Establishment and investigation of tendon-derived cell lines immortalized by the human telomerase reverse transcriptase gene.

Development of the musculosceletal system requires coordinated
formation of distinct types of tissues, including bone, cartilage,
muscle and tendon. Compared to muscle, cartilage and bone,
molecular, cellular and developmental biology of tendon have not
been well understood due to the lack of tendon cell lines. In
addition tissue engineering of tendon is hampered by the rather
difficult retrieval of tenocytes and their senescence-associated
growth arrest during culture. Therefore the purpose of this study
was to establish and characterize human tendon cell lines. Two
tendon cell lines (HTD2 hTERT and HTD5 hTERT) were established
using lentiviral gene transfer to ectopically express hTERT. The
cell lines stably expressed hTERT on RNA and protein level.
Untransduced cultured tenocytes show only a background level of
telomerase activity, but it was significantly increased by hTERT
transduction. Ectopic expression of hTERT led to an extended
lifespan and prevented senescence while the cells kept their
typical spindle-shaped morphology of young primary human tenocytes.
Moreover, in comparison to untransduced tenocytes the cells
possessed significantly lesser β-galactosidase activity indicating
that they had not entered a senescent state. Throughout the entire
culturing period the hTERT transduced tenocytes expressed
tendon-related genes such as those encoding collagen I, collagen
III, Tenascin C, EphA4, Eya1, scleraxis, Six and COMP. Using soft
agar assay, no malignant transformation was shown by the hTERT
expressing tenocytes. In conclusion, extending the lifespan of
human tenocytes by ectopic expression of hTERT using lentiviral
gene transfer may be an attractive and safe way to generate cells
allowing extensive molecular characterization and development of
novel tissue engineering applications.