A literature search was conducted on 17 May 2025 in PubMed (National Center for Biotechnology Information, 2025) to identify publications involving Huh-7 cells. The search term “Huh7 or Huh-7” was used to encompass all relevant papers, regardless of minor variations in cell line nomenclature. The retrieved articles were then assessed for their relevance to our study, as well as for information on experimental techniques, results, and applications involving HuH-7 cells.

The human cell line HuH-7 (#JCRB0403, RRID: CVCL_0336) was obtained from the Japanese Collection of Research Bioresources (JCRB) via the FUJIFILM Wako Chemicals Europe GmbH representative. The cells were cultured in a humidified incubator at 37 °C with 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM, high glucose #6171, Sigma-Aldrich, Merck, Taufkirchen, Germany) with 1.5 g/L sodium bicarbonate. This medium was supplemented with 10% fetal bovine serum (FBS, #F7524, Sigma-Aldrich), 4 mM L-glutamine (#G7513, Sigma-Aldrich), and 1× penicillin-streptomycin antibiotic solution (DE17-602E, Lonza, Cologne, Germany). The cells were passaged when they reached 80%–90% confluence, using an Accutase solution (A6964-100 ML, Sigma-Aldrich), and seeded at the appropriate density for subsequent analyses. All experiments were conducted using cells from low passage numbers to minimize phenotypic drift.

To confirm the authenticity of the HuH-7 cell line and identify any Mycoplasma spp. contamination, we performed short tandem repeat (STR) profiling and a sensitive real-time PCR assay to detect even trace amounts of the bacterium. For this purpose, we utilized the cell line authentication service provided by IDEXX (Kornwestheim, Germany) through the CellCheck™ 16 Human PLUS (test code: 42–00098). This test system includes the 13 human reference STR loci recommended for human cell line authentication: CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and vWA (Almeida and Korch, 2023; Korch et al., 2021), two additional variant markers (Penta D and Penta E), and the amelogenin (AMEL) locus, which is used to determine gender. Additionally, we conducted regular mycoplasma testing in our laboratory. Cell culture supernatants from 80%–90% confluent cultures were directly processed with the Venor®GeM OneStep kit for conventional PCR (#11–8050, Minerva Biolabs GmbH, Berlin, Germany). The resulting amplicons were analyzed on a 2% standard agarose gel and stained with Midori Green, in accordance with an established standard protocol (Supplementary Figure S1) (Weiskirchen et al., 2023).

For the protein expression studies, cells were lysed in an ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentration was determined using the DC Protein Assay (#5000116, Bio-Rad Laboratories, CA, USA). Subsequently, 60 µg of cell lysate was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as previously described (Liehr et al., 2025). The proteins were then transferred onto 0.45 µm nitrocellulose membranes, blocked with a 5% non-fat dry milk solution, and incubated overnight at 4 °C with primary antibodies specific to the targets of interest. After washing, the membranes were incubated with the relevant secondary antibodies conjugated to horseradish peroxidase (HRP). Protein bands were visualized using the Supersignal™ West Dura Extended duration Substrate (#34076, Thermo Fisher Scientific, Schwerte, Germany). Information on the antibodies, including their RRID identifiers, used in this study is provided in Table 1. In the Western blot analysis we used protein extracts generated from human livers as controls. These were obtained from the centralized RWTH Biomaterial Bank (https://www.ukaachen.de/kliniken-institute/institut-fuer-pathologie/biobank/) with approval from the Ethics Committee of the Medical Faculty of RWTH Aachen University (permit number EK 206/09). Human liver tissue was homogenized in a MM400 mixer mill (Retsch GmbH, Haan, Germany) using established protocols (Liehr et al., 2025). Following protein quantification, 80 µg of protein tissue lysate was utilized for Western blot analysis.

For the combined karyotype and M-FISH analysis, metaphase chromosome spreads were prepared by treating HuH-7 cells with colcemid to arrest them in metaphase, following the detailed protocol previously described (Liehr et al., 2024). After hypotonic treatment and fixation in methanol-acetic acid, the chromosome spreads were placed dropwise onto glass slides and hybridized with a M-FISH probe mixture containing 24 whole chromosome painting probes specific to the 24 different human chromosomes (#D-0125–120-DI, XCyting, MetaSystems Probes, Altlussheim, Germany). Chromosome banding was performed using inverted 4′,6-diamidino-2-phenylindole (DAPI, #D1306, Thermo Fisher Scientific) staining; 20 metaphases were analyzed in detail. We analyzed karyotypes across all 20 metaphases observed by mFISH using whole chromosome paints and also confirmed and refined the results with 20 chromosome-specific multicolor banding (MCB) experiments. For each MCB experiment 20–25 metaphases were analyzed. Additionally, 24 homemade chromosome-specific MCB probe sets were applied as previously described (Weise et al., 2008), and 20–25 metaphases were analyzed for each. All FISH analyses were conducted using a Zeiss Axioplan fluorescence microscope (Carl Zeiss Jena, Jena, Germany) equipped with a CCD camera and image processing software (ISIS, MetaSystems, Altlussheim, Germany.

