Development of a biologically immortalized equine stem cell line

Area of Science:

• Veterinary Medicine
• Regenerative Medicine
• Stem Cell Biology

Background:

• Equine bone repair often requires invasive surgery, increasing costs.
• Cell-based therapies using mesenchymal stromal cells (MSCs) show promise for equine musculoskeletal disorders.
• Equine bone marrow-derived MSCs (BMMSCs) have limited in vitro proliferation capacity.

Purpose of the Study:

• To develop a biologically immortalized equine stem cell line from bone marrow.
• To achieve unlimited in vitro proliferation and osteogenic differentiation of equine BMMSCs.
• To overcome the limitations of in vitro lifespan for clinical applications.

Main Methods:

• Equine BMMSCs were transfected with the human telomerase reverse transcriptase (hTERT) gene for immortalization.
• Stemness markers (CD90) and pluripotency genes (OCT-4, SOX2, NANOG) were analyzed.
• Osteogenic differentiation was induced and assessed via alkaline phosphatase, von Kossa staining, and gene expression (osteocalcin, Runx2, osterix).
• Telomerase activity was measured using the telomeric repeat amplification technique.

Main Results:

• Immortalized equine BMMSCs replicated in vitro up to passage 50 while retaining stem cell characteristics.
• Cells expressed key stemness markers and pluripotency genes.
• Successful differentiation into bone cells was confirmed by osteogenic staining and gene expression.
• Telomerase activity was successfully established in the immortalized cells.

Conclusions:

• Equine BMMSCs can be successfully immortalized using the hTERT gene.
• The immortalized cells maintain stem cell properties and osteogenic differentiation potential.
• This advancement offers a potential "off the shelf" source of MSCs for equine orthopedic surgery, revolutionizing treatment options.