Hypoxic Preconditioning Protects Cord Blood-Mesenchymal Stromal Cells from Ischemic Damage in Vitro

Abstract

Objectives: We have previously shown that cord blood mesenchymal stromal cell (CBMSC)-secreted factors act anti-apoptotic and pro-mitotic on endothelial cells and cardiomyocytes to ischemia-like conditions. However CB-MSCs themselves suffer ischemic damage in the infarcted heart, hampering their potential clinical efficacy. To increase CB-MSC tolerance to ischemia, we developed a specific hypoxic preconditioning (HP) protocol and investigated the molecular mechanisms involved. Methods: Human CBMSCs were subjected to different durations (12, 24 or 36 hour) of hypoxic preconditioning (1% O2 in glucose/serum-containing medium) prior to a final phase of simulated ischemia (24 hour, 1% O2 in glucose/serum-free medium). Cell loss, metabolic activity (WST-8 conversion), apoptosis (nuclear fragmentation and shrinking) and proliferation (BrdU incorporation) were measured. The effects of HP on Akt-, ERK1/2 and STAT3-phosphorylation and Bcl-2, Bcl-XL and Bag-1 mRNA expression were determined by Western blot and quantitative real time PCR. Results: Ischemic damage of CBMSCs was lowest after 24 hour HP. Here, the percentage of remaining cells was higher (74 ± 7% versus non-HP 39 ± 11%, p < 0.05) and metabolic activity was enhanced (35 ± 2% versus non-HP 18 ± 6%, p < 0.05). Nuclear fragmentation index was lower (11.9 ± 0.2 versus non-HP 13.8 ± 0.3, p < 0.05), nuclear shrinking was less pronounced (mean nuclear area 179 ± 5 µm2 versus non-HP 155 ± 5 µm2, p < 0.05), and the proliferation index was enhanced (49 ± 1 versus non-HP 16 ± 3, p < 0.05). While phosphorylation of Akt and STAT3 was increased in preconditioned cells, ERK1/2 phosphorylation and Bcl-2 mRNA expression were downregulated in response to HP, and mRNA expression of Bcl-XL and Bag-1 was not altered. Conclusions: Hypoxic preconditioning protects CBMSCs from the deleterious impact of combined oxygen/glucose/serum-deprivation via STAT3-dependent signaling. This might by a useful strategy to increase their tolerance to ischemia and improve their therapeutic efficacy in clinical applications.