Preconditioning of Mesenchymal Stem Cells with Non-Toxic Concentration of Hydrogen Peroxide Against Oxidative Stress Induced Cell Death: The Role of Hypoxia-Inducible Factor-1.

Fatemeh Nouri,Seyed Noureddin Nematollahi-Mahani,Ali Mohammad Shariﬁ

doi:10.15171/apb.2019.010

Abstract

Purpose: To investigate the protective effect of preconditioning with non-toxic dose of hydrogen peroxide (H2O2) as a possible cell signaling molecule, against cell death induced by toxic concentration of H2O2 or by serum deprivation in human Wharton’s jelly-derived mesenchymal stem cells (HWJ-MSCs) and underlying mechanisms. Methods: HWJ-MSCs were isolated and identified using flow cytometry. After finding non-toxic concentration of H2O2, cells preconditioning was performed by H2O2 (20 μM) for 12 h and cell tolerance against serum deprivation or toxic levels of H2O2 was assayed by MTT test. Effect of preconditioning on mRNA and protein expression of Akt-1, Bcl-2 and Bax were examined using reverse transcription polymerase chain reaction (RT-PCR) and western blotting respectively. Role of hypoxia-inducible factor (HIF)-1α was explored in presence HIF-1α inhibitor. Results: Preconditioning with 20 μM H2O2 for 12 h was non-toxic and decreased cell death induced by oxidative stress and serum deprivation in MSC cultures. However, the increased tolerance reversed in the presence of inhibitor of HIF-1α. By regards to RT-PCR and western blotting data, although expression of Akt-1, Bcl-2 and Bax was not change considerably but phosphorylated Akt-1 (pAkt-1) was up regulated after treatment with 20 μM H2O2 compared to control group. Moreover after exposure to 100 μM H2O2, western blotting analysis showed that cell pretreatment with 20 μM H2O2, decremented Bax/Bcl2 ratio and up-regulated HIF-1α and pAkt-1 compared to the control group. Conclusion: Increased tolerance of H2O2-pretreated cells led to the suggestion that transplantation of H2O2 preconditioned MSCs may improve therapeutic potential of stem cells in cell therapy procedures.

Highlights

Stroke as a destructive disease and one of the world’s biggest causes of death has led to more than 5 million deaths in 2016.1 despite wide investigations on the mechanisms of stroke, efficient therapies to restore lost neurological function after stroke, has not been achieved yet
Preconditioning with 20 μM H2O2 for 12 h was non-toxic and decreased cell death induced by oxidative stress and serum deprivation in MSC cultures
After exposure to 100 μM H2O2, western blotting analysis showed that cell pretreatment with 20 μM H2O2, decremented Bax/Bcl[2] ratio and up-regulated hypoxia-inducible factor (HIF)-1α and phosphorylated Akt-1 (pAkt-1) compared to the control group

Summary

Introduction

Stroke as a destructive disease and one of the world’s biggest causes of death has led to more than 5 million deaths in 2016.1 despite wide investigations on the mechanisms of stroke, efficient therapies to restore lost neurological function after stroke, has not been achieved yet. Several studies have underlined the potential of stem cell transplantation as a novel therapeutic strategy for stroke recovery. Stem cells may migrate into damaged regions of brain and differentiate into neural cells or may promote endogenous neurogenesis and angiogenesis through the secretion of trophic factors in the injured regions of central nervous system.[2,3,4]. High degree of transplanted cell mortality is a limiting factor for the successful clinical cell therapy.[5,6] development of strategies that lead to increase survival of transplanted cells may improve therapeutic potential of stem cell-based therapies.[7] Earlier investigations indicated that ischemic preconditioning can obviously increase the resistance of treated cells or tissues following stricter conditions.[8]

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Conclusion