Development of a Biologically Immortalized Equine Stem Cell Line

Introduction: Cell based treatment continues to be an expanding area for regenerative medical therapy in veterinary medicine including the field of bone healing. Stem cell lines that have a perpetual lifespan and maintain appropriate features may be useful as cell-based therapy ‘off the shelf’. The purpose of the study was to generate and evaluate in vitro a biologically immortalized stem cell line for potential use in promoting equine bone healing.

Materials and Methods: All animal study protocols were approved by the Animal Care Committee. Equine bone marrow-derived mesenchymal stem cells (BMMSCs) were immortalized by transfection with the pBABE-neo-hTERT retroviral vector plasmid. Immortalized cells in passages 2, 10, 20, 30, 40 and 50 were evaluated for stem cell characteristics including the ability to differentiate into osteoblasts.

Results: PCR analysis revealed the hTERT gene was successfully integrated into the genome DNA of BMMSCs. Immortalized cells maintained a spindle shaped morphology, and positively expressed surface markers CD90 and CD44 while lacking expression of CD45 and CD34, representative of MSCs. Bone nodules were produced in vitro by cells from all passages. RT PCR revealed that gene expression of bone-related genes RUNX2, Osterix and Osteocalcin was not significantly different from that of primary equine stem cells that were considered the gold standard.

Discussion/Conclusion: Immortalized equine BMMSCs maintained properties of stem cells and readily differentiated into osteoblasts. An equine immortalized stem cell line may be beneficial as a readily available source of MSCs for cell-based therapy for bone lesions.

Acknowledgement: Funding source: Atlantic Canada Opportunity Agency.