Induction of differentiation and metabolic reprogramming in human hepatoma cells by adult human serum

Tissue culture medium routinely contains fetal bovine serum (FBS). Here we show that culturing human hepatoma cells in their native, adult serum (human serum, HS) results in the restoration of key morphological and metabolic features of normal liver cells. When moved to HS, these cells show differential transcription of 22-32% of the genes, stop proliferating, and assume a hepatocyte-like morphology. Metabolic analysis shows that the Warburg-like metabolic profile, typical for FBS-cultured cells, is replaced by a diverse metabolic profile consistent with in vivo hepatocytes. We demonstrate the formation of large lipid and glycogen stores, increased glycogenesis, increased β-oxidation, increased ketogenesis, and decreased glycolysis. Finally, organ-specific functions are restored, including xenobiotics degradation and secretion of bile, very low density lipoprotein, and albumin. Thus, organ-specific functions are not necessarily lost in cell cultures, but might be merely suppressed in FBS. Together, we showed that cells that are representative of normal physiology can be produced from cancer cells simply by replacing FBS by HS in culture media. The effect of serum is often overseen in cell culture and we provide a detailed study in the changes that occur, provide insight in some of the serum components that may play a role in the establishment of the different phenotypes, and discuss how these finding might be beneficial to a variety of research fields.

particles that are more representative of the particles that are circulating in the serum of HCV infected patients 4 . In the current study, we further investigated the cellular changes that occur in HCC cells that are cultured in HS instead of FBS. We used a combination of microarray analysis, microscopic techniques, and biological assays to show that the limitations of standard HCC cultures can be overcome by changing the serum. By replacing FBS with HS in the cell culture medium, Huh7.5 cells (i) become growth arrested, obtain an epithelial, cuboid morphology and become polarized; (ii) undergo complete metabolic reprogramming, with a reversal of the cancer metabolic profile (Warburg effect and glutaminolyis); (iii) diversify other metabolic pathways, with a reduction in glycolysis, an increase in glycogen storage (glycogenesis) and higher reliance on β-oxidation; and (iv) increase mRNAs of many CypP450 enzymes and CypP450 metabolic rates and increase or restore secretory processes, like VLDL, albumin and bile secretion.
Summarizing, we show that by simply placing cells in their native adult serum, extensive reprogramming of Huh7.5 takes place, and the morphology and functions that were considered lost in cancer cell lines can be restored. We discuss the relevance of these findings for in vitro research, given the central role metabolism plays in various physiological processes.

Polarization, cytoskeletal organization and other morphological changes.
We investigated the effect of replacing FBS by HS in tissue culture media, on cell morphology and the gene expression profile of the HCC cell line Huh7.5. We first examined overall morphological changes resulting from extended culturing in HS.
HS and FBS-cultured cells where grown on transwell dishes, prepared for electron microscopy and sectioned perpendicular to the membrane surface, so that a 'side view' of the cell is created ( Figure 1A). HS-cultured cells become cuboid, consistent with the in vivo hepatocyte phenotype. Moreover, their apical surface has a more pronounced epithelial character than FBS-cultured cells, with more and larger villi.
HS-cultured cells are also tightly interconnected, with no open space in between, unlike their FBS-cultured counterparts. This is confirmed in higher magnification images of the cell boundaries ( Figure 1B). Increased cytoplasm density and altered organelle organization were also noted in HS-cultured cells as further described in Supplemental Data 1.
Cytoskeletal organization plays an important role in establishing polarization and cell shape. Therefore, we visualized cytoskeletal reorganization of tubulin, a microfilament, and vimentin, an intermediate filament, by confocal microscopy. 6 Whereas vimentin is disorganized in FBS-cultured cells, a structured organization is seen in HS-cultured cells (d21). Tubulin appears more condensed in HS-cultured cells than in FBS-cultured cells, where staining is faint and more dispersed.
We also visualized the organization of claudin-1, a major component of the tight junction complex ( Figure 1D). Claudin-1 is present in FBS, but the staining is patchy, whereas in HS-cultured cells claudin-1 is distributed evenly around the entire cell, pointing at better barrier function. To test if such a barrier exists, we measured the diffusion rate of fluorescently labeled 70 kDa dextran conjugates across confluent layers of FBS-cultured cells or HS-cultured cells grown on transwell dishes. HScultured cells were almost impermeable to these conjugates, showing that a barrier is indeed established, whereas FBS monolayers remained permeable ( Figure 1C).
In conclusion, morphological hallmarks of hepatocytes, including the formation of polarized cell layers consisting of tightly interconnected cuboid cells, can be achieved in Huh7.5 cells simply by culturing them in HS instead of FBS.

