Cloning and characterization of human vitreous tissue-derived cells.
OBJECTIVE
Previously, we established a porcine vitreous tissue-derived hyalocyte cell line (PH5) and investigated the regulation of hyaluronan synthesis in these cells by cytokines. The objective of the current study was to establish human vitreous tissue-derived cells and to compare their characteristics with those of PH5 cells.


METHODS
Human vitreous specimens from two patients were cultured in the presence of 10% foetal bovine serum and immortalized by infection with human papilloma virus 16 genes E6 and E7. We used reverse transcription polymerase chain reaction (RT-PCR) to analyse and compare the expression profiles for several genes in the human vitreous tissue-derived cells and PH5 cells. To investigate the regulation of hyaluronan production in response to cytokine stimulation, the expression of hyaluronan synthase isoforms was examined using RT-PCR, and hyaluronan production was measured using enzyme-linked immunosorbent assay (ELISA).


RESULTS
Two types of cells, HV64 and HV65, were derived from human vitreous tissue. The HV64 and HV65 cell-doubling times were 58 r and 76 hr, respectively. The cells expressed messenger RNA (mRNAs) encoding collagen type I α1 (COL1A1), collagen type II α1 (COL2A1), CD11b, CD14, CD68, CD204 and CD206 but did not express mRNA for glial fibrillary acidic protein (GFAP). Cytokine stimulation did not induce the expression of hyaluronan synthase mRNA or the production of hyaluronan. In contrast, mRNAs for GFAP and hyaluronan synthase-2 were expressed in the porcine PH5 cells, and treatment with transforming growth factor-β1 and/or platelet-derived growth factor-BB induced the production of hyaluronan in PH5 cells.


CONCLUSIONS
The new human vitreous tissue-derived cells have macrophage-like characteristics and are different from our previously developed porcine hyalocyte cells. These human vitreous tissue-derived cells might be useful for studies of human intraocular diseases.
Kashiwagi
Yoshiko
Y
Department of Ocular Cellar Engineering, Yamagata University Hospital, Japan. kasiwagi@med.id.yamagata-u.ac.jp
Nishitsuka
Koichi
K
Takamura
Hiroshi
H
Yamamoto
Teiko
T
Yamashita
Hidetoshi
H
D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D000077214:Becaplermin; D018929:Cell Culture Techniques; D002455:Cell Division; D002999:Clone Cells / Q000187:drug effects; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D024043:Collagen Type II / Q000235:genetics; D003470:Culture Media / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D006820:Hyaluronic Acid / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D014822:Vitreous Body / Q000166:cytology* / Q000378:metabolism*
D015703:Antigens, CD; C499177:COL2A1 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D024043:Collagen Type II; D003470:Culture Media; D017931:DNA Primers; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D000077214:Becaplermin; D006820:Hyaluronic Acid