Effect of Cytokine Priming on Equine Stem Cells

Equine bone marrow—derived mesenchymal stem cells (eBM-MSCs) demonstrate reduced proliferation and differentiation when primed with the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (INF-γ), according to a study recently published in the Journal of Veterinary Science.

Joint pathologies such as osteoarthritis are painful and complex conditions that commonly affect horses and people. The release of proinflammatory mediators into the synovial fluid plays an important role in osteoarthritis progression, which ultimately results in cartilage degradation.

Current osteoarthritis treatments focus on decreasing pain and inflammation; ideal treatment goals, the researchers wrote, are the prevention of progressive cartilage loss and stimulation of tissue repair.

MSCs have become increasingly important in joint pathology treatment because of their immunoregulatory and regenerative abilities. This immunoregulation is reportedly regulated by proinflammatory cytokines like TNF-α and INF-γ, particularly when they are in combination. Given the influence of proinflammatory cytokines on MSC function, it is possible that cytokine priming could enhance MSCs’ therapeutic potential, according to one previous study. To date, the effect of proinflammatory cytokines on equine MSC proliferation and differentiation are unknown.

Study Design

Researchers used 4 geldings with orthopedic injuries. From 3 geldings, sternal bone marrow was collected to isolate eBM-MSCs; synovial fluid was collected from the fourth gelding, which had aseptic synovitis in a tarso-crural joint. Researchers then performed cell culture during which eBM-MSCs were exposed to 3 inflammatory conditions for 72 hours:

• Condition #1 (SF): Synovial fluid
• Condition #2 (CK50): 50 ng/mL each of TNF-α and INF-γ
• Condition #3 (CK20): 20 ng/mL each of TNF-α and INF-γ

Next, researchers performed proliferation and differentiation assays and used polymerase chain reaction (PCR) to measure expression levels of genes associated with proliferation, apoptosis, and 2 forms of differentiation (osteogenesis, adipogenesis).

Results

Proliferation

Cytokine exposure, regardless of concentration, significantly reduced proliferation compared with control cells. For example, cell doubling time was significantly longer in cytokine-exposed eBM-MSCs than in control cells. Also, expression of the proliferation-associated genes COX-1 and cyclin D2 was significantly downregulated in both cytokine conditions.

Exposure to SF did not significantly affect proliferation or expression levels of proliferation-associated genes.

Apoptosis

Expression of apoptosis-associated genes varied according to experimental condition:

• SF: No significant changes
• CK20 and CK50: Significant BAX downregulation, nonsignificant CASP8 upregulation
• Only CK20: Significant BCL-2 downregulation, significant IFN-γ upregulation
• Only CK50: Significant heat shock protein 27 (HSP-27) downregulation, significant TNF-α upregulation

BAX and CASP8 genes are proapoptotic, and BCL-2 and HSP-27 genes are antiapoptotic.

These results suggest the role of apoptotic pathways, particularly the apoptosis death receptor pathway, in the reduced viability of cytokine-exposed eBM-MSCs, the researchers noted.

Differentiation

To evaluate tri-lineage differentiation—osteogenic, adipogenic, chondrogenic—researchers first cultured the eBM-MSCs in differentiation-inducing media, then stained the cells. Next, they measured expression levels of osteogenic- and adipogenic-associated genes.

Cell culture revealed that only SF-exposed eBM-MSCs demonstrated tri-lineage differentiation potential. PCR results indicated that, compared with control cells, cytokine-exposed eBM-MSCs demonstrated significant downregulation of the osteogenic- and adipogenic-associated genes. Given these findings, the researchers determined that exposure to high levels of proinflammatory cytokines inhibited tri-lineage eBM-MSC differentiation.

Gene expression levels did not significantly differ between SF-exposed eBM-MSCs and control cells, suggesting that moderate inflammation due to SF exposure did not affect differentiation.

Conclusions

Taken together, these study results indicate that cytokine priming can impair, rather than enhance, the therapeutic potential of eBM-MSCs. Such findings have important implications for cellular-based treatment of joint pathologies.

Dr. JoAnna Pendergrass received her Doctor of Veterinary Medicine degree from the Virginia-Maryland College of Veterinary Medicine. Following veterinary school, she completed a postdoctoral fellowship at Emory University’s Yerkes National Primate Research Center. Dr. Pendergrass is the founder and owner of JPen Communications, a medical communications company.