Immortalized Chondrocyte Cell Lines Research Articles

Generation of human immortalized chondrocytes from osteoarthritic and healthy cartilage

After a few passages of in vitro culture, primary human articular chondrocytes undergo senescence and loss of their phenotype. Most of the available chondrocyte cell lines have been obtained from cartilage tissues different from diarthrodial joints, and their utility for osteoarthritis (OA) research is reduced. Thus, the goal of this research was the development of immortalized chondrocyte cell lines proceeded from the articular cartilage of patients with and without OA. Using telomerase reverse transcriptase (hTERT) and SV40 large T antigen (SV40LT), we transduced primary OA articular chondrocytes. Proliferative capacity, degree of senescence, and chondrocyte surface antigen expression in transduced chondrocytes were evaluated. In addition, the capacity of transduced chondrocytes to synthesize a tissue similar to cartilage and to respond to interleukin (IL)-1β was assessed. Coexpression of both transgenes (SV40 and hTERT) were observed in the nuclei of transduced chondrocytes. Generated chondrocyte cell lines showed a high proliferation capacity and less than 2% of senescent cells. These cell lines were able to form 3D aggregates analogous to those generated by primary articular chondrocytes, but were unsuccessful in synthesizing cartilage-like tissue when seeded on type I collagen sponges. However, generated chondrocyte cell lines maintained the potential to respond to IL-1β stimulation. Through SV40LT and hTERT transduction, we successfully immortalized chondrocytes. These immortalized chondrocytes were able to overcome senescence in vitro, but were incapable of synthesizing cartilage-like tissue under the experimental conditions. Nonetheless, these chondrocyte cell lines could be advantageous for OA investigation since, similarly to primary articular chondrocytes, they showed capacity to upregulate inflammatory mediators in response to the IL-1β cytokine.Cite this article: Bone Joint Res2023;12(1):46-57.

Generation of an immortalized chondrocyte cell line from osteoarthritis articular cartilage

Purpose: Primary human articular chondrocytes tend to senesce and lose their phenotype in vitro after a few passages. Chondrocyte cell lines available have been generated from cartilage sources different from synovial joints, and their usefulness for articular cartilage research is limited. For these reasons, the aim of this study was the generation of an immortalized chondrocyte cell line derived from the articular cartilage of a patient with osteoarthritis (OA).

Chondrosarcoma-derived Mutation in IDH1 Promotes Tumorigenesis by Inducing HIF-1α

IDH mutations lead to the inability of IDH to convert isocitrate into α-ketoglutarate (α-KG). Instead, α-KG is reduced into an oncometabolite, D-2-hydroxyglutarate (D-2HG). IDH mutations occur in more than 50% of patients with conventional or dedifferentiated chondrosarcoma. However, the function of these mutations in the pathogenesis of chondrosarcoma is still unclear. In this study, we aimed to establish a mutant IDH1 knockout model in a chondrosarcoma cell line and determine the role of mutant IDH1 in this malignancy. In our study, we employed a human chondrosarcoma cell line, JJ012 which harbors an endogenous IDH1R132G mutation. As a control, we also used an immortalized chondrocyte cell line, C28 which expresses wildtype IDH1 only. IDH1 was knocked out via CRISPR/Cas9 technology. Quantitative analyses of D-2HG were conducted by tandem mass spectrometry. RNA-Seq analysis was performed to identify differentially expressed genes between the mutant IDH1 knockout (KO) cells and the parental cells. RNA-Seq data were validated by real time PCR. Colony-forming ability of KO cells under hypoxia and normoxia conditions was evaluated using soft agar assay. The effect of mutant IDH1 on chondrosarcoma growth was analyzed in murine xenograft models. The expression of HIF1-1α and its targeted genes were examined in cells and tumor tissues by western blot and Immunohistochemistry (IHC). Mutant IDH1 was successfully knocked out in JJ012 cells. Single cell-derived KO clones were then identified and developed. As expected, D-2HG production is almost completely depleted in these KO clones. Using these genetically modified cells, we found that knockout of mutant IDH1 remarkably inhibited colony formation by 40% under normoxia condition compared to the parental control (p<0.05). Intriguingly, we observed that the HIF1-α levels were significantly reduced in the JJ012 KO cells compared with the parental cells. Moreover, RNA-seq data showed a widespread downregulation of HIF1-1α targeted genes such as VEGFA, VEGFC, SLC2A3, EDN1 in response to the knockout (FDR-adjusted p value < 0.01). These data were confirmed by qRT-PCR. The soft-agar colony formation assay further showed a significantly increased colony number and diameter for JJ012 KO cells under hypoxia condition compared with those under normoxia condition (P<0.05). Furthermore, we observed that loss of mutant IDH1 led to a marked attenuation of chondrosarcoma formation in the murine xenograft model. Immunoblotting and IHC revealed that HIF-1α and its targeted genes were dramatically downregulated in the KO cell-derived tumors. We found that mutant IDH1 contributes to tumorigenicity of chondrosarcoma cells, at least partially through the upregulation of HIF1-α. This suggests that targeting HIF1-α signaling pathway is promising for combinatorial regimens with mutant IDH inhibitors for patients with IDH1-mutant chondrosarcoma.