Gene expression changes
Next, we used genome-wide expression arrays to investigate the overall gene expression changes of cells cultured in HS for 8, 15 and 23 days compared to cells cultured in FBS. These time points were chosen because: (i) HS-cultured cells 7 become growth arrested after 7-10 days, (ii) around day 15 the first morphological signs of differentiation become apparent and (iii) after 21 days the differentiation process appears complete 4 . Indeed, gene expression changed significantly (p<0.05 after multiple testing correction) in 22 to 32% of the genes (11,000-16,000 of 49,000 probes), revealing a complete cellular reprogramming upon shifting to HS media (Supplemental Data 2). Principal component analysis (PCA, Figure 2A) showed a good experimental replicability, and a clear change in expression profile upon shifting the cells from FBS to HS. To determine the similarity in gene expression between HS-cultured cells and primary hepatocytes, published hepatocyte expression profiles were projected onto the PCA, by applying the gene weights associated with PC1 and PC2 to the gene expression levels. This showed close proximity of primary hepatocytes to the later stages of the HS-cultured HCC cells, suggesting differentiation towards a hepatocyte phenotype ( Figure 2).

PAM clustering analysis
To obtain better insight into the specific genetic processes that changed as a result of culturing cells in HS, we identified clusters of co-expressed genes by applying hierarchical clustering using z-scores (Supplemental Data 3) followed by PAM 8 (partitioning around mediods) clustering ( Figure 3). Six gene expression patterns emerged, and Gene Ontology (GO) terms associated with these six patterns where determined ( Figure 3, Supplemental Data 4). Changes in cell cycle and lipid metabolism gave the strongest signals related to the shift to HS. Most biological processes associated with the six clusters are consistent with our current and previous analyses 4 : cells become growth arrested (cluster 1, 4, 5) and lipid droplets, as well as VLDL secretion were increased (cluster 1, 6). We also observed a derepression of apoptosis (cluster 2). Cancer cells often repress apoptosis, and the observed changes may reflect the loss of their proliferative character.
Thus, we further examined the metabolic changes occurring in HS-cultured cells that are predicted to accompany the transition from a proliferating to a differentiated and growth-arrested state of the cell.

