Effect of antioxidant supplementation on the total yield, oxidative stress levels, and multipotency of bone marrow-derived human mesenchymal stromal cells

Abstract

Bone marrow-derived multipotent mesenchymal stromal cells (MSCs) are the most frequently investigated cell type for potential regenerative strategies because they are relatively easy to isolate and are able to differentiate into several mesenchymal lineages. Unfortunately, during ex vivo culture, MSCs present gradual loss of differentiation potential and reduced clinical efficacy. Reactive oxygen species (ROS) are associated with oxidative damage and accumulate during MSC expansion. Because ROS are believed to be involved in the loss of multipotency, we hypothesized that compounds with antioxidant activity have the capacity to scavenge ROS, prevent cellular damage, and rescue culture-induced loss of multipotency. In this manuscript, we show that antioxidant supplementation can partially rescue the loss of alkaline phosphatase expression induced by oxidizing agents and increases the yield of hMSCs, when supplemented to a fresh bone marrow aspirate. Concomitantly, oxidative DNA damage and ROS levels in hMSCs were reduced by antioxidants. We conclude that antioxidant supplementation during MSC expansion reduces the DNA damage load and increases the MSC yield.

FIG. 1.

Effect of oxidative damage inducers and scavengers on proliferation and differentiation of human mesenchymal stromal cells (hMSCs). hMSCs were cultured in basic medium (BM) or osteogenic medium (OM) in the presence of antioxidants (Trolox 50 μM or D-mannitol 5 mM) (A, B) or oxidative damage inducers (peroxynitrite 30 μM; tert-butyl hydroperoxide (t-BHP) 50 μM, ascorbate/Fe²⁺ 250 μM and hydrogen peroxide 30 μM) (C, D). Proliferation (A, C) and alkaline phosphatase (ALP) expression (B, D) was measured after 6 days of culture. All conditions were performed at the same time, and the conditions were split for interpretation purposes only. For ALP, data are expressed as total ALP activity normalized for cell number. Proliferation is presented as the fluorescence intensity in arbitrary units (Alamar blue). Error bars represent standard deviation. Statistical analysis was performed using Student's t-test with a significance of p<0.05. Asterisks represent *p<0.05, **p<0.01, ***p<0.001. ns, not significant.

FIG. 2.

Antioxidant supplementation prevents reactive oxygen species (ROS) induced loss of differentiation. ALP expression was measured after 6 days of culture in BM or OM and the antioxidant effect (Trolox 50 μM and D-mannitol 5 mM) was measured in two-model systems of oxidative stress induction: t-BHP (50 μM) (A) or peroxynitrite (30 μM) (B). All conditions were performed at the same time, and the conditions were split for interpretation purposes only. Data are expressed as total ALP activity normalized for cell numbers. Error bars represent standard deviation. Statistical analysis was performed using Student's t-test with a significance of p<0.05. Asterisks represent *p<0.05, **p<0.01, ***p<0.001. ns, not significant.

FIG. 3.

Effect of antioxidant supplementation on DNA damage accrual. Effect of antioxidants (D-mannitol 5 mM, Trolox 50 μM, and sodium selenite [NaSel] 100 nM) on DNA damage load (A, B). DNA damage load was assessed by quantifying the percentage of 53BP1-positive cells (A, C) and the number of 53BP1 foci per cell (B, D) either in the absence of oxidative damage inducer (A, B) or in the presence of the oxidizing agent t-BHP (50 μM) (C, D). All conditions were performed at the same time, and the conditions were split for interpretation purposes only. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA and Tukey's post-test with a significance of p<0.05. Asterisks represent *p<0.05, **p<0.01. ns, not significant.

FIG. 4.

Effect of antioxidant supplementation on cellular parameters of hMSCs (passage 1). The effect of antioxidant supplementation (D-mannitol 5 mM, Trolox 50 μM, and NaSel 100 nM) on DNA damage load (A, B), on proliferation in vitro (P0) (C) and differentiation potential (P1) (D, E) were evaluated. Oxidative damage accrual was assessed by quantifying 8-oxoguanine adducts (A) and intracellular ROS production (CM-H₂DCFDA) (B) and expressed as percentage reduction of the mean intensity, relative to the control. Proliferation is expressed as thousands of cells/mL of culture medium (C). Percentage of ALP-positive cells was analyzed by FACS after 6 days, both in the presence or absence of the osteogenic inducer (dexamethasone) (D). Mineralization capacity was also measured and calcium accumulation was quantified and expressed as mg/dL (E). Error bars represent standard deviation. Statistical analysis was performed using Student's t-test with a significance of p<0.05. Asterisks represent *p<0.05, **p<0.01, ***p<0.001. Dex, dexamethasone.

FIG. 5.

Effect of continuous antioxidant supplementation on cellular parameters of expanded hMSCs (passage 3). Effect of antioxidants (D-mannitol 5 mM, Trolox 50 μM, and NaSel 100 nM) on differentiation potential of in vitro expanded hMSCs in the presence of antioxidants. Adipogenic differentiation (A) and mineralization (B) were assessed in passage 3 cells. Error bars represent standard deviation.