ALIX increases protein content and protective function of iPSC-derived exosomes.

Nature of exosome-secreting cells determines exosome content and function. ALIX, involved in exosome biogenesis, promotes cell degeneration. Here, ALIX was knocked out (iPSC-ALIX-/-) and overexpressed (iPSC-ALIX3+) in induced pluripotent stem cells (iPSCs) using CRISPR-Cas9 and lentiviral transduction, respectively, and the secreted exosomes were analyzed. Exosomes from iPSC-ALIX-/- (exosome-KO), iPSC-ALIX3+ (exosome-over), and their corresponding controls contained 176, 529, 431, and 351 proteins, respectively. Exosome-over showed increased protein levels, while exosome-KO contained fewer protein types without differing in total protein content. ALIX knockout did not affect exosome uptake by endothelial cells. Exosome-over more effectively promoted cell viability than exosome-GFP, in a dose-dependent manner. All exosomes were protective for endothelial cells injured by hydrogen peroxide or cisplatin, as demonstrated by promotion of cell viability, horizontal migration, angiogenic sprouting from aortic rings, and formation of capillary-like structures, inhibition of apoptosis, and maintenance of permeability of endothelial monolayer, although exosome-over and exosome-KO had stronger and weaker effects, respectively. SNX2 was important for ALIX-mediated exosomal function. Beneficial functions of the exosomes were independent of experimental models, targeted cell types, causes of injury, exosome-producing iPSC passages, clones of ALIX knockout, and transfection batches of ALIX overexpression. Thus, we present a novel strategy to manipulate iPSCs for production of exosomes with beneficial ALIX-regulated protein composition for varied exosome functions. KEY MESSAGES: ALIX knockout and overexpression regulate protein profile in iPSC-derived exosome. ALIX knockout decreases therapeutic function of iPSC-derived exosomes. ALIX overexpression increases therapeutic function of iPSC-derived exosomes. Manipulating iPSCs can produce exosomes with more beneficial protein content.