The losses and gains of chromosomal regions in human chromosomes were identified using chromosomal microarray as array comparative genomic hybridization (aCGH). DNA was isolated from the cell line and aCGH analysis was performed using 4 × 180 K SurePrint G3 Human CGH Microarray slides (Agilent Technologies). These slides cover the entire human genome with a 13-kb overallmedian probe spacing (11 kb in refseq genes). The probes were combined with reference DNA (male) provided by Promega as controls and processed as previously reported in GRCh37/hg19 (Aust et al., 2013). The data obtained was compared with CGH-results obtained in human hepatocellular carcinoma (Guan et al., 2000).

For the transcriptomic analysis, total RNA was extracted from HuH-7 cells that had been grown to 80% confluency using an established CsCl density gradient protocol (Liehr et al., 2024). Quantity and quality/integrity were evaluated via UV spectroscopy and on the Agilent 4200 TapeStation instrument (Agilent Technologies Inc., Waldbronn, Germany). Library preparation involved mRNA enrichment or rRNA depletion, followed by fragmentation, synthesis of first- and second-strand cDNA synthesis, adapter ligation, and PCR amplification according to established protocols (Liehr et al., 2025). The libraries were then sequenced on a high-throughput Illumina sequencing platform with prefilled cartridges and the downstream analysis was performed using standardized pipelines at the IZKF Genomic Facility of the University Hospital Aachen. The obtained sequence data were aligned to the human reference genome (Genome assembly GRCh38: https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001405.26/), and quantified at the gene and transcript levels. This resulted in length-normalized transcripts per million (TPM) values.

Cellular morphology was routinely monitored using phase-contrast or bright-field microscopy to assess growth patterns and overall health. Images were captured using a Leica EC3 digital camera connected to a Leica DM IL LED microscope fitted with the Leica Application Suite (LAS) software (version 3.4.0, Leica Microsystems GmbH, Wetzlar, Germany).

For Phalloidin Staining, 30,000 cells were seeded in four-chamber culture sides (#354104, BD Falcon™, BD Biosciences, Erembodegem, Belgium). After 72 h, microfilament staining was conducted as previously described (Liehr et al., 2025). The cells were stained with a 1× diluted Rhodamine-Phalloidin solution (#R415, Invitrogen, Thermo Fisher Scientific, Schwerte, Germany) for 20 min in the dark. Nuclear counterstain was performed with 4′,6-diamidino-2-phenylindole (DAPI, #D1306, Thermo Fisher Scientific) for 30 min in the dark. After staining, the slides were embedded in Vectashield Antifade mounting medium (#H-1000, Burlingame, California, United States) and stored at 4 °C. For analysis, a Nikon Eclipse E80i fluorescence microscope with NIS-Elements Vis software (version 3.22.01) was used.

For ultrastructure studies, the cells were harvested and fixed in a solution of 2.5% glutaraldehyde solution in a 0.1 M phosphate buffer. They were then post-fixed in 1% osmium tetroxide. Following dehydration using a graded ethanol series, the samples were embedded in epoxy resin. Ultrathin sections (approximately 70–90 nm thick) were cut using an ultramicrotome and mounted on copper grids. These sections were subsequently post-stained with uranyl acetate and lead citrate prior to examination using a Zeiss Leo 906 transmission electron microscope (Carl Zeiss AG, Oberkochen, Germany) operating at 60 kV. High-resolution micrographs were taken at various magnifications ranging from ×2,784 to 12,930× in order to elucidate the organelle integrity of organelles and the morphology of mitochondria and other subcellular features that are characteristic of HuH-7 cells.

