEK293 was based on the consensus score calculated based on GO term
rank aggregation in Piano for each directionality from all pairwise gene set statistics calculations with Wilcoxon
rank-sum test. The heatmap for suspension cells (293-H, 293-F) vs. adherent cells (293E, 293T) was based on the
consensus score from gene set statistics calculations with mean, median, sum, Stouffer and tailStrength tests and
was calculated with Piano’s consensusHeatmap function. To produce the network plot, gene sets were exported

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from ­HMR289. For finding differentially expressed pathways of genes between adherent and suspension cell
lines, we used the Wilcoxon statistical test and filtered gene sets with adjusted p-value lower than 0.05 as signifi-
cantly changed. In addition, for gene set analysis of 38 common DE genes between adherent and suspension cell
lines we used EnrichR and GO biological process as gene set c­ ollection36.

Ingenuity pathway analysis.  In order to predict the pathway changes between cell lines based on dif-
ferentially expressed genes from pairwise comparisons, ingenuity pathway analysis (IPA, QIAGEN Inc.,) was
performed. To consider a gene as differentially expressed we used log2 fold change > 1 or < 1 and adjusted
p-value < 0.05. For filtering results of gene set analysis by IPA we used Benjamini–Hochberg multiple testing
corrected p-values lower than 0.05 to find gene sets with a different expression pattern.

Data availability
Genomics and transcriptomics data is available at Sequence Read Archive (SRA)—BioProject: PRJNA565658.

Received: 11 February 2020; Accepted: 22 October 2020

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Acknowledgements
This work was supported by the Knut and Alice Wallenberg Foundation, AstraZeneca, Swedish Foundation for
Strategic Research (SSF), Swedish innovation agency Vinnova through AAVNova, CellNova and AdBIOPRO
and the Novo Nordisk Foundation (grant no. NNF10CC1016517).

Author contributions
Conceptualization, R.F., P.V., J.N., and J.R; Methodology, M.M., R.S. and J.R. Formal analysis, M.M., R.S., and
M.L.; Investigation, M.M., R.S., V.C., M.L. M.G., and J.R., Writing—Original Draft, M.M, R.S., M.L., and J.R.;
Writing—Review & Editing, M.M., M.L., D.H., J.N., L.G., V.C., and J.R.; Visualization, M.M., R.S., and M.L.,
Supervision, R.F., P.V., D.H., T.S., J.N., and J.R., Funding Acquisition, R.F., P.V., J.N, and J.R.

Funding
Open Access funding provided by Kungliga Tekniska Hogskolan.

Competing interests 
The authors declare no competing interests.

Additional information
Supplementary information is available for this paper at https​://doi.org/10.1038/s4159​8-020-76137​-8.
Correspondence and requests for materials should be addressed to J.N. or J.R.
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