STAT3 isoforms produced by the C-28/I2 human immortalized chondrocyte cell line

STAT3 isoforms produced by the C-28/I2 human immortalized chondrocyte cell line

Matrix Metalloproteinase-9 Production by Immortalized Human Chondrocyte Lines.

We reported at the Keynote Forum of Immunology Summit-2015 that recombinant human (rh) TNF-α or rhIL-6 stimulated production of matrix metalloproteinase-9 (MMP-9) in the T/C28a2 and C-28/I2 human immortalized chondrocyte cell lines. Furthermore, we reported that tocilizumab (TCZ), a fully humanized monoclonal antibody which neutralizes IL-6-mediated signaling, inhibited the rhIL-6-mediated increase in the production of MMP-9. IL-6 is also a known activator of the JAK/STAT signaling pathway. In that regard, we evaluated the effect of rhIL-6 on total and phosphorylated Signal Transducer and Activator of Transcription by these chondrocyte lines which showed that whereas STAT3 was constitutively phosphorylated in T/C28a2 chondrocytes, rhIL-6 activated STAT3 in C-28/I2 chondrocytes. The finding that rhIL-6 increased the production of MMP-9 by human immortalized chondrocyte cell lines may have important implications with respect to the destruction of articular cartilage in rheumatoid arthritis and osteoarthritis. Thus, the markedly elevated level of IL-6 in rheumatoid arthritis and osteoarthritis sera and synovial fluid would be expected to generate significant MMP-9 to cause the degradation of articular cartilage extracellular matrix proteins. The finding that TCZ suppressed rhIL-6-mediated MMP-9 production suggests that TCZ, currently employed in the medical therapy of rheumatoid arthritis, could be considered as a drug for osteoarthritis.

Articular cartilage chondrocytes express aromatase and use enzymes involved in estrogen metabolism.

IntroductionSex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17β-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue.MethodsWe analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes.ResultsAromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10−11 M to 10−7 M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-β. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole.ConclusionsWe demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-β. In addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated.

THU0022 Enhanced expression of suppressor of cytokine signaling 3 in arthritic cartilage dysregulates human chondrocyte function

BackgroundThe intracellular suppressor of cytokine signaling (SOCS)-proteins are inducible and negative regulators in receptor signaling pathways of several cytokines, toll-like receptor ligands and some growth factors. We previously have shown...

Lentiviral shRNA knock-down of ADAMTS-5 and -9 restores matrix deposition in 3D chondrocyte culture.

Aggrecan is one of the two major constituents of articular cartilage, and during diseases such as osteoarthritis (OA) it is subject to degradation by proteolytic enzymes. The primary proteases responsible for aggrecan cleavage are the aggrecanases, identified as members of the ADAMTS family of proteases, which are upregulated in response to inflammatory stimuli. It is uncertain which of the six aggrecanases (ADAMTS-1, -4, -5, -8, -9 and -15) are primarily responsible for the degradation of aggrecan in human cartilage. Here we show that four of the six aggrecanases are expressed in immortalized chondrocyte cell-lines and can be upregulated in response to inflammatory cytokines. Using RNA interference, we demonstrate robust knock-down of ADAMTS-5 and -9 expression in these cells and, by culturing them on three-dimensional (3D) scaffolds, show that reduction in expression of ADAMTS-5 enzyme results in an increase in matrix deposition. These data suggest that the quality of tissue-engineered cartilage matrix might be improved by targeted depletion of aggrecanase expression. Moreover, this work also provides further evidence that ADAMTS-5 may be a therapeutic target in the treatment of arthritic disease.