The Huh-7 cell line was first established in 1982 by Hidekazu Nakabayashi and his team at Kagawa University. It originated from a well-differentiated hepatocyte carcinoma that was removed during surgery from a 57-year-old Japanese male (Nakabayashi et al., 1982). A PubMed search conducted on 17 May 2025 using the search term “HuH-7 or HuH7” revealed that this cell line has been used in 8,319 studies. Furthermore, HuH-7 cells have been referenced over 5,000 times in the RRID portal (https://rrid.site/data/source/SCR_013869-1/search?q=CVCL_0336&l=CVCL_0336), which underscores the extensive use of this cell line in biomedical research.

When observed under a light microscope, HuH-7 cells exhibit an epithelial morphology. They grow as adherent, polygonal cells with a relatively uniform appearance (Figure 1). They often form monolayers in culture, with rounded or oval nuclei that have prominent nucleoli and abundant cytoplasm. Binucleation and characteristic intercellular contacts may occasionally be observed. These morphological features are consistent with the cells being derived from a well-differentiated hepatocellular carcinoma tissue.

For authentication purposes, we performed short tandem repeat (STR) profiling on our Huh-7 cells. We analyzed 15 specific STR markers, including Penta E, D18S51, D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, vWA, Penta D, CSF1PO, D16S539, D7S820, D13S317, and D5S818, as well as the amelogenin gene to determine sex (Figure 2; Table 2). The resulting profile showed a 100% match to the published reference data (Kasai et al., 2018), thus confirming the authenticity and genetic consistency of our HuH-7 cell line.

A molecular cytogenetic analysis on the HuH-7 cell line was conducted to obtain a comprehensive understanding of its chromosomal composition. Utilizing inverted DAPI-banding in conjunction with M-FISH allowed for each chromosome to be visualized in a distinct color, adding in the identification and mapping of intrachromosomal genetic alterations and/or aneuploidies within the cells. Our analysis indicated that HuH-7 cells have an almost triploid karyotype (59<3n>), showcasing both numerical and highly complex structural aberrations in some parts (Figure 3).

To clarify the complex chromosomal rearrangements in HuH-7 cells, we performed a high-resolution, multicolor banding (MCB) analysis. This technique enables DNA-specific color banding along one chromosome at a time (Figures 4, 5). Notably, the Y-chromosome is absent, with only one derivative X chromosome present, although parts of X-chromosome(s) are also found on derivatives of chromosomes 11, 14, and 19. Chromosome 1 shows multiple rearrangements, including a deletion and an insertion involving chromosome 14, as well as a large derivative containing chromosome 15. Chromosome 2 displays deletions and a translocation to chromosome 4, leading to the complete loss of one copy. Several other chromosomes (3, 4, 5, 7, 9, 10, 11, 14 and 19) exhibit complex derivative forms, with merged, inverted, or inserted segments from multiple sources. For example, chromosome 3 has a derivative that incorporates multiple rearrangements and a neochromosome. Isochromosomes are observed in chromosomes 8, and 18. Chromosome 13 is absent, with some fragments replaced by translocated segments. Chromosome 14 shows significant rearrangements, while the remaining derivatives contain interspersed chromosome arms. Additional abnormalities include large deletions, missing chromosomes, and recurring translocations, such as with chromosomes 15, 16, 21, and 22. Moreover, several aberrations are so complex that they could not be fully resolved by FISH or aCGH (see below). Therefore, one derivative each of chromosome 5 and 11 were reported as having been going through ‘cha’ = chromoanasynthesis, which is a special form of chromothripsis.

