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, 11 (1), 4

Age-dependent Impairment of Adipose-Derived Stem Cells Isolated From Horses

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Age-dependent Impairment of Adipose-Derived Stem Cells Isolated From Horses

Michalina Alicka et al. Stem Cell Res Ther.

Abstract

Background: Progressive loss of cell functionality caused by an age-related impairment in cell metabolism concerns not only mature specialized cells but also its progenitors, which significantly reduces their regenerative potential. Adipose-derived stem cells (ASCs) are most commonly used in veterinary medicine as an alternative treatment option in ligaments and cartilage injuries, especially in case of high-value sport horses. Therefore, the main aim of this study was to identify the molecular alternations in ASCs derived from three age-matched horse groups: young (< 5), middle-aged (5-15), and old (> 15 years old).

Methods: ASCs were isolated from three age-matched horse groups using an enzymatic method. Molecular changes were assessed using qRT-PCR, ELISA and western blot methods, flow cytometry-based system, and confocal and scanning electron microscopy.

Results: Our findings showed that ASCs derived from the middle-aged and old groups exhibited a typical senescence phenotype, such as increased percentage of G1/G0-arrested cells, binucleation, enhanced β-galactosidase activity, and accumulation of γH2AX foci, as well as a reduction in cell proliferation. Moreover, aged ASCs were characterized by increased gene expression of pro-inflammatory cytokines and miRNAs (interleukin 8 (IL-8), IL-1β, tumor necrosis factor α (TNF-α), miR-203b-5p, and miR-16-5p), as well as apoptosis markers (p21, p53, caspase-3, caspase-9). In addition, our study revealed that the protein level of mitofusin 1 (MFN1) markedly decreased with increasing age. Aged ASCs also displayed a reduction in mRNA levels of genes involved in stem cell homeostasis and homing, like TET-3, TET-3 (TET family), and C-X-C chemokine receptor type 4 (CXCR4), as well as protein expression of DNA methyltransferase (DNMT1) and octamer transcription factor 3/4 (Oct 3/4). Furthermore, we observed a higher splicing ratio of XBP1 (X-box binding protein 1) mRNA, indicating elevated inositol-requiring enzyme 1 (IRE-1) activity and, consequently, increased endoplasmic reticulum (ER) stress. We also observed reduced levels of glucose transporter 4 (GLUT-4) and insulin receptor (INSR) which indicated impaired insulin sensitivity.

Conclusions: Obtained data suggest that ASCs derived from horses older than 5 years old exhibited several molecular alternations which markedly limit their regenerative capacity. The results provide valuable information that allows for a better understanding of the molecular events occurring in ASCs in the course of aging and may help to identify new potential drug targets to restore their regenerative potential.

