Human articular chondrocytes immortalized by HPV-16 E6 and E7 genes: Maintenance of differentiated phenotype under defined culture conditions.
OBJECTIVE
To establish an immortalized normal human articular chondrocyte line which could be useful for a better understanding of cell molecular mechanisms relevant for the development of new therapeutic approaches in rheumatic diseases.


METHODS
Chondrocytes from human adult articular healthy cartilage were transfected in primary culture with a plasmid containing two human papilloma virus type 16 (HPV-16) early function genes: E6 and E7, using the highly efficient cationic liposome-mediated (lipofection) procedure. The transfection was verified by reverse transcriptase-polymerase chain reaction analysis of E7 mRNA and by immunofluorence localization of the E7 protein in the cell cytoplasm. The established chondrocyte cell line was examined in monolayer and in two culture conditions that were described to re-induce differentiated characteristics: culturing in a serum-free defined medium supplemented with an insulin-containing serum substitute and seeding on a hyaluronan-based non-woven structured biomaterial. The expression of markers characteristic of cartilage was shown in the mRNA by reverse transcriptase-polymerase chain reaction. Immunohistological staining and Western blotting analysis were performed to evaluate type II collagen synthesis. Proteoglycans deposition was detected by Alcian Blue staining. A Field Emission In Lens Scanning Microscopy was used to look at the morphology of the immortalized cells at very high magnification.


RESULTS
Normal human articular chondrocytes were efficiently transfected leading to the establishment of an immortalized cell line as confirmed by HPV-16 E7 mRNA and protein detection. These cells were able to re-express type II collagen both at mRNA and protein levels under the two defined cultured conditions we used, still maintaining type I collagen expression. Collagen IX mRNA was present only in early primary culture while collagen type X and aggrecan transcripts were always detected. Alcian Blue staining showed a proteoglycan-rich matrix production. The ultrastructural analysis of the immortalized cells revealed that their morphology strictly resembled that of normal chondrocytes.


CONCLUSIONS
The cell line that we obtained may be a useful tool for increasing our knowledge of the genetic and biochemical events involved in the processes of cartilage growth and differentiation. Moreover, it appears to be a suitable model for pharmacological and toxicological studies related to rheumatic diseases relevant to humans.
Grigolo
B
B
Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA.
Roseti
L
L
Neri
S
S
Gobbi
P
P
Jensen
P
P
Major
E O
EO
Facchini
A
A
D015153:Blotting, Western; D002358:Cartilage, Articular / Q000473:pathology*; D002460:Cell Line / Q000473:pathology*; D019902:Chondrocytes / Q000473:pathology*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D008297:Male; D008855:Microscopy, Electron, Scanning; D008875:Middle Aged; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D012367:RNA, Viral