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Utility of a Mouse Model of Osteoarthritis to Demonstrate Cartilage Protection by IFN γ -Primed Equine Mesenchymal Stem Cells

View Article: PubMed Central - PubMed

ABSTRACT

Objective: Mesenchymal stem cells isolated from adipose tissue (ASC) have been shown to influence the course of osteoarthritis (OA) in different animal models and are promising in veterinary medicine for horses involved in competitive sport. The aim of this study was to characterize equine ASCs (eASCs) and investigate the role of interferon-gamma (IFNγ)-priming on their therapeutic effect in a murine model of OA, which could be relevant to equine OA.

Methods: ASC were isolated from subcutaneous fat. Expression of specific markers was tested by cytometry and RT-qPCR. Differentiation potential was evaluated by histology and RT-qPCR. For functional assays, naïve or IFNγ-primed eASCs were cocultured with peripheral blood mononuclear cells or articular cartilage explants. Finally, the therapeutic effect of eASCs was tested in the model of collagenase-induced OA (CIOA) in mice.

Results: The immunosuppressive function of eASCs on equine T cell proliferation and their chondroprotective effect on equine cartilage explants were demonstrated in vitro. Both cartilage degradation and T cell activation were reduced by naïve and IFNγ-primed eASCs, but IFNγ-priming enhanced these functions. In CIOA, intra-articular injection of eASCs prevented articular cartilage from degradation and IFNγ-primed eASCs were more potent than naïve cells. This effect was related to the modulation of eASC secretome by IFNγ-priming.

Conclusion: IFNγ-priming of eASCs potentiated their antiproliferative and chondroprotective functions. We demonstrated that the immunocompetent mouse model of CIOA was relevant to test the therapeutic efficacy of xenogeneic eASCs for OA and confirmed that IFNγ-primed eASCs may have a therapeutic value for musculoskeletal diseases in veterinary medicine.

No MeSH data available.


Related in: MedlinePlus

Characterization of equine ASCs. (A) Representative photomicrograph of eASCs. (B) Percentage of eASCs positive for the indicated membrane markers. Results are percentage of positive cells and are expressed as mean ± SEM for three separate experiments. (C) Gene expression level of indicated markers in eASCs. Results are relative expression (2−ΔCT) and are expressed as mean ± SEM for three separate experiments. (D) Differentiation of eASCs: adipogenesis is characterized by the expression of peroxysome proliferator-activated receptor (PPAR)-γ at day 21 (d21) versus d0 and by the visualization of lipid droplets stained with HCS LipidTOX™ Green neutral lipid stain and counterstained with DAPI (lower panel) versus control proliferative medium (upper panel) at d21 (scale bar is 25 μm). Osteogenesis is characterized by the expression of Runx2 at d21 versus d0 and by Alizarin Red S positive staining in differentiation versus proliferative medium at d21 (scale bar is 100 μm). Chondrogenesis is characterized by the expression of Sox9, collagen IIA1 (col IIA1), and aggrecan (Agg) at d21 versus d0 and by Safranin O staining (lower panel) versus undifferentiated control (upper panel) at d21 (scale bar is 100 μm). Results are expressed as relative expression (2−ΔCT) and represented as mean ± SEM for three to six independent biological replicates. Data were analyzed using the Mann–Whitney test. *p < 0.05. ND: not detected.
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Figure 1: Characterization of equine ASCs. (A) Representative photomicrograph of eASCs. (B) Percentage of eASCs positive for the indicated membrane markers. Results are percentage of positive cells and are expressed as mean ± SEM for three separate experiments. (C) Gene expression level of indicated markers in eASCs. Results are relative expression (2−ΔCT) and are expressed as mean ± SEM for three separate experiments. (D) Differentiation of eASCs: adipogenesis is characterized by the expression of peroxysome proliferator-activated receptor (PPAR)-γ at day 21 (d21) versus d0 and by the visualization of lipid droplets stained with HCS LipidTOX™ Green neutral lipid stain and counterstained with DAPI (lower panel) versus control proliferative medium (upper panel) at d21 (scale bar is 25 μm). Osteogenesis is characterized by the expression of Runx2 at d21 versus d0 and by Alizarin Red S positive staining in differentiation versus proliferative medium at d21 (scale bar is 100 μm). Chondrogenesis is characterized by the expression of Sox9, collagen IIA1 (col IIA1), and aggrecan (Agg) at d21 versus d0 and by Safranin O staining (lower panel) versus undifferentiated control (upper panel) at d21 (scale bar is 100 μm). Results are expressed as relative expression (2−ΔCT) and represented as mean ± SEM for three to six independent biological replicates. Data were analyzed using the Mann–Whitney test. *p < 0.05. ND: not detected.

