Induced pluripotent stem cell-derived mesenchymal stem cells attenuate cigarette smoke-induced mitochondrial dysfunction and apoptosis in airway smooth muscle cells

Abstract

INTRODUCTION: Cigarette smoking is the primary cause of chronic obstructive pulmonary disease (COPD). Oxidative stress-mediated mitochondrial dysfunction is believed to contribute to lung inflammation and remodelling. Mesenchymal stem cells were reported to attenuate lung damage in various animal models of COPD. The mechanisms are still unclear. We investigated the effect of human induced–pluripotent stem cell–derived mesenchymal stem cells (iPSC-MSCs) on cigarette smoke medium (CSM)–induced mitochondrial damage in primary airway smooth muscle cells (ASMCs). Mitochondrial transfer and paracrine effect were examined. METHODS: ASMCs were pre-stained with CellTrace Violet and were cultured alone or in the presence of iPSCMSCs stained with the mitochondrial-specific dye MitoTracker. After treatment with CSM, mitochondrial transfer was determined by flow cytometry or fluorescence microscopy. In ASMCs, we determined mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and apoptosis by MitoSOX, JC-1, and Annexin V staining, respectively. To study paracrine effects, the co-cultured cells were separated by Transwell inserts. RESULTS: CSM (25%) increased mitochondrial ROS levels, reduced MMP, and increased apoptosis in ASMCs. Co-culture with iPSC-MSCs attenuated CSM-induced mitochondrial ROS levels (4-fold vs 6.71-fold; P<0.05) and apoptosis (29.8% vs 40.6%; P<0.01), whilst it partially reversed the reduction in MMP (0.78-fold vs 0.52-fold; P<0.01). CSM treatment increased the percentage of ASMCs positive for iPSC-MSC-derived (MitoTrackerstained) mitochondria (75.5% vs 18.8%; P<0.001). Mitochondrial transfer was also confirmed by observation of iPSC-MSC–derived mitochondria in ASMCs by fluorescence microscopy. Co-culture using the Transwell system also led to attenuation of CSM-induced mitochondrial ROS in ASMCs. However, the CSM-induced drop in MMP and increased apoptosis were not attenuated by the Transwell co-culture. CONCLUSIONS: Co-culture of iPSC-MSCs and ASMCs leads to protection of ASMCs against CSM-induced mitochondrial dysfunction and apoptosis. Mitochondrial transfer from iPSC-MSCs to ASMCs may be a mechanism mediating this protective effect. Paracrine regulation may also be a contributing factor. Our findings illustrate the potential of iPSC-MSCs as a novel therapy for airway diseases and demonstrate that their action might be driven by multiple mechanisms.