Metabolic reprogramming: reversal of the Warburg effect and metabolic diversification.
Proliferating cells often display a 'cancer metabolism' profile, first described by Otto Warburg in 1924, which includes reduced levels of oxidative phosphorylation and mitochondrial activity, higher dependence on aerobic glycolysis and glutaminolysis 9 for ATP production, and increased generation of biosynthetic intermediates that are essential for the production of macromolecules (phospholipids, nucleotides, proteins) to support cell proliferation (reviewed in 3, 5-7 ). The metabolic reprogramming that occurs during the Warburg effect is tightly regulated. Key regulators include pyruvate dehydrogenase kinase 1 (PDK1), the lactate dehydrogenase A/B ratio (LDHa/LDHb), and monocarboxylic acid transporter 4 (MCT4), as further explained in Supplemental Data 5.
To test our hypothesis that cancer metabolism is reversed in HS-cultured Huh7.5 cells we compared HS-to FBS-cultured cells using a combination of gene expression analyses, measurement of end-point metabolites, electron microscopy and biological assays. The first indication that HS-cultured cells rely less on aerobic glycolysis than their FBS-cultured counterparts, is the much slower acidification of the cell culture media, as indicated by medium colour. In line with this, and with the reversal of the Warburg effect, mRNA of LDHa was decreased and LDHb was increased, shifting the reaction away from lactate production ( Figure 4A). Additionally, mRNA of lactate transporter MCT4 was also decreased in HS-cultured cells, as well as strong downregulation of PDK1, which regulates pyruvate uptake by mitochondria ( Figure   4A, Supplemental Data 6) Changes in mitochondrial morphology also might suggest increased rates of oxidative phosphorylation in HS-cultured cells. In FBS-cultured cells, the mitochondrial matrix appeared lighter than in HS, where it was condensed and dark with large spaces between the cristae ( Figure 4B). Hackenbrock 8-10 described similar mitochondrial transitions in isolated mitochondria, and linked them to electron transport chain activity: condensed mitochondria are indicative of high activity, whereas orthodox mitochondria indicate low activity. Other studies have since confirmed the link between mitochondrial activity and mitochondrial morphology [11][12][13] . High oxidative phosphorylation rates are also associated with mitochondrial narrowing 13 , and we made similar observations in HS-cultured cells ( Figure 4C). Although the physiological role of the mitochondrial heterogeneity remains largely unclear, matrix density may influence diffusion rates of metabolites in and out of the mitochondria [14][15][16] , for example of ATP and ADP.
We investigated which metabolic pathways changed as Huh7.5 cells shifted away from their cancer metabolism. Figure 5A shows that fermentation and glutaminolysis were decreased in HS-cultured cells, consistent with the reversal of cancer metabolism. Additionally, (i) all but one glycolysis enzymes were downregulated, including the rate-limiting step. The one enzyme that is up-regulated is also involved in gluconeogenesis, and the mRNAs of enzymes in the gluconeogenesis pathway were generally up-regulated (data not shown). (ii) Most glycogenesis enzymes, including the rate-limiting step were up-regulated, indicating that HScultured cells convert large amounts of glucose to glycogen, relying less on glucose for ATP production. This is supported by the presence of large glycogen deposits within the cell, as is shown in Figure 5B, bottom image (marked G). Glucose use was not altered in HS-cultured cell compared to FBS-cultured cells ( Figure 5D, left panel). (iii) In HS-cultured cells lipid droplets size was increased ( Figure 5B, top two panels 4,17 ). β-oxidation is in part regulated by the availability of lipid stores and metabolic analysis supports an increase in β-oxidation rate: many enzymes involved in TG (Triacylglycerol) degradation and β-oxidation are increased, including the rate regulating step of β-oxidation, CPT-1 (carnitine-palmitoyl transferase 1). Increased protein levels of CPT-1 (approximately 7-fold) were confirmed by western blot ( Figure 5C). (iv) Acetyl CoA produced by β-oxidation is partially converted to ketone bodies in the liver of healthy individuals, which play a critical role in normal energy homeostasis 18 . Formation of ketone bodies is therefore often used to estimate the rate of β-oxidation. Ketogenesis was also increased in HS-cultured Huh7.5 cells ( Figure 5A), which was confirmed by NMR analysis of metabolic end-products in HScultured cells. Acetoacetate and 3-hydroxybuyrate, the two main ketone bodies produced during ketogenesis, where significantly increased in HS cultured cells ( Figure 5D). This further supports increased levels of β-oxidation in HS serum cultured cells.
Finally, consistent with the reversal of a proliferative metabolic profile, mRNAs of pathways that produce 'building blocks' for cell growth and proliferation, specifically cholesterol synthesis and lipogenesis, are decreased ( Figure 5E). In these pathways, citrate that is produced in the mitochondria is exported to the cytosol, where it is converted to Acetyl-CoA. Acetyl-CoA can then be incorporated into cholesterol and fatty acids, as depicted in Figure 5E. mRNAs of most genes in these pathways are decreased in HS-cultured cells. Malonyl-CoA is also produced in the lipogenesis pathway and inhibits CPT-1, and thereby β-oxidation. Thus, its decreased production in HS-cultured cells is in line with the activation of βoxidation in HS-cultured cells, and metabolic reprogramming in general.
Summarizing, our analyses show that the metabolism in HS-cultured cells shifts away from cancer metabolism (glutaminolysis and the Warburg effect) and glycolysis, in favor of glycogen storage. Lipid stores are increased, as are β-oxidation and ketogenesis. The increase in lipid droplet size is probably the result of the activation of TG biosynthesis and storage pathways, that are regulated by LXRα and PPARγ, which in turn are both upregulated in HS-cultured cells 4 . The presence of 13 native lipoproteins in the human serum may also play a role: when we used lipoprotein free human serum, differentiation proceeded as expected, but the increase in lipid droplet size was largely prevented (not shown).

