Age-dependent impairment of adipose-derived stem cells isolated from horses

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 (>15years 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 beta -galactosidase activity, and accumulation of gamma H2AX 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-1 beta, tumor necrosis factor alpha (TNF-alpha), 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 5years 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