Enhanced viability and function of mesenchymal stromal cell spheroids is mediated via autophagy induction

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 via the HIF1A-HMOX1 axis play pivotal roles in enhancing the viability of MSC_3D.Abbreviations: 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.