Immortalized Mesenchymal Stromal Cells Overexpressing Alpha-1 Antitrypsin Protect Acinar Cells from Apoptotic and Ferroptotic Cell Death

Abstract

Abstract Chronic pancreatitis (CP) is a progressive inflammatory disorder that impairs endocrine and exocrine function. Our previous work suggests that mesenchymal stem/stromal cells (MSCs) and MSCs overexpressing alpha-1 antitrypsin (AAT-MSCs) could be therapeutic tools for CP treatment in mouse models. However, primary MSCs have a predisposition to undergo senescence during culture expansion which limits their therapeutic applications. Here we generated and characterized immortalized human MSCs (iMSCs) and AAT-MSCs (iAAT-MSCs) and tested their protective effect on 2,4,6-Trinitrobenzenesulfonic acid (TNBS) -induced acinar cell death in an in vitro cell culture system. Primary MSCs were immortalized by transduction with simian virus 40 large T antigen (SV40LT), and the resulting iMSC and iAAT-MSC lines were analyzed for proliferation, senescence, phenotype, and multi-differentiation potential. Subsequently, the impact of these cells on TNBS-induced cell death was measured and compared. Both apoptosis and ferroptosis pathways were investigated by assessing changes of critical factors before and after cell treatment. Coculture of iMSCs and iAAT-MSCs with acinar cell lines inhibited early apoptosis induced by TNBS, reduced ER stress, and reversed TNBS-induced protein reduction at tight junctions. Additionally, iMSCs and iAAT-MSCs exerted such protection by regulating mitochondrial respiration, ATP content, and ROS production in TNBS-induced acinar cells. Furthermore, iMSCs and iAAT-MSCs ameliorated ferroptosis by regulating the ferritin heavy chain 1 (FTH1)/protein disulfide isomerase (PDI)/glutathione peroxide 4 (GPX4) signaling pathways and by modulating ROS function and iron generation in acinar cells. These findings identified ferroptosis as one of the mechanisms that leads to TNBS-induced cell death and offer mechanistic insights relevant to using stem cell therapy for the treatment of CP.