Overall, the karyotype (also detailed by chromosome in Table 3) can be summarized as: 56<3n>,der(X) (Xpter- > Xq12::20p13->20p13::13q11.2->13q12::14q23->14q24.3::3q26->3qter),-X,-X,del (1) (q12),ins (1; 14) (q12; p1?2q11.2),der (1) (15qter->15q26.1::1q12->1q24::1q24->1q12::1q24->1qter),del (2) (p21),der (2)t (2; 4) (q14.1; q31),-2,3,der (3) (qter- > q28::p12- > q11.2::q28- > q29::q11.2- > qter),neo (3) (pter- > p21.3::p21.3- > pter),der (4) (3pter->3p21.3::4p16->4q12),der (4)t (4; 8) (q33; p23.1),-4,der (5)cha (q12->?qter),del (5) (q13),-5,6,6,der (6)t (4; 6) (p12; p11.2),del (7) (q22),der (7)t (4; 7) (q31; p11.2),der (7) (7p12->7q11.2::10p13->10pter),i (8) (p10),i (8) (q10),-8,9,del (9) (q12),der (9) (9pter->9q34.3::19q13->19q13::10q25.3->10qter),-9,del (10) (q23),der (10)t (9; 10) (q31; p11.2),-10,der (11)cha (pter- > q1?2),der (11)t (X; 11) (q26; p11.2),der (11)t (3; 11) (q26; p11.11),+der (11)t (10; 11) (q11.2; p14),12,12,der (12) (21qter->21q22.2 :: 12p11.2->12q23 ::16q11.2-> 16qter), -13, -13,-13,14,der (14) (10pter->10p11.2::7q11.21->7q11.21::14q11.2->14p12::Xp11.21- > Xp11.21::9q12->9q21::9q33->9q34::2p16->2p16::19p12->19p12::10q25.1->10qter),der (14)t (13; 14) (q11.2; q23),+der (14)t (14; 15) (p13; q11.2),del (15) (q22),-15,-16,17,i (17) (q10),der (17)t (2; 17) (p21; q12),18,i (18) (p10),der (18) (7qter->7q11.2::18q12->18p11.1::7q11.2->7q11.2::18q21->18q12::7q11.2->7qter),der (19) (19pter->19q13.4::Xq11.2- > Xq12::7p11.2->7pter),der (19)t (10; 19) (q24; q13.4),der (19)t (13; 19) (q11.2; q13.4),+der (20)t (5; 20) (q13; q12),der (21)t (1; 21) (p33; p13),-21,der (22) (22pter->22q11.2::5p13.2->5q35::2q14.1->2qter)[cp∼500].

Array comparative genomic hybridization (aCGH) was conducted on HuH-7 cells to visually map chromosomal gains and losses throughout the genome. This revealed the unique and complex genomic landscape of HuH-7 cells. Through aCGH analysis, we identified gains on the short arms of chromosomes 3, 5, 18, and 20, as well as on the long arms of chromosomes 15, 16, and 20. Conversely, losses were observed on the short arms of chromosomes 5, 6, 8, 11, 12, and 17, and on the long arms of chromosomes 4, 8, 11, 12, 13, 14, 21, and 22 (Figure 6). Overall, the results obtained here were consistent with the copy number alterations seen in molecular cytogenetic analyses. However, the complexity revealed in the molecular cytogenetic analyses was not fully captured in the aCGH profile.

As shown in Table 4, 15 out of 19 major copy number variations (CNVs) (approximately 80%) observed in HuH-7 are also found in human hepatocellular carcinoma, indicating that this cell line is a suitable model for advanced hepatocellular carcinoma.

To gain an unbiased overview of the transcriptomic landscape of HuH-7 cells and accurately assess their mRNA expression capacity, we conducted next-generation mRNA bulk sequencing. This approach involves isolating and sequencing the entire mRNA pool, providing a comprehensive snapshot of gene expression in the cells when cultured in a standard medium. Capturing both abundant and lowly expressed transcripts provides valuable insight into the molecular pathways, potential biomarkers, and regulatory networks underlying cell function and disease mechanisms. This information is particularly important for understanding the susceptibility of HuH-7 cells to the hepatitis C virus (HCV).

Our analysis revealed several groups of highly expressed genes in HuH-7 cells. Firstly, there was strong representation of mitochondrial transcripts, including subunits of the electron transport chain (e.g., MT-CO1, MT-CO2, MT-CO3, MT-ND1, MT-ND2, MT-ATP6, and MT-ATP8), reflecting the active metabolic state of these cells. Secondly, numerous ribosomal proteins (e.g., RPL41, RPS12, RPL37, RPS17, and RPS27) and translation factors, such as eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) and eukaryotic translation elongation factor 2 (EEF2), exhibited robust expression, suggesting significant protein synthesis activity (Supplementary Table S1). A further notable category comprised essential housekeeping and cytoskeletal genes, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), tubulin alpha 1b (TUBA1B), vimentin (VIM), the biliary/hepatic progenitor cell marker keratin 19 (KRT19), and keratins 18 (KRT18), and 8 (KRT8). These genes are indicative of fundamental cellular processes. Similarly, HuH-7 cells exhibited high mRNA expression of the transferrin receptor 1 (TFRC), which is a hepatitis C virus entry factor (Martin and Uprichard, 2013). HuH-7 cells also express scavenger receptor class B member 1 (SCARB1), which has also been reported to act as a hepatitis C virus receptor that and facilitates its entry into cells (Arandhara et al., 2023). Other genes that are thought to act as hepatitis C virus entry factors such as the CD81 molecule, occludin (OCLN), and claudin-1 (CLDN1), as well as the cofactor epidermal growth factor receptor (EGFR) (Carriquí-Madronal et al., 2023), are expressed at high levels (CD81, CLDN1, and EGFR) or median levels (OCLN) in HuH-7 cells (Supplementary Table S1).