Xenobiotics biodegradation and metabolism
As cells move away from the state of proliferation and cancer metabolism, surplus nutrients become available for storage, and to reinstate secretory processes like VLDL secretion. Indeed, we showed previously that VLDL secretion is completely absent in FBS-cultured cells, but as cells become growth arrested and differentiate, VLDL secretion is gradually restored 4 . Here, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis supports the re-establishment of lipid and carbohydrate metabolism as described in the previous sections, as well as re-establishment of secretory processes ( Ascorbate and aldarate metabolism is central to many conversions of glucose, including nucleoside synthesis and pentose interconversions, which itself was 14 significantly increased after 15 days in HS media. The most notable changes involved 'Xenobiotics biodegradation and metabolism'. Three pathways in this cluster were significantly increased (Table 1)  Transcriptional levels of other proteins involved in degradation and removal of xenobiotics were also determined, and findings were generally consistent with increased degradation and removal of toxic compounds (Supplemental Data 9).
To functionally confirm the changes observed at a transcriptional level, we examined the rates of xenobiotics degradation in FBS-and HS-cultured cells ( Figure   6B). The intrinsic, non-induced rates of degradation by Cyp3A4 (testosterone), 15 Cyp1A2 (methoxy-resorufin) and Cyp2B6 (buproprion) were increased approximately 4-, 6-and 6-fold respectively.
Many products resulting from CypP450 activity are removed from the liver in the bile, through the bile canalicular surface, which is only established in polarized cells.
Bile synthesis is also dependent on the availability of excess cholesterol. Figure 6C shows the formation of structures that have a high resemblance to bile canaliculi in HS-cultured cells. Most mRNAs of enzymes involved in bile synthesis are increased ( Figure 6D), indicating higher levels of bile acid secretion. Similar observations were reported elsewhere in HepG2 cells which were cultured in human serum 19 .

