Hypoxia and Serum Deprivation in Human Mesenchymal Stem Cells, and the Protective Role of Thymosin [Beta]-4

Abstract

Background: Current literature suggests nutrient deprivation and hypoxia affect human mesenchymal stem cell (hMSC) survival after cell transplantation. Wound-healing protein Thymosin [beta]-4 (T[beta]-4) has been shown to improve hMSC proliferation and prevent apoptosis. This study examines the effects of nutrient deprivation and hypoxia on hMSC survival, and the pro-survival effect of pre-treatment with T[beta]-4. Methods: Early hMSC cultures, at 48 hours, were grown under nutrient deprivation (1.5% FBS media), a 24-hour, 1.0 [mu]g/mL [beta]-4 pre-treatment, and 24 hours of hypoxia (1% O[subscript]2). Proliferation of hMSCs was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, and intracellular stress was examined by a Reactive Oxygen Species (ROS) assay. In addition, hMSCs were grown for 2 weeks in complete media (16.5% FBS), then nutrient-deprived (1.5% FBS media) with a 24-hour, 1.0 [mu]g/mL T[beta]-4 pre-treatment, followed by 24 hours of hypoxia (1% O[subscript]2). Proliferation was measured with immunocytochemistry using a Ki-67 antibody. Protein expression of hypoxia inducible factor-1 alpha (HIF-1[alpha]), Vascular Endothelial Growth Factor- alpha (VEGF-[alpha]), caspase-3, B cell lymphoma-2 (Bcl-2), and Connexin 43 (Cx43) were measured with western blot. Results: In 48-hour cell cultures, nutrient-deprived hMSCs had significantly increased ROS production (p< .01), and decreased proliferation compared to hMSCs in complete media; hypoxia had no effect. T[beta]-4-treated hMSCs showed decreased ROS production and increased proliferation compared to non-treated hMSCs. In 2-week hMSC cultures, nutrient deprivation and hypoxia resulted in elevated caspase-3 protein expression. Hypoxia increased HIF-1[alpha] and VEGF-[alpha] expression in complete media and nutrient-deprived media. Additionally, T[beta]-4-treated hMSCs showed increased HIF-1[alpha] and VEGF-[alpha] expression under normoxia and hypoxia. In T[beta]-4-treated hMSCs, expression of Bcl-2 increased under normoxia, and Cx43 expression increased under hypoxia. Discussion: These findings suggest that nutrient deprivation is the predominant factor affecting hMSC survival in early cultures. However, T[beta]-4 may promote hMSC survival in a nutrient-deprived environment in early and expanded cultures. Further studies regarding T[beta]-4 effectiveness will shed insight into its potential as a therapeutic agent in hMSC transplantation.