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. 2023 May 25:10:1203012.
doi: 10.3389/fvets.2023.1203012. eCollection 2023.

Feline umbilical cord-derived mesenchymal stem cells: isolation, identification, and antioxidative stress role through NF-κB signaling pathway

Zhu-Hui Zhai  1 , Jun Li  1 , Zhao You  1 , Yang Cai  1 , Jie Yang  2 , Jie An  2 , Di-Peng Zhao  1 , He-Jie Wang  1 , Min-Min Dou  1 , Rong Du  1 , Jian Qin  1   2   3
Affiliations

Feline umbilical cord-derived mesenchymal stem cells: isolation, identification, and antioxidative stress role through NF-κB signaling pathway

Zhu-Hui Zhai et al. Front Vet Sci. .

Abstract

At present, the differentiation potential and antioxidant activity of feline umbilical cord-derived mesenchymal stem cells (UC-MSCs) have not been clearly studied. In this study, feline UC-MSCs were isolated by tissue adhesion method, identified by flow cytometry detection of cell surface markers (CD44, CD90, CD34, and CD45), and induced differentiation toward osteogenesis and adipogenesis in vitro. Furthermore, the oxidative stress model was established with hydrogen peroxide (H2O2) (100 μM, 300 μM, 500 μM, 700 μM, and 900 μM). The antioxidant properties of feline UC-MSCs and feline fibroblasts were compared by morphological observation, ROS detection, cell viability via CCK-8 assay, as well as oxidative and antioxidative parameters via ELISA. The mRNA expression of genes related to NF-κB pathway was detected via quantitative real-time polymerase chain reaction, while the levels of NF-κB signaling cascade-related proteins were determined via Western Blot. The results showed that feline UC-MSCs highly expressed CD44 and CD90, while negative for CD34 and CD45 expression. Feline UC-MSCs cultured under osteogenic and adipogenic conditions showed good differentiation capacity. After being exposed to different concentrations of H2O2 for eight hours, feline UC-MSCs exhibited the significantly higher survival rate than feline fibroblasts. A certain concentration of H2O2 could up-regulate the activities of SOD2 and GSH-Px in feline UC-MSCs. The expression levels of p50, MnSOD, and FHC mRNA in feline UC-MSCs stimulated by 300 μM and 500 μM H2O2 significantly increased compared with the control group. Furthermore, it was observed that 500 μM H2O2 significantly enhanced the protein levels of p-IκB, IκB, p-p50, p50, MnSOD, and FHC, which could be reversed by BAY 11-7,082, a NF-κB signaling pathway inhibitor. In conclusion, it was confirmed that feline UC-MSCs, with good osteogenesis and adipogenesis abilities, had better antioxidant property which might be related to NF-κB signaling pathway. This study lays a foundation for the further application of feline UC-MSCs in treating the various inflammatory and oxidative injury diseases of pets.

Keywords: NF-κB signaling pathway; feline; fibroblasts; oxidative stress; umbilical cord-derived mesenchymal stem cells.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Morphological characteristics of feline UC-MSCs isolated and cultured with the tissue adhesion method. Feline UC-MSCs migrated from adherent tissue on the tenth day (A). Morphology of feline UC-MSCs after passaging for 48 h (B). Morphological characteristics of the third-generation feline UC-MSCs (C). Scale bar: 200 μm.
Figure 2
Figure 2
Analysis by flow cytometry of the expression levels of cell surface markers CD44, CD90, CD34, and CD45 in feline UC-MSCs. The purple histograms represent isotype controls and red histograms represent cell surface marker staining.
Figure 3
Figure 3
Osteogenic differentiation of three types of cells. No osteogenic cells were detected in C3H10T1/2 (A), feline UC-MSCs (D), and feline fibroblasts (G) under the uninduced control treatment. Morphological changes were detected in C3H10T1/2 (B), feline UC-MSCs (E), and feline fibroblasts (H) during osteogenesis. Images of Alizarin red staining in C3H10T1/2 (C), feline UC-MSCs (F), and feline fibroblasts (I). Scale bar: 200 μm.
Figure 4
Figure 4
Adipogenic differentiation of three types of cells. No adipogenic cells were detected in C3H10T1/2 (A), feline UC-MSCs (D), and feline fibroblasts (G) under the uninduced control treatment. Morphological changes were detected in C3H10T1/2 (B), feline UC-MSCs (E), and feline fibroblasts (H) during adipogenesis. Images of Oil red O staining in C3H10T1/2 (C), and feline UC-MSCs (F), and negative results of adipogenic differentiation in feline fibroblasts (I). Scale bar: 100 μm.
Figure 5
Figure 5
Optimization for osteogenic and adipogenic differentiation of feline UC-MSCs. Comparison of Alizarin red staining areas in feline UC-MSCs under previous conditions (A) and optimized conditions (B). Comparison of calcium nodules in feline UC-MSCs under previous conditions (C) and optimized conditions (D). Comparison of the accumulation of lipid droplets in feline UC-MSCs under previous conditions (E) and optimized conditions (F). Comparison of lipid droplets staining in feline UC-MSCs under previous conditions (G) and optimized conditions (H). Scale bar: 100 μm, 200 μm.
Figure 6
Figure 6
Effects of different concentrations of H2O2 on cell morphology in feline UC-MSCs and feline fibroblasts. Scale bar: 200 μm.
Figure 7
Figure 7
Effects of different concentrations of H2O2 on cell viability in feline UC-MSCs and feline fibroblasts. All data are presented as mean ± SD and n = 4 in each group. **p < 0.01.
Figure 8
Figure 8
Effects of different concentrations of H2O2 on ROS production in feline UC-MSCs and feline fibroblasts. (A) Observation of ROS fluorescence. Scale bar: 200 μm. (B) Quantitative analysis of the mean fluorescence intensity (MFI). All data are presented as mean ± SD and n = 3 in each group. *p < 0.05, **p < 0.01.
Figure 9
Figure 9
Effects of different concentrations of H2O2 on intracellular MDA, CAT, SOD2, and GSH-Px levels in feline UC-MSCs and feline fibroblasts. All data are presented as mean ± SD and n = 3 in each group. *p < 0.05, **p < 0.01.
Figure 10
Figure 10
Morphological changes of feline UC-MSCs treated by H2O2 alone or together with BAY 11-7,082. Scale bar: 200 μm.
Figure 11
Figure 11
Effects of H2O2 alone or together with BAY 11-7,082 on mRNA expression of NF-κB pathway-related genes including IκB, p50, MnSOD, and FHC in feline UC-MSCs. All data are presented as mean ± SD and n = 6 in each group. *p < 0.05, **p < 0.01.
Figure 12
Figure 12
Effects of H2O2 alone or together with BAY 11-7,082 on NF-κB pathway in feline UC-MSCs. (A) Western blot analysis of p-IκB, IκB, p-p50, p50, MnSOD, FHC, and GAPDH. (B) Quantitative analysis of p-IκB, IκB, p-IκB/IκB, p-p50, p50, p-p50/p50, MnSOD, and FHC relative levels. All data are presented as mean ± SD and n = 3 in each group. *p < 0.05, **p < 0.01.
Figure 13
Figure 13
Schematic diagram illustrating the isolation and identification of feline umbilical cord-derived mesenchymal stem cells (UC-MSCs), and antioxidant mechanism by NF-κB signaling pathway under H2O2 stimulation. This graphic was created with BioRender.com.
See this image and copyright information in PMC

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