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.