Mentions: Equine ASCs isolated from subcutaneous AT exhibited a classic fibroblastic morphology at passage 1 (Figure 1A). The majority of cells were positive for CD29, CD44, CD90, and ELA class I markers and negative for ELA class II as detected by flow cytometry (Figure 1B). In absence of available antibodies, expression of ASC markers CD34, CD73, and CD105 was confirmed at the mRNA level, while CD45 hematopoietic and von Willebrand factor (vWF) endothelial markers were not detected (Figure 1C). Under adipogenic conditions, eASCs stored triglycerides in lipid droplets as shown by HCS LipidTOX™ Green neutral lipid staining and the expression of the transcription factor regulating adipogenesis PPARγ, which was significantly increased after 21 days of differentiation compared to day 0 (Figure 1D). eASCs differentiated into osteoblasts, as shown by Alizarin Red S staining and increased Runx2 expression at day 21 (Figure 1D). They also differentiated toward chondrocytes, as suggested by the presence of sulfated GAG evidenced by Safranin O staining and demonstrated by an enhanced expression of Sox9, Aggrecan, and Collagen type II markers (Figure 1D). These results indicated that eASCs possess all the characteristics of mesenchymal stem cells (33).


Utility of a Mouse Model of Osteoarthritis to Demonstrate Cartilage Protection by IFN γ -Primed Equine Mesenchymal Stem Cells
Characterization of equine ASCs. (A) Representative photomicrograph of eASCs. (B) Percentage of eASCs positive for the indicated membrane markers. Results are percentage of positive cells and are expressed as mean ± SEM for three separate experiments. (C) Gene expression level of indicated markers in eASCs. Results are relative expression (2−ΔCT) and are expressed as mean ± SEM for three separate experiments. (D) Differentiation of eASCs: adipogenesis is characterized by the expression of peroxysome proliferator-activated receptor (PPAR)-γ at day 21 (d21) versus d0 and by the visualization of lipid droplets stained with HCS LipidTOX™ Green neutral lipid stain and counterstained with DAPI (lower panel) versus control proliferative medium (upper panel) at d21 (scale bar is 25 μm). Osteogenesis is characterized by the expression of Runx2 at d21 versus d0 and by Alizarin Red S positive staining in differentiation versus proliferative medium at d21 (scale bar is 100 μm). Chondrogenesis is characterized by the expression of Sox9, collagen IIA1 (col IIA1), and aggrecan (Agg) at d21 versus d0 and by Safranin O staining (lower panel) versus undifferentiated control (upper panel) at d21 (scale bar is 100 μm). Results are expressed as relative expression (2−ΔCT) and represented as mean ± SEM for three to six independent biological replicates. Data were analyzed using the Mann–Whitney test. *p < 0.05. ND: not detected.
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Figure 1: Characterization of equine ASCs. (A) Representative photomicrograph of eASCs. (B) Percentage of eASCs positive for the indicated membrane markers. Results are percentage of positive cells and are expressed as mean ± SEM for three separate experiments. (C) Gene expression level of indicated markers in eASCs. Results are relative expression (2−ΔCT) and are expressed as mean ± SEM for three separate experiments. (D) Differentiation of eASCs: adipogenesis is characterized by the expression of peroxysome proliferator-activated receptor (PPAR)-γ at day 21 (d21) versus d0 and by the visualization of lipid droplets stained with HCS LipidTOX™ Green neutral lipid stain and counterstained with DAPI (lower panel) versus control proliferative medium (upper panel) at d21 (scale bar is 25 μm). Osteogenesis is characterized by the expression of Runx2 at d21 versus d0 and by Alizarin Red S positive staining in differentiation versus proliferative medium at d21 (scale bar is 100 μm). Chondrogenesis is characterized by the expression of Sox9, collagen IIA1 (col IIA1), and aggrecan (Agg) at d21 versus d0 and by Safranin O staining (lower panel) versus undifferentiated control (upper panel) at d21 (scale bar is 100 μm). Results are expressed as relative expression (2−ΔCT) and represented as mean ± SEM for three to six independent biological replicates. Data were analyzed using the Mann–Whitney test. *p < 0.05. ND: not detected.
