Abstract

BackgroundProgressive 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).MethodsASCs 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.ResultsOur 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.ConclusionsObtained 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.Graphical abstract

Highlights

  • Mesenchymal stem cells (MSCs) are adult stem cells with the ability to differentiate into cells of mesodermal lineage such as adipocytes, osteocytes, and chondrocytes

  • We found that the transcript levels of Ten-eleven translocation methylcytosine dioxygenase (TET)-2 and TET-3 were significantly upregulated in Adipose-derived stem cells (ASCs)< 5 compared with the oldest group (p < 0.05, p < 0.01) (Fig. 3g, h)

  • We found that ASCs derived from the oldest individuals exhibited significant upregulation of miR-203b-3p and miR-16-5p, which are involved in the pro-inflammatory response and miR-146a-5p, miR-21, and miR-24-3p that act as anti-inflammatory factors

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Summary

Introduction

Mesenchymal stem cells (MSCs) are adult stem cells with the ability to differentiate into cells of mesodermal lineage such as adipocytes, osteocytes, and chondrocytes. MSC exhibits expression of specific surface antigen, such as CD105, CD73, and CD90, and lacks expression of hematopoietic and endothelial markers (CD45, CD34, CD11, CD14) [3]. They express pluripotency markers like Oct-4 and SOX-2 [4]. Extensive evidence has shown that MSC exhibits low expression of MHC class I and lack of MHC II along with the secretion of soluble immunomodulatory agents that regulate B cell and T cell function, which makes them a promising tool in regenerative medicine [5, 6]. The main aim of this study was to identify the molecular alternations in ASCs derived from three age-matched horse groups: young (< 5), middleaged (5–15), and old (> 15 years old)

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