Declined adipogenic potential of senescent MSCs due to shift in insulin signaling and altered exosome cargo

Abstract

Multipotent mesenchymal stromal cells (MSCs) maintain cellular homeostasis and regulate tissue renewal and repair both by differentiating into mesodermal lineage, e.g., adipocytes, or managing the functions of differentiated cells. Insulin is a key physiological inducer of MSC differentiation into adipocytes, and disturbances in MSC insulin sensitivity could negatively affect adipose tissue renewal. During aging, regulation and renewal of adipose tissue cells may be disrupted due to the altered insulin signaling and differentiation potential of senescent MSCs, promoting the development of serious metabolic diseases, including metabolic syndrome and obesity. However, the potential mechanisms mediating the dysfunction of adipose-derived senescent MSC remains unclear. We explored whether aging could affect the adipogenic potential of human adipose tissue-derived MSCs regulated by insulin. Age-associated senescent MSCs (isolated from donors older than 65 years) and MSCs in replicative senescence (long-term culture) were treated by insulin to induce adipogenic differentiation, and the efficiency of the process was compared to MSCs from young donors. Insulin-dependent signaling pathways were explored in these cells. We also analyzed the involvement of extracellular vesicles secreted by MSCs (MSC-EVs) into the regulation of adipogenic differentiation and insulin signaling of control and senescent cells. Also the microRNA profiles of MSC-EVs from aged and young donors were compared using targeted PCR arrays. Both replicatively and chronologically senescent MSCs showed a noticeably decreased adipogenic potential. This was associated with insulin resistance of MSCs from aged donors caused by the increase in the basal level of activation of crucial insulin-dependent intracellular effectors ERK1/2 and Akt. To assess the impact of the paracrine cross-talk of MSCs, we analyzed microRNAs profile differences in MSC-EVs and revealed that senescent MSCs produced EVs with increased content of miRNAs targeting components of insulin-dependent signaling cascade PTEN, MAPK1, GAREM1 and some other targets. We also confirmed these data by differentiation of control MSCs in the presence of EVs from senescent cells and vice versa. Thus, aging attenuated the adipogenic potential of MSCs due to autocrine or paracrine-dependent induction of insulin resistance associated with the specific changes in MSC-EV cargo.

Keywords: MSCs (mesenchymal stromal cells); adipogenic potential; extracellular vesicles (EVs); insulin signaling; senescence.

FIGURE 1

Senescent biomarkers in MSCs isolated from young and aged donors. (А)—population doubling time (PDT, hours) and lag phase duration (hours) measured in Incucyte ZOOM system; (B)—telomere length (bp); (C)—proportion of cells with positive staining for beta-galactosidase (b-gal); (D–F)–representative microphotographs of MSCs isolated from young and aged donors: (D)—cell morphology in culture, phase-contrast images; (E)—b-gal staining, scale bar = 100 μm; (F)—immunocytochemical staining for p21 expression (green), nuclei are stained with DAPI). Control–MSCs isolated from young donors, senescent–MSCs isolated from aged donors. Mean ± SE, *p < 0.05, **p = 0.057.

FIGURE 2

Senescent MSCs demonstrated reduced adipogenic potential. (A,B), Representative phase-contrast images of the time dynamics of differentiation of control (A) and senescent (B) cells on 0, 7, 10, and 14 days in the same fields of view, and staining of lipid droplets in these cells with Nile Red dye in the end point (14th day). (C). Dynamics of increase in the number of cells accumulating fat drops during adipogenic differentiation of MSCs, n = 6–7; (D). The tangent of the slope of the increase in the number of differentiating cells (rate of differentiation, increase in percent of differentiated cells per day), n = 7–9; (E,F). Expression level of markers of adipogenic differentiation PPARγ (PPARG) (E) and adiponectin (ADIPOQ) (F) on 14 days of adipogenic differentiation, n = 3–7. Mean ± SE. *p < 0.05, **p < 0.01.

FIGURE 3

Insulin-dependent signaling in control and senescent MSCs. (A,B). Representative results of Western-blot analysis of Akt (Thr308) and Erk1/2 phosphorylation under the action of insulin in different time points in control (A) and senescent (B) MSCs. The results of densitometric analysis marked under the protein bands; (C). Time dynamics of Akt (Thr308) phosphorylation in control and senescent MSCs under insulin stimulation, n = 6 Y-axis represent ratio in Western-blot band volume between p-Akt (T308) and total Akt; (D). Time dynamics of Erk1/2 (Thr202/Tyr204) phosphorylation in control and senescent cells under insulin stimulation, n = 6 Y-axis represent ratio in Western-blot band volume between p-Erk1/2 (Thr202/Tyr204) and total Erk; (E,F). Basal level of Akt (E) and Erk1/2 (F) phosphorylation in control and senescent cells, n = 4–5. Mean ± SE, *p < 0.05.

FIGURE 4

Selected miRNAs differentially expressed in EVs secreted by control and senescent MSCs along with their targets. Interaction maps illustrated differently expressed miRNAs as edges and their potential target mRNA as nodes. Coloured miRNAs are shown if significantly reduced (red) or enhanced (blue) in MSC-EVs from aged donors compared to young ones. The degree of color saturation shows the value of the target score level (relative units). A more saturated color indicates a higher value, and therefore a high reliability of the relationship between the miRNA and its target.

FIGURE 5

Extracellular vesicles affect the adipogenic potential of MSCs. (A). Phase-contrast images of the dynamics of differentiation of control cells for 14 days with staining with neutral lipid dye NileRed on the 14th day; (B). Phase-contrast images of the dynamics of differentiation of control cells with stimulation by EVs from senescent cells for 14 days with staining with neutral lipid dye NileRed on the 14th day; (С). Phase-contrast images of the dynamics of differentiation of senescent cells for 14 days with staining with neutral lipid dye NileRed on the 14th day; (D). Phase-contrast images of the dynamics of differentiation of senescent cells with stimulation by EVs from control cells for 14 days with staining with neutral lipid dye NileRed on the 14th day; (E). Dynamics of increase in the number of cells accumulating fat drops during adipogenic differentiation of control MSCs in the presence of EVs from senescent cells and without, n = 7; (F). The tangent of the slope of the increase in the number of differentiating control cells (differentiation rate) in the presence of EVs from senescent cells and without EVs, n = 7–11; (G). Dynamics of increase in the number of cells accumulating fat drops during adipogenic differentiation of senescent MSCs in the presence of EVs from control cells and without EVs, n = 6; (H). The tangent of the slope of the increase in the number of differentiating senescent cells (differentiation rate) in the presence of EVs from control cells and without, n = 5–9. Mean ± SE, *p < 0.05, **p < 0 01.