Beta-cell (β-cell) injury is the hallmark of autoimmune diabetes. However, the mechanisms by which autoreactive responses are generated in susceptible individuals are not well understood. Extracellular vesicles (EV) are produced by mammalian cells under normal and stressed physiological states. They are an important part of cellular communication, and may serve a role in antigen processing and presentation. Researchers at the McGill University Health Centre hypothesized that isolated human islets in culture produce EV that contain diabetes autoantigens (DAA) from these otherwise normal, non-diabetic donors. Here they report the caspase-independent production of EV by human islets in culture, and the characterization of DAA glutamic acid decarboxylase 65 (GAD65) and zinc transporter 8 (ZnT8), as well as the β-cell resident glucose transporter 2 (Glut2), present within the EV.
Schema describing the workflow to generate and analyze
human islet conditioned media extracellular vesicles
Islet isolation was performed after the surgical retrieval of a donor pancreas. The pancreas was enzymatically digested in a closed loop circuit until free islets were observed. The islets were then purified, and cultured for up to 72 hours. Islet conditioned medium (ICM) was harvested, centrifuged at 1,200 × g for 15 min. and analyzed by small particle flow cytometry for DAA and markers of EV, or serially centrifuged a 50,000 × g and 200,000 × g for analysis by transmission electron microscopy or mass spectrometry on the ICM-50 K and ICM-200 K fractions.