miR-212/132-Enriched Extracellular Vesicles Promote Differentiation of Induced Pluripotent Stem Cells Into Pancreatic Beta Cells

Extracellular Vesicles

Pancreatic beta cell transplantation is the ideal method for treatment of type 1 diabetes mellitus (T1DM), and the generation of beta cells from induced pluripotent stem cells (iPSCs) of patients is a promising strategy. In this study, we improved a previous strategy to produce beta cells using extracellular vesicles (EVs) derived from mature beta cells and differentiated beta cells from iPSCs (i-Beta cells), which secreted insulin under glucose stimulation in vitro and ameliorated hyperglycemia in vivo. Mechanistic analyses revealed that EV-carried microRNA (miR)-212/132 (EV-miR-212/132) directly bound to the 3' UTR of FBW7 to prevent its translation and FBW7 combined with NGN3 to accelerate its proteasomal degradation. EV-miR-212/132 stabilized NGN3 expression to promote differentiation of endocrine cells from induced iPSCs. Moreover, NGN3 bound to PDX1 to enhance transcription of endogenous miR-212/132 and formed a positive regulatory circuit that maintained the functions of mature pancreatic beta cells. CONCLUSION: This study describes a novel approach for beta cell production and supports the use of iPSCs for cell replacement therapy of T1DM.

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Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

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