Expression and modulation of ghrelin O‐acyltransferase in cultured chondrocytes

To use reverse transcription-polymerase chain reaction to detect ghrelin O-acyltransferase (GOAT) transcripts in both murine and human chondrocytes, to evaluate the effect of pharmacologic in vitro treatments with lipopolysaccharide (LPS), growth hormone, ghrelin, and dexamethasone on GOAT messenger RNA (mRNA) expression, and to study the GOAT mRNA profile during chondrocyte differentiation. Murine and human GOAT and ghrelin mRNA levels were determined by the SYBR Green-based quantitative real-time polymerase chain reaction method. GOAT mRNA was expressed in murine cartilage explants as well as in the cultured murine chondrogenic ATDC-5 cell line. GOAT was also expressed in human immortalized chondrocyte cell lines and in human cultured primary chondrocytes. In addition, GOAT mRNA expression in differentiating ATDC-5 cells was lower at the early stage of differentiation (days 3-7), whereas GOAT mRNA levels increased progressively at the late stages. Finally, among the drugs and hormones tested, only LPS was able to strongly decrease GOAT mRNA expression. These data indicate that chondrocytes are equipped with biochemical machinery for the synthesis of acylated ghrelin and suggest a novel role of the ghrelin axis in prehypertrophic and hypertrophic chondrocyte differentiation during endochondral ossification.

Lectin binding patterns reflect the phenotypic status of in vitro chondrocyte models

In vitro studies using chondrocyte cell cultures have increased our understanding of cartilage physiology and the altered chondrocytic cell phenotype in joint diseases. Beside the use of primary cells isolated from cartilage specimens of donors, immortalized chondrocyte cell lines such as C-28/I2 and T/C-28a2 have facilitated reproducible and standardized experiments. Although carbohydrate structures appear of significance for cartilage function, the contribution of the chondrocyte glycocalyx to matrix assembly and alterations of the chondrocyte phenotype is poorly understood. Therefore, the present study aimed to evaluate the glycoprofile of primary human chondrocytes as well as of C-28/I2 and T/C-28a2 cells in culture. First, the chondrocytic phenotype of primary and immortalized cells was assessed using real-time reverse transcriptase polymerase chain reaction, immunofluorescence, and glycosaminoglycans staining. Then, a panel of lectins was selected to probe for a range of oligosaccharide sequences determining specific products of the O-glycosylation and N-glycosylation pathways. We found that differences in the molecular phenotype between primary chondrocytes and the immortalized chondrocyte cell models C-28/I2 and T/C-28a2 are reflected in the glycoprofile of the cells. In this regard, the glycocalyx of immortalized chondrocytes was characterized by reduced levels of high-mannose type and sialic acid-capped N-glycans as well as increased fucosylated O-glycosylation products. In summary, the present report emphasizes the glycophenotype as an integral part of the chondrocyte phenotype and points at a significant role of the glycophenotype in chondrocyte differentiation.

Matrix degradation inhibitory effect of Schisandra fructus on human articular cartilage and chondrocytes

The purpose of this study is to assess the protective effects exerted by Schisandra fructus (SF) on hyaluronidase (HAase) and the recombinant human interleukin-1β (IL-1β) induced matrix degradation in human articular cartilage and chondrocytes. The effect of SF on the matrix gene expression of immortalized chondrocyte cell line C-28/I2 treated with HAase was investigated using a reverse transcription polymerase chain reaction (RT-PCR). In addition, the effects of SFEA on HAase and IL-1β induced matrix degradation in human articular cartilage were assessed using a staining method, and the quantity of glycosaminoglycan (GAG) degraded was calculated from the cultured media. In HAase treatment group, the released GAGs content increased significantly to 15.8 ± 0.7 μg, compared with control levels (5.0 ± 0.2 μg), whereas co-treatment with SFEA (100 μg/ml) reduced the GAG release to 10.8 ± 0.7 μg ( P < 0.05). Also, in the group treated only with IL-1β (11.9 ± 0.3 μg), the amount of released GAG increased significantly compared to the control (7.9 ± 0.1 μg) and the SFEA-treated group (7.8 ± 0.4 μg). SFEA at 100 μg/ml inhibited PG degradation (9.0 ± 0.5 μg, P < 0.05). However, no concentration-dependent increases in inhibitory activity were observed. Therefore, since SFEA treatment resulted in no pathological impact on either the chondrocytes or cartilage, we suggest that SF can be safely used as an effective material for the prevention of proteoglycan (PG) degradation.

