Enhanced viability and function of mesenchymal stromal cell spheroids is mediated <i>via</i> autophagy induction

Abstract

Mesenchymal stromal cells (MSCs) have received attention as promising therapeutic agents for the treatment of various diseases. However, poor post-transplantation viability is a major hurdle in MSC-based therapy, despite encouraging results in many inflammatory disorders. Recently, three dimensional (3D)-cultured MSCs (MSC_3D) were shown to have higher cell survival and enhanced anti-inflammatory effects, although the underlying mechanisms have not yet been elucidated. In this study, we investigated the molecular mechanisms by which MSC_3D gain the potential for enhanced cell viability. Herein, we found that macroautophagy/autophagy was highly induced and ROS production was suppressed in MSC_3D as compared to 2D-cultured MSCs (MSC_2D). Interestingly, inhibition of autophagy induction caused decreased cell viability and increased apoptotic activity in MSC_3D. Furthermore, modulation of ROS production was closely related to the survival and apoptosis of MSC_3D. We also observed that HMOX1 (heme oxygenase 1) was significantly up-regulated in MSC_3D. In addition, gene silencing of HMOX1 caused upregulation of ROS production and suppression of the genes related to autophagy. Moreover, inhibition of HIF1A (hypoxia inducible factor 1 subunit alpha) caused suppression of HMOX1 expression in MSC_3D, indicating that the HIF1A-HMOX1 axis plays a crucial role in the modulation of ROS production and autophagy induction in MSC_3D. Finally, the critical role of autophagy induction on improved therapeutic effects of MSC_3D was further verified in dextran sulfate sodium (DSS)-induced murine colitis. Taken together, these results indicated that autophagy activation and modulation of ROS production mediated <i>via</i> the HIF1A-HMOX1 axis play pivotal roles in enhancing the viability of MSC_3D.<b>Abbreviations:</b> 3D: three dimensional; 3MA: 3 methlyadenine; AMPK: AMP-activated protein kinase; Baf A₁: bafilomycin A₁; CFSE: carboxyfluorescein succinimidyl ester; CoCl₂: cobalt chloride; CoPP: cobalt protoporphyrin; DSS: dextran sulfate sodium; ECM: extracellular matrix; FOXO3/FOXO3A: forkhead box O3; HIF1A: hypoxia inducible factor 1 subunit alpha; HMOX1/HO-1: heme oxygenase 1; HSCs: hematopoietic stem cells; IL1A/IL-1α: interleukin 1 alpha; IL1B/IL-1β: interleukin 1 beta; IL8: interleukin 8; KEAP1: kelch like ECH associated protein 1; LAMP1: lysosomal associated membrane protein 1; LAMP2: lysosomal associated membrane protein 2; MSC_2D: 2D-cultured MSCs; MSC_3D: 3D-cultured MSCs; MSCs: mesenchymal stromal cells; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; PGE2: prostaglandin E2; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PINK1: PTEN induced kinase 1; ROS: reactive oxygen species; siRNA: small interfering RNA; SIRT1: sirtuin 1; SOD2: superoxide dismutase 2; SQSTM1/p62: sequestosome 1; TGFB/TGF-β: transforming growth factor beta.