Keywords: Aging; Endoplasmic reticulum stress; Equine adipose-derived mesenchymal stem cells; Insulin resistance; Pro-inflammatory cytokines.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Analysis of proliferation of ASCs for the three age donor groups (0–5 years, 5–15 years, > 15 years). a Cell proliferation was assessed, and b PDT was quantified using resazurin-based TOX-8 assay. The red asterisk refers to the comparison of the < 5 and 5–15 groups to > 15, whereas the green asterisk refers to the comparison of the < 5 and < 15 groups to 5–15. c Relative expression of proliferation-associated miRNAs was detected by the qRT-PCR method. d Representative images of immunofluorescence staining for Ki-67 (red) and nuclei (blue). Ki-67 expression in cells was presented as Ki-67-positive cell percentage. A positive colocalization of Ki-67 in the nucleus is indicated by pink signals (merge) due to overlap of Atto 590 (Ki-67) and DAPI staining. Results expressed as mean ± SD. Statistical significance is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA (and non-parametric) test
Fig. 2
Fig. 2
Morphological alternations caused by aging in ASCs. a Representative images of senescence-associated SA-β-gal assay. Black arrows indicate SA-β-gal-positive cells (blue), whereas white arrows show binucleated cells. b Quantification of SA-β-gal-positive cells (blue). c Representative images of Phalloidin Atto 590 staining for F-actin. d SEM images of ASCs isolated from the three age groups (ASC< 5, ASC5–15, ASC15< ). e Mean diameter of the cell nuclei measured on the basis of SEM images. Results expressed as mean ± SD. Statistical significance is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA (and non-parametric) test
Fig. 3
Fig. 3
Age-associated changes in the cell cycle distribution (a, b), protein level of Oct 3/4 (c), mRNA level of CXCR4 (d), accumulation of γH2AX (c), expression of DNMT1 (f), and transcript levels of TET-2 and TET-3 (g, h). Cell cycle distribution was determined using a flow cytometry-based system Muse™ Cell Analyzer. The levels of Oct 3/4, γH2AX, and DNMT1 were estimated with the western blot method. Relative quantity of proteins was estimated using Image Lab software after normalization with β-actin (loading control). Representative blots are shown on the bottom panel. Alternations in the mRNA expressions of CXCR4 (c), TET-2 (g), and TET-3 (h) were determined using qRT-PCR. Results expressed as mean ± SD. Statistical significance is indicated with asterisks:*p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA (and non-parametric) test
Fig. 4
Fig. 4
Relative expressions of apoptosis-related genes in ASCs of the three age groups. Transcript levels of Casp-3 (a), Casp-9 (b), p53 (c), p21 (d), BAX (e), and BCl-2 (f) were estimated using the qRT-PCR method. g BAX/BCl-2 ratio was determined using the relative expression values of both BAX and BCl-2. h Protein levels of p53 were determined with ELISA method. Results expressed as mean ± SD. Statistical significance is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA (and non-parametric) test
Fig. 5
Fig. 5
Age-related decline in oxidative stress of ASCs. a Evaluation of ROS accumulation in ASCs using Muse™ Cell Analyzer. b Percentage of ROS(−) live cells and ROS(+) cells. c Catalase activity was detected by Catalase (CAT) Assay Kit. d, e Transcript levels of SOD 1 (Cu/Zn SOD) and SOD2 (Mn SOD) were determined using the qRT-PCR method. Results expressed as mean ± SD. Statistical significance is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA (and non-parametric) test
Fig. 6
Fig. 6
Age-related changes in the mitochondria network. a Mitochondria visualization using MitoRed staining (red). Cells’ nuclei were counterstained with DAPI (blue). b Mitochondria were analyzed morphometrically to evaluate the mitochondria area and mitochondria volume using Bitplane Imaris software. Results expressed as mean ± SD. Statistical significance is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA (and non-parametric) test
Fig. 7
Fig. 7
Analysis of mitochondria dynamics in ASCs using qRT-PCR and western blot techniques. Relative mRNA expression of MFN1 (a), FIS1 (b), PINK1 (c), and PARKIN (d) determined by qRT-PCR. e, f Protein levels of MFN1 and MFF were estimated using western blot. Relative quantity was determined using Image Lab software after normalization with β-actin as a loading control. Representative blots are shown in the bottom panels. Results expressed as mean ± SD. Statistical significance is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA (and non-parametric) test
Fig. 8
Fig. 8
Changes in the expressions of pro- (a) and anti-inflammatory (b) cytokines. Relative expressions of inflammation-associated miRNA (c, d). Each analysis was performed using the qRT-PCR method. Results expressed as mean ± SD. Statistical significance is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA (and non-parametric) test
Fig. 9
Fig. 9
Age-associated alternations in the expressions of insulin resistance-related markers. Relative transcript levels of IRS (a), SREBP-1C (b), SIRT1 (c), GLUT-4 (d), and FOXO1 (e) were determined using the qRT-PCR technique. Protein contents of GLUT-4 (f) and INSR (g) were evaluated with the western blot method. β-Actin was used as a loading control. Results expressed as mean ± SD. Statistical significance is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA (and non-parametric) test. h Representative images of immunofluorescence staining for GLUT-4 in equine ASCs from the three age-matched groups
Fig. 10
Fig. 10
Age-associated changes in the expressions of UPR-linked markers. Expressions of CHOP (a), PERK (b), eIF2α (c), BiP (d), ATF6, (e), and IRE1 (f) were determined using qRT-PCR. g To evaluate the mRNA levels of uXBP1 and sXBP1, RT-PCR was performed with XBP1 primers, and the PCR products were run in the 2% agarose gel. Relative quantity of uXBP1 and sXBP1 was determined using Image Lab software after normalization with GAPDH as a reference gene. Results expressed as mean ± SD. Statistical significance is indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 using one-way ANOVA (and non-parametric) test. h Representative confocal microscopy images of PDIA3 staining

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