Phenotypic Characterization of Human Chondrocyte Cell Line C-20/A4: A Comparison between Monolayer and Alginate Suspension Culture

DNA microarray analysis was used to investigate the molecular phenotype of one of the first human chondrocyte cell lines, C-20/A4, derived from juvenile costal chondrocytes by immortalization with origin-defective simian virus 40 large T antigen. Clontech Human Cancer Arrays 1.2 and quantitative PCR were used to examine gene expression profiles of C-20/A4 cells cultured in the presence of serum in monolayer and alginate beads. In monolayer cultures, genes involved in cell proliferation were strongly upregulated compared to those expressed by human adult articular chondrocytes in primary culture. Of the cell cycle-regulated genes, only two, the CDK regulatory subunit and histone H4, were downregulated after culture in alginate beads, consistent with the ability of these cells to proliferate in suspension culture. In contrast, the expression of several genes that are involved in pericellular matrix formation, including MMP-14, COL6A1, fibronectin, biglycan and decorin, was upregulated when the C-20/A4 cells were transferred to suspension culture in alginate. Also, nexin-1, vimentin, and IGFBP-3, which are known to be expressed by primary chondrocytes, were differentially expressed in our study. Consistent with the proliferative phenotype of this cell line, few genes involved in matrix synthesis and turnover were highly expressed in the presence of serum. These results indicate that immortalized chondrocyte cell lines, rather than substituting for primary chondrocytes, may serve as models for extending findings on chondrocyte function not achievable by the use of primary chondrocytes.

Molecular phenotyping of human chondrocyte cell lines T/C-28a2, T/C-28a4, and C-28/I2.

Because the immortalized chondrocyte cell lines C-28/I2, T/C-28a2, and T/C-28a4 have become a common tool in cartilage research, permitting investigations in a largely unlimited and standardized manner, we investigated the molecular phenotype of these cell lines by gene expression profiling. Complementary DNA-array analysis as well as online quantitative polymerase chain reaction were used to identify the gene expression profiles of the 3 cell lines cultured in monolayer and alginate beads, as compared with the expression profiles of cultured human adult primary chondrocytes. A similar, but not identical, gene expression profile was established for all 3 cell lines. SOX9 was expressed at a significant level in all 3 cell lines. Extracellular matrix proteins and matrix-degrading proteases were rarely expressed. In contrast, genes involved in the cell cycle were strongly up-regulated, as compared with the expression levels in physiologic chondrocytes. The expression of SOX9, the master gene of chondrocytic cell differentiation, reflects the basically chondrocytic phenotype of these cells. However, the major issue appears to be that these cell lines mainly proliferate and show less expression of genes involved in matrix synthesis and turnover. In this respect, C-28/I2 cells display the highest levels of matrix-anabolic and matrix-catabolic genes and thus are presumably preferable for use in investigating chondrocyte anabolic and catabolic activity and its regulation. None of the 3 cell lines appears to be a direct substitute for primary chondrocytes. A successful approach will have to validate the findings obtained with chondrocyte cell lines by using primary chondrocytes or cartilage-tissue cultures. This would permit the establishment of reproducible in vitro models and subsequently allow investigators to relate the findings to the physiologic situation.

Human chondrocyte culture models for studying cyclooxygenase expression and prostaglandin regulation of collagen gene expression

ObjectiveSince articular chondrocytes and synovial fibroblasts are particularly responsive to interleukin-1 (IL-1) with respect to stimulation of prostaglandin E2(PGE2) biosynthesis, we have used them as models to examine feedback modulatory effects of PGE2, which blocks or attenuates the direct effects of IL-1β on cell-specific collagen gene expression.MethodsImmortalized human chondrocytes were developed for studying responses to cytokines and prostaglandins. Regulatory sequences of the type II collagen gene (COL2A1) in reporter gene constructs were analyzed in transient transfection experiments. Endogenous expression of COL2A1 mRNA, as well as aggrecan, biglycan, and decorin mRNAs, and IL-1-inducible cyclooxygenase (COX-2), phospholipase A2 (PLA2), and inducible nitric oxide synthetase (iNOS) mRNAs were analyzed by RT-PCR.ResultsPrevious work has shown that IL-1β inhibits, while prostaglandins stimulate COL2A1 expression. In different immortalized chondrocyte cell lines, the ability to respond to IL-1β with increased levels of COX-2, PLA2, and iNOS mRNAs depends upon expression of the differentiated chrondrocyte phenotype.ConclusionsOur studies suggest that some IL-1-induced responses in chondrocytes may require differentiation-specific transcription factors that could serve as therapeutic targets for arthritis.

Expression of type X collagen and matrix calcification in three-dimensional cultures of immortalized temperature-sensitive chondrocytes derived from adult human articular cartilage.

Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.