Furthermore, a high expression of α1-antitrypsin, encoded by the serpin family A member 1 (SERPINA1) gene, was observed. This confirms previous data showing that HuH-7 cells can secrete large quantities of this protein (Ke et al., 2022). Considerable amounts of mRNA encoding the hepatocyte differentiation marker nuclear factor 4α (HNF4A) were also found. HNF4A regulates many genes involved in lipid and carbohydrate metabolism, including those that control very low-density lipoprotein (VLDL) secretion and gluconeogenesis (Huck et al., 2021). Ferritin heavy chain 1 (FTH1) and ferritin light chain (FTL) were expressed at high mRNA levels. These proteins form a complex that stores iron in a non-toxic form, and are predominantly expressed in the liver (Gao et al., 2022). Lastly, markers predominantly expressed in hepatocytes were found among the most highly expressed transcripts, which is consistent with the hepatocellular carcinoma origin of HuH-7 cells. Notably, HuH-7 cells exhibited high levels of expression of both albumin (ALB) and α-fetoprotein (AFP) (Table 5). However, cytochrome P450 (CYP) genes, such as CYP3A4, which are mainly responsible for the phase I metabolism of xenobiotics in hepatocytes, were found to be expressed at low levels. This confirms a previous report demonstrating that HuH-7 cells express negligible amounts of CYP450 enzymes (Bulutoglu et al., 2019). Under the chosen condition, HuH-7 cells also express mRNA of several transporters relevant for drug metabolism at low level, including organic anion transporter polypeptides (OATP1A2/SLCO1A2, OATP1B1/SLCO1B1, OATPB3/SLC01B3), organic cation transporters (OCT1/SLC22A1), multidrug-resistance-associated proteins (MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4), bile salt export pumps (ABCB11), and many others (Supplementary Table S1).

To ensure the accuracy of our next-generation mRNA sequencing results, we performed Western blot analyses on selected proteins of interest (Figure 7), using a protein extract from human liver tissue as a control. In most cases, the Western blot results were consistent with our NGS findings, confirming that many of the transcripts detected at the RNA level were indeed translated into proteins. Specifically, we observed strong expression of fibronectin, HNF4α, β-actin, FTH1, cyclophilin A, collagen type III, AFP, GAPDH, FTL, and (cyto)keratin 19. In line with our NGS data, we demonstrated that CYP3A4 expression was rather low. However, despite the presence of relatively high quantities of fatty acid binding protein 1 (FABP1) mRNA, the FABP1 protein was either undetectable or very low. Interestingly, HuH-7 cells also expressed the tumor marker p53, consistent with the mRNA data (Supplementary Table S1). Overall, the Western blot data predominantly confirmed the transcript-level findings from our NGS analysis, demonstrating that most of the identified mRNAs were translated into proteins. This strong alignment underscores the reliability of our NGS data.

Electron microscopy was used to characterize the ultrastructure of HuH-7 cells (Figure 8). As expected for a hepatocyte-derived cell line, the cells exhibited numerous well-organized organelles associated with protein synthesis and detoxification. The large number of mitochondria reflects the high metabolic demand of these cells. Additionally, cells also exhibited prominently developed rough endoplasmic reticulum and a distinct Golgi apparatus. Similarly, the nuclei of the cells have a lot of euchromatin and pronounced, often two, nucleoli, which also indicate a high metabolic activity of the cells. Some cells also showed evidence of glycogen granules and lipid droplets in certain areas, both of which are characteristic features of hepatocytes involved in energy storage and lipid metabolism. Fibers typical of keratin bundles were also found inside the cells. Moreover, cell junctions were visible between neighboring cells, suggesting preserved epithelial characteristics. However, these junctions may be less extensive in vitro than in primary liver tissue. Overall, these ultrastructural traits corroborate the hepatocellular identity of HuH-7 cells and align with the known morphology of parenchymal liver cells.

