Cloning and characterization of cell strains derived from human corneal stroma and sclera.
OBJECTIVE
To establish human corneal stroma- and sclera-derived cells as models for studying diseases of the anterior segment of the eye.


METHODS
Using a recombinant retrovirus system, we transfected human papilloma virus 16 E6 and E7 (HPV16 E6/E7) into human corneal stroma- and sclera-derived cells. The primary cells and established cell strains were characterized by assessing the mRNA expression of collagen, matrix metalloproteinase, and tissue inhibitor of metalloproteinase by reverse transcription-polymerase chain reaction. We also examined the effects of inflammatory cytokines on hyaluronan synthase expression and hyaluronan products.


RESULTS
Both a corneal stroma-derived cell strain, Cs3, and a sclera-derived cell strain, Sc1, were obtained, and both cell strains could be passaged up to 25 times. The mRNA expression pattern observed in the primary cells was identical to that observed in the cell strains. Hyaluronan synthase 1 and 2 mRNAs were increased by transforming growth factor beta and platelet-derived growth factor BB. Significant differences were observed between the hyaluronan products with and without cytokine treatment.


CONCLUSIONS
Cell strains derived from corneal stroma and sclera fibroblast cells can be established using HPV16 E6/E7 immortalized genes of the same origin. The phenotypic cell characteristics did not change after transfection, immortalization, or successive passages in culture.
Department of Ocular Cellar Engineering, Yamagata University, Yamagata, Japan. kasiwagi@med.id.yamagata-u.ac.jp
Yoshiko
Y
Kashiwagi
Koichi
K
Nishitsuka
Hiroyuki
H
Namba
Masaru
M
Kamiryo
Hiroshi
H
Takamura
Hidetoshi
H
Yamashita
D000368:Aged; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003094:Collagen / Q000235:genetics; D003319:Corneal Stroma / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D014453:Glucuronosyltransferase / Q000235:genetics; D006801:Humans; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics; D009856:Oncogene Proteins, Viral / Q000235:genetics; D050725:Papillomavirus E7 Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012590:Sclera / Q000166:cytology* / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases / Q000235:genetics; D014162:Transfection
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D019714:Tissue Inhibitor of Metalloproteinases; C059731:oncogene protein E7, Human papillomavirus type 16; D006820:Hyaluronic Acid; D003094:Collagen; D014453:Glucuronosyltransferase; C523794:HAS1 protein, human; C101754:HAS2 protein, human; D000076002:Hyaluronan Synthases; D020782:Matrix Metalloproteinases