Mesenchymal Stem Cell Behavior under Microgravity: From Stress Response to a Premature Senescence

Abstract

Mesenchymal stem cells are undifferentiated cells able to acquire different phenotypes under specific stimuli. Wharton's jelly is a tissue in the umbilical cord that contains mesenchymal stromal cells (MSCs) with a high plasticity and differentiation potential. Their regeneration capability is compromised by cell damage and aging. The main cause of cell damage is oxidative stress coming from an imbalance between oxidant and antioxidant species. Microgravity represents a stressing condition able to induce ROS production, ultimately leading to different subcellular compartment damages. Here, we analyzed molecular programs of stemness (Oct-4; SOX2; Nanog), cell senescence, p19, p21 (WAF1/CIP1), p53, and stress response in WJ-MSCs exposed to microgravity. From our results, we can infer that a simulated microgravity environment is able to influence WJ-MSC behavior by modulating the expression of stress and stemness-related genes, cell proliferation regulators, and both proapoptotic and antiapoptotic genes. Our results suggest a cellular adaptation addressed to survival occurring during the first hours of simulated microgravity, followed by a loss of stemness and proliferation capability, probably related to the appearance of a molecular program of senescence.

Keywords: cell senescence; cellular mechanisms; mesenchymal stem cells; microgravity; stress response.

Figure 1

Expression of stemness genes. The expression of the stemness-related genes Oct-4 (A), SOX2 (B) and NANOG (C) was assessed in WJ-MSCs cultured under simulated microgravity (µg) for 6 h-µg (orange bars), 12 h-µg (green bars), 24 h-µg (red bars) or 48 h-µg (blue bars). The mRNA levels for each gene were normalized against glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and were expressed as fold of change (2^−ΔΔCt) in mRNA levels observed in controls WJ-MSCs (Ctrl). Controls WJ-MSCs (gray bars) are defined as 1 (mean ± SD; n = 6). Data are expressed as mean ± SD referenced to control (* p ≤ 0.05), (** p ≤ 0.01).

Figure 2

Analysis of sirtuins, heat shock protein and NADPH oxidase. Expression of SIRT1 (A), HSP60 (B), HSP70 (C), and NOX4 (D) was assessed in WJ-MSCs cultured under simulated microgravity for microgravity (µg) for 6 h-µg (orange bars), 12 h-µg (green bars), 24 h-µg (red bars) or 48 h-µg (blue bars). The mRNA levels for each gene were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and were expressed as fold of change (2^−ΔΔCt) in the mRNA levels observed in controls WJ-MSCs (Ctrl). Controls WJ-MSCs (gray bars) are defined as 1 (mean ± SD; n = 6). Data are expressed as mean ± SD referred to control (* p ≤ 0.05), (** p ≤ 0.01).

Figure 3

Expression of specific senescence-related markers p16, p19ARF, p21 and p53. Expression of p16 (A), p19ARF (B), p21 (C) and p53 (D) was assessed in WJ-MSCs cultured under simulated microgravity (µg) for 6 h-µg (orange bars), 12 h-µg (green bars), 24 h-µg (red bars) or 48 h-µg (blue bars). The mRNA levels for each gene were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and were expressed as fold of change (2^−ΔΔCt) in the mRNA levels observed in controls WJ-MSCs (Ctrl). Controls WJ-MSCs (gray bars) are defined as 1 (mean ± SD; n = 6). Data are expressed as mean ± SD referred to control (* p ≤ 0.05), (** p ≤ 0.01).

Figure 4

Expression of the apoptotic markers BAX and Bcl-2. Expression of BAX (A) and Bcl-2 (B) was assessed in WJ-MSCs cultured under simulated microgravity for microgravity (µg) for 6 h-µg (orange bars), 12 h-µg (green bars), 24 h-µg (red bars) or 48 h-µg (blue bars). The mRNA levels for each gene were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and were expressed as fold of change (2^−ΔΔCt) in the mRNA levels observed in controls WJ-MSCs (Ctrl). Controls WJ-MSCs (gray bars) are defined as 1 (mean ± SD; n = 6). Data are expressed as mean ± SD referred to control (* p ≤ 0.05).

Figure 5

Expression of the principal cytoskeleton markers β-Actin and β-Tubulin. Expression of β-Actin (A) and β-Tubulin (B) was assessed in WJ-MSCs cultured under simulated microgravity for microgravity (µg) for 6 h-µg (orange bars), 12 h-µg (green bars), 24 h-µg (red bars) or 48 h-µg (blue bars). The mRNA levels for each gene were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and were expressed as fold of change (2^−ΔΔCt) in the mRNA levels observed in controls WJ-MSCs (Ctrl). Controls WJ-MSCs (gray bars) are defined as 1 (mean ± SD; n = 6). Data are expressed as mean ± SD referred to control (* p ≤ 0.05), (** p ≤ 0.01).

Figure 6

Analysis of Cyt C. Immunofluorescence analysis of Cyt C (green) was assessed in controls WJ-MSCs (Ctrl), and WJ-MSCs incubated from 6 to 48 h under microgravity conditions (µg). The figures are representative of different independent experiments. Nuclei are labelled with 4,6-diamidino-2-phenylindole (DAPI, blue). Magnification 40×. Scale bars: 40 µm.

Figure 7

Optical microscope analysis of WJ-MSC morphology after exposure to microgravity (µg). Figure shows morphological changes in cells treated for 6, 12, 24 and 48 h in µg as compared to controls (Ctrl). Scale bar = 100 µm.

Figure 8

Western blot analysis. Levels of Oct-4, SOX2 and NANOG proteins shown in arbitrary units (A). (B): levels of p16, p19, p21 and p53 protein shown in arbitrary units. (C): levels of Hsp60, Hsp70, Sirt1, Bax, Bcl2 and Nox4 proteins shown in arbitrary units. (D): levels of β-Actin and β-Tubulin protein shown in arbitrary units was assessed in WJ-MSCs cultured under simulated microgravity (µg) for 6 h-µg (orange bars), 12 h-µg (green bars), 24 h-µg (red bars) or 48 h-µg (blue bars). The protein levels for each gene were compared against glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Controls WJ-MSCs (gray bars) are defined as 1 (mean ± SD; n = 6). Data are expressed as mean ± SD referenced to control (* p ≤ 0.05), (** p ≤ 0.01).