J Vet Sci.  2017 Mar;18(1):39-49. 10.4142/jvs.2017.18.1.39.

Inflammation affects the viability and plasticity of equine mesenchymal stem cells: possible implications in intra-articular treatments

• Barrachina L
• Remacha AR
• Romero A
• Vázquez F
• Albareda J
• Prades M
• Ranera B
• Zaragoza P
• Martín-Burriel I
• Rodellar C

• KMID: 2412586
• DOI: http://doi.org/10.4142/jvs.2017.18.1.39

Abstract

Mesenchymal stem cells (MSCs) are gaining relevance for treating equine joint injuries because of their ability to limit inflammation and stimulate regeneration. Because inflammation activates MSC immunoregulatory function, proinflammatory priming could improve MSC efficacy. However, inflammatory molecules present in synovial fluid or added to the culture medium might have deleterious effects on MSCs. Therefore, this study was conducted to investigate the effects of inflammatory synovial fluid and proinflammatory cytokines priming on viability and plasticity of equine MSCs. Equine bone marrow derived MSCs (eBM-MSCs) from three animals were cultured for 72 h in media supplemented with: 20% inflammatory synovial fluid (SF); 50 ng/mL IFN-γ and TNF-α (CK50); and 20 ng/mL IFN-γ and TNF-α (CK20). Proliferation assay and expression of proliferation and apoptosis-related genes showed that SF exposed-eBM-MSCs maintained their viability, whereas the viability of CK primed-eBM-MSCs was significantly impaired. Tri-lineage differentiation assay revealed that exposure to inflammatory synovial fluid did not alter eBM-MSCs differentiation potential; however, eBM-MSCs primed with cytokines did not display osteogenic, adipogenic or chondrogenic phenotype. The inflammatory synovial environment is well tolerated by eBM-MSCs, whereas cytokine priming negatively affects the viability and differentiation abilities of eBM-MSCs, which might limit their in vivo efficacy.

Keyword

horses; mesenchymal stromal cells; joint diseases; proinflammatory cytokines; synovial fluid

MeSH Terms

Animals
Horse Diseases/*immunology/metabolism
Horses
Inflammation/immunology/metabolism/*veterinary
Injections, Intra-Articular/veterinary
Interferon-gamma/*metabolism
Male
Mesenchymal Stromal Cells/*cytology
Synovial Fluid/*cytology
Tumor Necrosis Factor-alpha/*metabolism
Tumor Necrosis Factor-alpha
Interferon-gamma

Figure

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  Fig. 1 Proliferation related enzymes of control and inflammatory-exposed eBM-MSCs from Experiment 1 (A), 2 (B) and 3 (C) over 7 days. eBM-MSC proliferation potential was unaltered after SF exposure; however, this property was diminished after culturing cells with both CK20 and CK50 media. Proliferation was evaluated by MTT assay. The means ± SEM (n = 3) of the cell count are shown for each experiment at each time-point in both linear graphs (*p < 0.05) and a data table.

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  Fig. 2 eBM-MSCs expression of proliferation related enzymes is influenced by inflammatory stimulation. Gene expression of COX-1 and CyclinD2 in inflammatory exposed eBM-MSCs is expressed as the mean ± SEM (n = 3) fold increase or decrease compared to the corresponding control from each experiment (*p < 0.05).

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  Fig. 3 eBM-MSCs expression of certain genes encoding apoptosis-related molecules can be affected by proinflammatory cytokines. Gene expression of each gene in every experiment is represented as the mean ± SEM (n = 3) fold increase or decrease compared to the corresponding control from each experiment. (A) Expression of genes encoding molecules directly participating in the apoptosis mechanisms BAX, BCL-2, BCL-XL, CASP8, and HSP27. (B) Expression of genes encoding proinflammatory cytokines related to apoptosis TNF-α and IFN-γ. NE, no expression. *p < 0.05 and **p < 0.01.

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  Fig. 4 Staining for osteogenic, adipogenic and chondrogenic differentiation of control and inflammatory-stimulated eBM-MSCs from three experiments. (A) Alizarin red staining of eBM-MSCs cultured for 7 days in osteogenic differentiation medium from control and Experiment 1–3. (B) Oil red O staining of eBM-MSCs cultured for 15 days in adipogenic differentiation medium from control and Experiment 1–3. (C) Haematoxylin and Alcian blue staining of pellets cultured for 21 days in chondrogenic medium from control and Experiment 1–3. Scale bars = 500 µm (A and B), 2 µm (C). 10× (A), 4× (B), 20×(C).

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  Fig. 5 Effect of inflammatory conditions on gene expression of differentiation markers. Influence of the three inflammatory conditions tested on eBM-MSC expression of genes encoding osteogenesis (ALP and RUNX2) and adipogenesis (LPL and PPARγ) markers. The results are expressed as the mean ± SEM (n = 3) fold increase or decrease of differentiated control cells (CTRL.DIF), non-differentiated SF or CK50 exposed eBM-MSCs (SF.NON-DIF and CK50.NON-DIF) and differentiated SF or CK50 exposed eBM-MSCs (SF.DIF and CK50.DIF) over control unstimulated and non-differentiated cells (CTRL.NON-DIF). The white bar in all graphs (1 ± 0) represents the CTRL.NON-DIF fold change over itself relative to the fold increase or decrease under other conditions. (A and B) Gene expression of osteogenesis markers in cells from Experiment 1 (A) and Experiment 2 (B). (C and D) Gene expression of adipogenesis markers in cells from Experiment 1(C) and Experiment 2 (D).

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