2. Exosomes from Refeed?-supplemented hFM-MSCs enhance their migration ability without modifying vasculogenic properties. decrease was mainly due to a different rate of exosomal exocytosis rather than to an effect of the lipid supplement on the endocytic pathway. Endoplasmic reticulum homeostasis was modified by supplementation, through the upregulation of PKR-like ER kinase (PERK) and inositol-requiring enzyme 1 (IRE1). Increased expression of these proteins did not lead to stress-induced, unfolded protein response (UPR)-mediated apoptosis, nor did it affect phosphorylation of p38 kinase, suggesting that PERK and IRE1 overexpression was due to augmented metabolic activities mediated by optimization of a cellular feeding network afforded through lipid supplementation. In summary, these results demonstrate how tailored lipid supplementation can successfully modify the paracrine features in hFM-MSCs, impacting both intracellular vesicle trafficking and secreted exosome Amicarbazone number and function. different mesenchymal lineage-derived cells, such as osteoblasts, chondrocytes, and adipocytes1, but also cardiac-like cells2, endothelial cells3,4, and even ectodermal lineage cells5. Often, however, therapeutic benefits mediated by MSC transplantation appear to be mainly due to a secretome-based paracrine activity, rather than a substantial MSC differentiation6,7. Secretome-mediated MSC beneficial Amicarbazone effects are well documented in several clinical conditions8, such as cardiac diseases9C12, central nervous system disorders13C15, renal injury16, articular cartilage defects17C21, spontaneous tendon lesions22, and rheumatic diseases23. We have already demonstrated that transplantation of human MSCs (hMSCs) into infarcted rat hearts enhanced cardiac repair, increasing capillary density, normalizing left ventricular function, and decreasing scar tissue7. These pleiotropic effects were partially due to hMSC secretion of trophic mediators, such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), acting in a paracrine way on different cellular elements of the heart. Its now clear that MSCs secrete a wide range of bioactive molecules, with various effects on tissue-resident cells, such as promoting angiogenesis24, enhancing proliferative capability, and inhibiting apoptosis25 and fibrosis26 and many Amicarbazone others27. The secretome released from MSCs is not only formed by naked molecules (cytokines, chemokines, growth factors, and metabolites) but also by different kinds of extracellular membrane vesicles including exosomes, microvesicles, microparticles, nanovesicles, and others. Exosomes are a Mouse monoclonal antibody to Hexokinase 1. Hexokinases phosphorylate glucose to produce glucose-6-phosphate, the first step in mostglucose metabolism pathways. This gene encodes a ubiquitous form of hexokinase whichlocalizes to the outer membrane of mitochondria. Mutations in this gene have been associatedwith hemolytic anemia due to hexokinase deficiency. Alternative splicing of this gene results infive transcript variants which encode different isoforms, some of which are tissue-specific. Eachisoform has a distinct N-terminus; the remainder of the protein is identical among all theisoforms. A sixth transcript variant has been described, but due to the presence of several stopcodons, it is not thought to encode a protein. [provided by RefSeq, Apr 2009] characterized population of extracellular vesicles (EVs), with a diameter ranging from 30 to 150 nm28,29, and their protein, RNA, and lipid compositions are catalogued in a dedicated database, ExoCarta30. Unlike microvesicles, that originate at the cellular surface and are released by direct budding of plasma membrane, exosomes are generated within multivesicular bodies (MVBs) through an endolysosomal pathway and released by membrane fusion of MVBs with plasma membrane. Due to its origin, exosome membrane presents endosomal proteins, such as CD9, CD63, and CD81, frequently used for immunoaffinity isolation31. The exact mechanism and regulation of exosome secretion is not yet clear32. There is some evidence that secretion is not completely constitutive but can be modulated by different endogenous and exogenous stimuli33. Furthermore, the exact mechanism of exosome internalization by neighboring cells has not been not fully elucidated. EVs released in the environment can be incorporated into recipient cells by different mechanisms including phagocytosis, endocytosis, pinocytosis, and fusion with plasma membrane34. Once englobed, exosomes could be led to different fates. In one way, exosomes merge into endosomes, undergo transcytosis, and are released into the extracellular space without any processing. In another way, fusion of endosomes with lysosomes compels exosomes to degradation35,36. Unfortunately, Amicarbazone there is little evidence about regulatory mechanisms involved in exosome internalization even if exosome uptake appears to be cell typeCspecific37,38. In recent years, MSC-derived exosomes have received an increasing scientific interest due to their emerging regenerative potential. Furthermore, bypassing problems concerning.