Int J Stem Cells.  2024 Feb;17(1):80-90. 10.15283/ijsc23101.

Nervonic Acid Inhibits Replicative Senescence of Human Wharton’s Jelly-Derived Mesenchymal Stem Cells

• Kim SJ
• Kwon S
• Chung S
• Lee EJ
• Park SE
• Choi SJ
• Oh SY
• Ryu GH
• Jeon HB
• Chang JW

• KMID: 2552020
• DOI: http://doi.org/10.15283/ijsc23101

Abstract

Cellular senescence causes cell cycle arrest and promotes permanent cessation of proliferation. Since the senescence of mesenchymal stem cells (MSCs) reduces proliferation and multipotency and increases immunogenicity, aged MSCs are not suitable for cell therapy. Therefore, it is important to inhibit cellular senescence in MSCs. It has recently been reported that metabolites can control aging diseases. Therefore, we aimed to identify novel metabolites that regulate the replicative senescence in MSCs. Using a fecal metabolites library, we identified nervonic acid (NA) as a candidate metabolite for replicative senescence regulation. In replicative senescent MSCs, NA reduced senescence-associated β-galactosidase positive cells, the expression of senescence-related genes, as well as increased stemness and adipogenesis. Moreover, in non-senescent MSCs, NA treatment delayed senescence caused by sequential subculture and promoted proliferation. We confirmed, for the first time, that NA delayed and inhibited cellular senescence. Considering optimal concentration, duration, and timing of drug treatment, NA is a novel potential metabolite that can be used in the development of technologies that regulate cellular senescence.

Keyword

Nervonic acid; Replicative senescence; Metabolites; Mesenchymal stem cells

Figure

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  Fig. 1 Induction of replicative senescence in Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs). (A) SA-β-galactosidase staining was performed during sequential subculture. The staining ratio was expressed as the number of stained cells to the total number of cells. Scale bar=250 μm. (B) The protein expression of p16 and p21, according to the passage, was examined by western blot. Data are presented as mean±SEM. The significance of the differences was assessed by one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001).

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  Fig. 2 Nervonic acid (NA) suppressed the replicative senescence of Wharton’s jelly-derived mesenchymal stem cells. (A) Experimental scheme for identification of senescence inhibition through NA after induction of replicative senescent mesenchymal stem cells (MSCs). (B) Cell viability was assessed after treatment with various NA concentrations. (C) β-Galactosidase staining (scale bar=250 μm) and (D) area measurement of cells was performed with or without NA. Data are presented as mean±SEM. The significance of the differences was assessed by one-way ANOVA (***p<0.001). NS-MSCs: non-senescent MSCs; RS-MSCs: replicative senescent MSCs; CCK-8: Cell Counting Kit-8; DMSO: dimethyl sulfoxide.

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  Fig. 3 Nervonic acid (NA) treatment significantly reduced the expression of senescence markers in replicative senescent mesenchymal stem cells (RS-MSCs). (A) mRNA expression of p16 and p21, (B) protein expression of p16 and p21, and (C) mRNA expression of senescence-associated secretory phenotype, including IL-6, MMP3, IGFBP3, IGFBP5, and IGFBP7, were measured in non-senescent MSCs (NS-MSCs), RS-MSCs, and NA-treated RS-MSCs. Data are presented as mean±SEM. The significance of the differences was assessed by one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001).

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  Fig. 4 Effects of nervonic acid (NA) on stemness, differentiation, and secretion in replicative senescent mesenchymal stem cells (RS-MSCs). (A) Expression of cell surface markers determined using flow cytometry analysis. The x-axis depicts fluorescence intensity, and the y-axis depicts cell count. (B) Osteogenesis was confirmed using Alizarin Red S staining (scale bar=250 μm), and adipogenesis was identified using Oil Red O staining (scale bar=50 μm) in non-senescent mesenchymal stem cells (NS-MSCs), RS-MSCs, and NA-treated RS-MSCs. (C) Heat map shows nine differentially expressed genes (fold change>1.5). (D) Violin plot was used to visualize the difference in expression levels and cell number of three genes in RS-MSCs and NA-treated RS-MSCs (fold change>2). (E) The differences in secretory proteins were confirmed using an antibody array. Heat map shows five differentially secreted proteins (fold change>1.5). Data are presented as mean±SEM. The significance of the differences was assessed by one-way ANOVA (*p<0.05, ***p<0.001).

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  Fig. 5 Nervonic acid (NA)-treated non-senescent mesenchymal stem cells (NS-MSCs) attenuated replicative senescence and increased proliferation in the continuous subculture. (A) Experimental schedule for measuring the effect of NA treatment in NS-MSCs. (B) The analysis of SA-β-galactosidase staining at each passage (scale bar=250 μm). (C) Relative mRNA expression of p16 and p21. (D) Relative mRNA expression of senescence-associated secretory phenotype. (E) Proliferation and doubling time confirmed by Cell Counting Kit-8 assay in NS-MSCs treated with dimethyl sulfoxide (DMSO) or NA at passage 10. Data are presented as mean±SEM and analyzed by two-tailed student’s t-test (*p<0.05, **p<0.01, ***p<0.001).

Reference

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