Generation and characterization of an immortalized equine osteoblast cell line

Bone is a dynamic tissue that is maintained through the co-ordination of bone resorption and bone formation. An imbalance of these processes can lead to bone disease. In vitro studies of osteoblasts can help to understand bone formation, but primary cells have a limited lifespan in culture. Herein, we report the successful generation of equine immortalized osteoblasts through the stable overexpression of human telomerase reverse transcriptase (hTERT) and Simian virus 40 (SV40) large T-antigen in osteoblasts isolated from trabecular bone taken from the third metacarpal of a two-year-old Thoroughbred horse. Primary osteoblasts displayed limited proliferation in culture, a decrease in the expression of osteogenic-associated genes and alkaline phosphatase activity with increasing passage and a failure to survive and produce a mineralised matrix after 21 days of osteogenic culture at high passage. In contrast, immortalized equine osteoblasts could be expanded for over 50 passages while retaining osteogenic gene expression, high alkaline phosphatase activity, a normal karyotype and the ability to produce a mineralised matrix after osteogenic culture. The immortalized equine osteoblasts therefore constitute a useful in vitro model to study equine bone formation. • Primary equine osteoblasts have limited proliferation in vitro , exhibit changes in gene expression and lose their ability to produce a mineralised matrix with increasing passage. • Stable co-expression of hTERT and SV40 large T-antigen was used to successfully generate immortalized equine osteoblasts. • Immortalized equine osteoblasts can be passaged extensively while retaining osteogenic gene expression, a normal karyotype and the ability to produce a mineralised matrix.