Finally, we used Rhodamine-Phalloidin staining to visualize the organization of the actin cytoskeleton in HuH-7 cells, revealing their characteristic epithelial morphology (Figure 9). As expected for a hepatocyte-derived cell line, the cells exhibited a prominent cortical actin belt. This helps maintain a polygonal cell shape and facilitates strong cell-to-cell contacts in order to form monolayers. Additionally, F-actin stress fibers were visible throughout the cytoplasm, reflecting the dynamic and contractile properties of the cellular actin network. Some cells also displayed peripheral ruffling, indicating ongoing membrane remodeling activities that support cell motility and adhesion. Overall, this cytoskeletal arrangement is consistent with the hepatocellular origin of HuH-7 cells, emphasizing their capacity to maintain robust structural integrity in culture.

In our analyses, markers specific to hepatic stellate cells (HSCs) (Supplementary Table S2), Kupffer cells (Supplementary Table S3), and liver sinusoidal endothelial cells (LSECs) (Supplementary Table S4) were either absent or present only minimally in HuH-7 cells. This finding supports the conclusion that HuH-7 cells predominantly exhibit a hepatocellular phenotype rather than that of non-parenchymal liver cells. For example, the HSC-associated genes such as those encoding α-smooth muscle actin (ACTA2, which is essential for the contractile phenotype of activated perisinusoidal cells) and fibroblast activation protein alpha (FAP, which is involved in tissue remodeling and fibrotic processes) were not detected at the transcript level. Other markers, such as desmin (DES, an intermediate filament characteristic of muscle and stellate cells), decorin (DCN, an extracellular matrix proteoglycan), glial fibrillary acidic protein (GFAP, an intermediate filament), and retinol binding protein 1 (RBP1, an intracellular retinoid transporter in stellate cells) were only found in trace amounts. The absence of these core stellate cell markers highlights the hepatocyte-like nature of HuH-7 cells (Supplementary Table S2).

Similarly, the following canonical Kupffer cell markers were either absent or present at extremely low levels in HuH-7 cells: CD163 (a hemoglobin-haptoglobin scavenger receptor); C-type lectin domain family member 1B (CLEC1B) and member CLEC4E (both are pattern-recognition lectins); folate receptor β (FOLR2); Spi-C transcription factor (SPIC, which guides macrophage differentiation), Toll like receptor 9 (TLR9, which mediates innate immune responses via endosomes), CLEC4G (which is involved in pathogen recognition and immune regulation); myeloperoxidase (MPO, which is a hallmark enzyme in myeloid cells) (Supplementary Table S3). These proteins are key mediators of macrophage or immune cell activity, which further supports the idea that HuH-7 cells do not exhibit macrophage-like characteristics.

Finally, genes that are usually found in liver sinusoidal endothelial cells (LSECs), such as stabilin-1 (STAB1) and stabilin-2 (STAB2) (which are important for clearing lymph and blood waste), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1, a hyaluronan receptor found in lymphatic and sinusoidal endothelium), scavenger receptor class F member 1 (SCARF1, another scavenger receptor), and von Willebrand factor (VWF, a glycoprotein that plays a key role pivotal in platelet adhesion), were not expressed, or were only marginally expressed, in HuH-7 cells (Supplementary Table S4). These genes normally confer specialized endothelial functions that are absent in hepatocytes. Together, these results strongly support the conclusion that HuH-7 cells are of hepatocyte origin, further confirming their utility as a model for hepatic biology and liver carcinoma research.

HuH-7 hepatoma cells and some of their derivatives, such as HuH-7.5, are the only continuous cell culture models that consistently support high-level replication of both sub-genomic replicons and fully infectious hepatitis C virus (Dächert et al., 2019). However, within this single lineage, the magnitude of viral RNA amplification varies significantly. Individual sub-clones or long-term passages can differ by up to three orders of magnitude in replication efficiency, highlighting the significant impact of subtle genetic or transcriptional drift on HCV permissiveness (Dächert et al., 2019). Specifically, seven host-dependency factors (ZNF512B, SFI1, LBHD1, CRYM, CRAMP1, THAP7, and NROB2) appear to play a crucial role in mediating HCV permissiveness (Dächert et al., 2019). In our bulk mRNA-sequencing analysis of our low-passage HuH-7 stock, we confirmed the expression of each of the HCV-permissiveness genes identified by Dächert and colleagues (Supplementary Table S5). This confirms that the molecular machinery previously associated with efficient HCV replication is intact in our HuH-7 cultures. Interestingly, we also detected the expression of THAP7 antisense RNA 1 (THAP7-AS1), a long noncoding RNA (lncRNA) that has been linked to oncogenic properties such as invasion and metastasis (Liu et al., 2022).