Equine Metabolic Syndrome Affects Viability, Senescence, and Stress Factors of Equine Adipose-Derived Mesenchymal Stromal Stem Cells: New Insight into EqASCs Isolated from EMS Horses in the Context of Their Aging

Abstract

Currently, equine metabolic syndrome (EMS), an endocrine disease linked to insulin resistance, affects an increasing number of horses. However, little is known about the effect of EMS on mesenchymal stem cells that reside in adipose tissue (ASC). Thus it is crucial to evaluate the viability and growth kinetics of these cells, particularly in terms of their application in regenerative medicine. In this study, we investigated the proliferative capacity, morphological features, and accumulation of oxidative stress factors in mesenchymal stem cells isolated from healthy animals (ASCN) and horses suffering from EMS (ASCEMS). ASCEMS displayed senescent phenotype associated with β-galactosidase accumulation, enlarged cell bodies and nuclei, increased apoptosis, and reduced heterochromatin architecture. Moreover, we observed increased amounts of nitric oxide (NO) and reactive oxygen species (ROS) in these cells, accompanied by reduced superoxide dismutase (SOD) activity. We also found in ASCEMS an elevated number of impaired mitochondria, characterized by membrane raptures, disarrayed cristae, and vacuole formation. Our results suggest that the toxic compounds, accumulating in the mitochondria under oxidative stress, lead to alternations in their morphology and may be partially responsible for the senescent phenotype and decreased proliferation potential of ASCEMS.

Figure 1

Immunophenotyping of EqASCs. Representative photographs showing the results of EqASC immunophenotyping. (a) The cells expressed the following mesenchymal markers CD44, CD73, CD90, and CD105 and showed no expression of hematopoietic marker CD45. Markers were detected with specific antibodies and visualized with secondary antibodies conjugated with atto-488; nuclei were counterstained with DAPI. Magnification ×100, scale bar: 250 μm. The presence of antigens CD44 (b), CD73 (c), CD90 (d), and CD105 (e) was compared using the relative fluorescent intensity. Results expressed as mean ± SD. ^∗ p value < 0.05; ^∗∗∗ p value < 0.001.

Figure 2

Results of multipotency assay. Morphology of EqASCs cultured under standard, osteogenic, chondrogenic, and adipogenic conditions. Osteogenic differentiation was visualized with Alizarin Red staining on the 11th day; chondrogenic differentiation was confirmed by Safranin O on the 11th day and adipogenesis by Oil Red O staining on the 9th day. Images included in the figure were selected as representative across investigated groups. Magnification ×100, scale bar: 250 μm.

Figure 3

Growth kinetics and clonogenic potential of EqASCs. Mean cells number in both test groups during the seven-day culture period (a) assessed with TOX-8 assay. Time required to double the population of cells expressed in hours (b). Images depicting the results of pararosaniline staining (c). The percentage of colonies consisting of more than 50 cells in the control and EMS group (d). Results expressed as mean ± SD. ^∗∗ p value < 0.01; ^∗∗∗ p value < 0.001.

Figure 4

Morphology of EqASCs. The architecture and morphology of the cultures were evaluated on the 1st and 7th day of culture (a). Actin filaments are shown in green and nuclei in white (i, iii, vi, viii). Cell morphology was assessed with a scanning electron microscope at the same time points (ii, iv, vii, ix). ASC_EMS showed decreased number of both, filopodia and lamellipodia, as well as decreased production of microvesicles in comparison to control group. Nuclei (n), microvesicles (mv), lamellipodia (lp), and filopodia (fp). TEM images of cell nuclei (V, X). Higher magnification images of boxed regions shown on the right depict heterochromatin underneath the nuclear envelope. EqASC_EMS exhibited accelerated breakdown of heterochromatin associated with the inner nuclear membrane. Moreover nuclei of those cells were visibly enlarged. Fluorescent images: magnification ×100, scale bar: 250 μm; SEM images: magnification ×5000, scale bar: 5 μm, TEM magnification ×24000, scale bars: 1 μm. Mean diameter of the nuclei (b), the percentage of enlarged nuclei (c), and the amount of heterochromatin (d). Results expressed as mean ± SD. ^∗ p value < 0.05.

Figure 5

Organelles of EqASCs. Cellular composition of EqASC_N and EqASC_EMS (a) observed under epifluorescent microscope. Morphological features are indicated with the following abbreviations: n: nucleus; t: tubulin; er: endoplasmic reticulum; g: Golgi apparatus. Images show a qualitative evaluation of the composition of cells in the cultures (a). Magnification ×100, scale bar: 100 μm. Evaluation of the quantitative difference using the relative fluorescent intensity for tubulin-RFP (b), ER-RFP (c), and Golgi apparatus (d). Results expressed as mean ± SD. ^∗ p value < 0.05.

Figure 6

Evaluation of apoptosis and senescence in EqASC_N and EqASC_EMS. Assessment of apoptosis and senescence (a). Pictures showing the results of calcein: live cells (i, iv); propidium iodide: dead cells (ii, v); and β-galactosidase: senescence cells (iii, vi) staining. Percentage of dead cells in the cultures was evaluated based on the calcein/propidium iodide staining (b). Differences in the accumulation of β-galactosidase were determined based on the percentage of stained area (c). lc: live cell, dc: dead cell, and β-gal: β-galactosidase. Magnification ×100, scale bar: 250 μm. Results expressed as mean ± SD. ^∗ p value < 0.05.

Figure 7

Oxidative stress factors and p53 levels. To evaluate oxidative stress in the culture supernatants, we assessed the levels of nitric oxide (a), reactive oxygen species (b), and superoxide dismutase (c) using commercially available kits on days 1, 4, and 7 of culture. p53 protein level was determined with ELISA (d). Results expressed as mean ± SD. ^∗ p value < 0.05, ^∗∗ p value < 0.01, and ^∗∗∗ p value < 0.001.

Figure 8

Mitochondria and reactive oxygen species in EqASCs. Images showing DAPI (i, vi), Mito Red (ii, vii), and ROS staining (iii, viii). Magnification ×100, scale bar: 250 μm. Analysis of mitochondrial morphology by a transmission electron microscope (iv, v, ix, x). Representative electron micrographs showing alternations in mitochondrial morphology in EqASC_EMS. Small structures with disarrayed cristae are indicated with black arrows; membrane raptures and vacuole formation are indicated with red arrows. Quantitative assessment of ROS by the relative fluorescent intensity (b). Percentage of abnormal mitochondria (c); the width (d); and length (e) of mitochondria. Fluorescent images: magnification ×100, scale bar: 250 μm; TEM magnification ×120000, scale bar: 200 μm. Results expressed as mean ± SD. ^∗ p value < 0.05; ^∗∗ p value < 0.01.

Figure 9

EqASC mRNA levels of p21, p53, BAX, caspase-9, and the BCL-2/BAX ratio. Gene expression in the EqASC culture. The upregulation of apoptotic-related genes observed in ASC_EMS results in decreased proliferation potential and senescence phenotype. BAX and CAS-9 increased expression suggest that those cells undergo apoptosis via mitochondrial pathway. mRNA levels of p21 (a), p53 (b), BAX (c), and caspase-9 (d). The ratio between BCL-2 and BAX expression in each group estimated by dividing Qn of BCL-2 by Qn of BAX (e). Results expressed as mean ± SD. ^∗ p value < 0.05; ^∗∗ p value < 0.01.