MK/T-1, an immortalized fibroblast cell line derived using cultures of mouse corneal stroma

PURPOSE: Immortalized cell lines representing fibroblast cells from corneal stroma would facilitate studies of corneal cell biology and injury respons e. METHODS: Primary cultures of cells derived from mouse corneal stroma were t ransfected with a human telomerase reverse transcriptase (hTERT) expression construct to maximize chances of cellular immortalization. A resulting cel l line was analyzed for telomerase activity, cell growth characteristics, s enescence and gene expression patterns. Specific responses to transforming growth factor beta (TGF-beta) were also analyzed. RESULTS: An immortalized cell line was derived and was named MK/T-1. MK/T-1 cells show no signs of cellular senescence or transformation at over 100 p assages. Telomerase activity was significantly higher in MK/T-1 cells as co mpared to the parental cell cultures. However, relative telomere length (RT L) in the MK/T-1 and parental cells was not significantly different. Senesc ence associated beta -galactosidase (SA-beta -Gal) activity was not detecte d in late passage MK/T-1 cells while the parental cells had already upregul ated SA-beta -Gal at high levels by passage 9. The MK/T-1 cells express vim entin, tubulin, lumican, mimecan, decorin and collagen I, but not keratocan . Exposure of the MK/T-1 cells to TGF-beta induces the expression of smooth muscle alpha -actin (ASMA), the activation of MAP Kinase (p38-MAPK) and mo rphological changes consistent with cytoskeletal reorganization. CONCLUSIONS: MK/T-1 cells represent an immortalized fibroblast cell line de rived using cultures from corneal stroma cell preparations. Expression of h TERT may contribute to immortalization of the MK/T-1 cells by a mechanism o ther than increases in RTL. MK/T-1 cells may be a useful model in which to study the responses of corneal fibroblast cells to cytokines and other dive rse environmental factors in vitro.