Mentions: Equine ASCs isolated from subcutaneous AT exhibited a classic fibroblastic morphology at passage 1 (Figure 1A). The majority of cells were positive for CD29, CD44, CD90, and ELA class I markers and negative for ELA class II as detected by flow cytometry (Figure 1B). In absence of available antibodies, expression of ASC markers CD34, CD73, and CD105 was confirmed at the mRNA level, while CD45 hematopoietic and von Willebrand factor (vWF) endothelial markers were not detected (Figure 1C). Under adipogenic conditions, eASCs stored triglycerides in lipid droplets as shown by HCS LipidTOX™ Green neutral lipid staining and the expression of the transcription factor regulating adipogenesis PPARγ, which was significantly increased after 21 days of differentiation compared to day 0 (Figure 1D). eASCs differentiated into osteoblasts, as shown by Alizarin Red S staining and increased Runx2 expression at day 21 (Figure 1D). They also differentiated toward chondrocytes, as suggested by the presence of sulfated GAG evidenced by Safranin O staining and demonstrated by an enhanced expression of Sox9, Aggrecan, and Collagen type II markers (Figure 1D). These results indicated that eASCs possess all the characteristics of mesenchymal stem cells (33).

View Article: PubMed Central - PubMed

ABSTRACT

Objective: Mesenchymal stem cells isolated from adipose tissue (ASC) have been shown to influence the course of osteoarthritis (OA) in different animal models and are promising in veterinary medicine for horses involved in competitive sport. The aim of this study was to characterize equine ASCs (eASCs) and investigate the role of interferon-gamma (IFN&gamma;)-priming on their therapeutic effect in a murine model of OA, which could be relevant to equine OA.

Methods: ASC were isolated from subcutaneous fat. Expression of specific markers was tested by cytometry and RT-qPCR. Differentiation potential was evaluated by histology and RT-qPCR. For functional assays, na&iuml;ve or IFN&gamma;-primed eASCs were cocultured with peripheral blood mononuclear cells or articular cartilage explants. Finally, the therapeutic effect of eASCs was tested in the model of collagenase-induced OA (CIOA) in mice.

Results: The immunosuppressive function of eASCs on equine T cell proliferation and their chondroprotective effect on equine cartilage explants were demonstrated in vitro. Both cartilage degradation and T cell activation were reduced by na&iuml;ve and IFN&gamma;-primed eASCs, but IFN&gamma;-priming enhanced these functions. In CIOA, intra-articular injection of eASCs prevented articular cartilage from degradation and IFN&gamma;-primed eASCs were more potent than na&iuml;ve cells. This effect was related to the modulation of eASC secretome by IFN&gamma;-priming.

Conclusion: IFN&gamma;-priming of eASCs potentiated their antiproliferative and chondroprotective functions. We demonstrated that the immunocompetent mouse model of CIOA was relevant to test the therapeutic efficacy of xenogeneic eASCs for OA and confirmed that IFN&gamma;-primed eASCs may have a therapeutic value for musculoskeletal diseases in veterinary medicine.

No MeSH data available.


Related in: MedlinePlus