Discussion
When tissue and cell culture was initially developed in the early to mid 1950s, the lack of cell growth was a major problem, which was overcome by the addition of fetal animal sera or embryo extracts. The first human cell line for example, HeLa cells, was grown in chicken plasma clot, mixed with saline, human umbilical cord serum and bovine embryo extract 20 . Eventually, fetal bovine serum (FBS) became the serum of choice, because of its abundant availability and excellent growth promoting capacities. Undeniably, great progress has been made since cell cultures were first developed, however, it has also become increasingly clear that rapidly dividing cells are not representative of normal functional cells in an organ or organism, including the morphology of the cell, metabolism and organ-specific functionality.
In this study we used a human hepatocellular carcinoma cell line, Huh7.5, and showed that culturing these cells in their native adult serum, instead of FBS, results in growth arrest and drastic changes in morphology and intracellular organization, metabolic reprogramming and re-establishment of organ-specific functions, like VLDL secretion, albumin secretion and detoxification of xenobiotics. Significant changes in transcription in 22-32% of the genes are indicative of extensive cellular reprogramming. We showed that metabolism shifts from a cancer-like profile, to a profile that is more representative of hepatic metabolism, that includes glycogenesis, higher β-oxidation rates, lower glycolysis rates, as well as restoration of processes like VLDL secretion 4 , degradation of xenobiotics by cytochrome P450s, and bile secretion (this study, 19 ). This refutes the notion that cell lines derived from cancers have lost their ability to undergo contact inhibition and much of their organspecific functionality. We showed in Huh7.5 cells, that most of these lost functions, Conversely, human umbilical cord blood serum (CBS), the closest human equivalent of FBS, induces high cell proliferation rates, but also increases Huh7.5 functionality as shown by increased levels of albumin secretion. However, VLDL secretion is not restored in CBS-cultured cells 25 . Likely, VLDL secretion co-depends on the presence of sufficiently large TG stores (lipid droplets), and may thus also depend on a nonproliferative metabolic profile/ growth arrest.
Growth factors that are remarkably higher in human serum (CBS and HS), compared to FBS, are IGF-1 (insulin like growth factor 1) and several IGFBPs (IGF binding proteins) 26 . IGF-1 is increased approximately 20 and 40-fold in HS and hUCBS respectively, relative to FBS. IGF-1 and IGFBPs both play a role in hepatocyte differentiation, with IGFBPs modulating and prolonging the activity of IGF-1.
Consistent with their role in differentiation, IGF-1 and IGFBPs impede the aggressive growth of certain liver cancers [27][28][29] . IGFBP-3 is one of the factors listed in the top-25 genes with the greatest increase in transcription in our microarray analysis (Supplemental Data 6).
In addition to differences in growth factor level and composition, the composition of the lipids in HS is also markedly different from FBS. Lipoproteins in bovine serum 20 mainly contain saturated fatty acids, whereas lipoproteins in human serum are enriched in lipids containing unsaturated fatty acids, particularly oleic acid (18:1) 30 .
The presence of oleic acid is significant, as oleic acid is often added to cells to stimulate lipoprotein secretion 31    Metabolites formed were analyzed on a liquid chromatographytandem mass spectrometry system, similar to a previously published method 41 . Selected reaction monitoring in the positive-ion electrospray ionization mode was performed for acetaminophen as an internal standard, resorufin (a metabolite of 7methoxyresorufin produced by CYP1A2 activity), hydroxybupropion (a metabolite of bupropion produced by CYP2B6 activity), 6α-hydroxypacliataxel (a metabolite of paclitaxel produced by CYP2C8 activity), 4′-hydroxymephenytoin (a metabolite of S-mephenytoin produced by CYP2C19 activity), dextrorphan (a metabolite of dextromethorphan produced by CYP2D6 activity), and 6β-hydroxytestosterone (a metabolite of testosterone produced by CYP3A activity).

Data availability, code availability
Microarray data were deposited in the GEO repository with Accession Number

Supplemental Data 1
other morphological changes in HS cultured cells Additional morphological changes that were observed in the elelctron microscopic analysis include: -the cytoplasm of HS cultured cells appears much more crowded than the cytoplasms of FBS cultured cells.
-mitochondiral morphology has drastically changed, as outlined in section 4 of the main document -in general. organelles appeared more structured and organized, for example, the endop[lasmic reticulum shows a higher degree of organization, particularly around the mitochondira. We assume these regions represent the mitochondria associated membranes or MAM.
-An increase in vesicle transport and changes in the lysosomal pathways were also observed on the electron micrographs. Whereas in FBS cells early lysosomes (dark structures in the top figure) are abundant, but other components of the endosomal/lysosomal pathway were not detected, lysosomal structures were not seen on EM images of HS cultured cells. Instead, in HS cultured cells late endosomes, multivesicular bodies and autophagosome, potentially indicating that a more complete endosomal/ lysosomal pathway is operational in HS cultured cells.