The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Abstract:Cell-based therapies have the potential to revolutionize current treatments for diseases with high prevalence and related economic and social burden. Unfortunately, clinical trials have made only modest improvements in restoring normal function to degenerating tissues. This limitation is due, at least in part, to the death of transplanted cells within a few hours after transplant due to a combination of mechanical, cellular, and host factors. In particular, mechanical stress during implantation, extracellular matrix loss upon delivery, nutrient and oxygen deprivation at the recipient site, and host inflammatory response are detrimental factors limiting long-term transplanted cell survival. The beneficial effect of cell therapy for regenerative medicine ultimately depends on the number of administered cells reaching the target tissue, their viability, and their promotion of tissue regeneration. Therefore, strategies aiming at improving viable cell engraftment are crucial for regenerative medicine. Here we review the major factors that hamper successful cell engraftment and the strategies that have been studied to enhance the beneficial effects of cell therapy. Moreover, we provide a perspective on whether mesenchymal stromal cell-derived extracellular vesicle delivery, as a cell-free regenerative approach, may circumvent current cell therapy limitations.

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50À200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to

Extracellular vesicles (EVs) are newly appreciated regulators of tissue homeostasis and a means of intercellular communication. Reports have investigated the role of EVs and their cargoes in cellular regulation and have tried to fine-tune their biotechnological use, but to date very little is known on their function in bone biology. To investigate the relevance of EV-mediated communication between bone cells, we isolated EVs from primary mouse osteoblasts and assessed membrane integrity, size, and structure by transmission electron microscopy (TEM) and fluorescence-activated cell sorting (FACS). EVs actively shuttled loaded fluorochromes to osteoblasts, monocytes, and endothelial cells. Moreover, osteoblast EVs contained mRNAs shared with donor cells. Osteoblasts are known to regulate osteoclastogenesis, osteoclast survival, and osteoclast function by the pro-osteoclastic cytokine, receptor activator of nuclear factor k-B ligand (Rankl). Osteoblast EVs were enriched in Rankl, which increased after PTH treatment. These EVs were biologically active, supporting osteoclast survival. EVs isolated from rankl -/-osteoblasts lost this pro-osteoclastic function, indicating its Rankldependence. They integrated ex vivo into murine calvariae, and EV-shuttled fluorochromes were quickly taken up by the bone upon in vivo EV systemic administration. Rankl -/-mice lack the osteoclast lineage and are negative for its specific marker tartrate-resistant acid phosphatase (TRAcP). Treatment of rankl -/-mice with wild-type osteoblast EVs induced the appearance of TRAcP-positive cells in an EV density-dependent manner. Finally, osteoblast EVs internalized and shuttled anti-osteoclast drugs (zoledronate and dasatinib), inhibiting osteoclast activity in vitro and in vivo. We conclude that osteoblast EVs are involved in intercellular communication between bone cells, contribute to the Rankl pro-osteoclastic effect, and shuttle anti-osteoclast drugs, representing a potential means of targeted therapeutic delivery.

ABSTRACT. Objective. The objective of this study was to evaluate the roles of production and conjugation of bilirubin, individually and in combination, in the mechanism of neonatal jaundice.Methods. A cohort of healthy, term male newborns was sampled on the third day of life, coincident with routine metabolic screening, for blood carboxyhemoglobin determination, a reflection of heme catabolism, and for serum unconjugated and conjugated bilirubin fractions, reflecting bilirubin conjugation. The former was determined by gas chromatography, corrected for inspired CO (COHbc), and expressed as percentage of total hemoglobin. Serum bilirubin fractions were quantified by alkaline methanolysis and reverse phase high performance liquid chromatography. The sum of all bilirubin fractions comprised serum total bilirubin (STB). Conclusions. Within the range of STB concentrations encountered, both increasing bilirubin production and diminishing bilirubin conjugation contributed to STB. The production/conjugation index confirmed that imbalance between production and conjugation of bilirubin plays an important role in the mechanism of neonatal bilirubinemia. Pediatrics 2002;110(4). URL: http://www. pediatrics.org/cgi/content/full/110/4/e47; alkaline methanolysis, bilirubin, bilirubin conjugation, carbon monoxide, carboxyhemoglobin, gas chromatography, hemolysis, high performance liquid chromatography, physiologic jaundice. Total conjugated bilirubin (TCB) was comprised of the sum of the conjugated fractions and was expressed as percentage of STB (TCB[%]). A "bilirubin production/conjugation index" (COHbc/[TCB(%)]ABBREVIATIONS. STB, serum total bilirubin; CO, carbon monoxide; COHb, carboxyhemoglobin; COHbc, COHb corrected for inspired CO; UGT, uridine diphosphoglucuronate glucuronosyltransferase 1A1; G-6-PD, glucose-6-phosphate dehydrogenase; TCB, serum total conjugated bilirubin. J aundice is common during the first days of postnatal life and affects almost two thirds of human newborns. The mechanism of this bilirubinemia is multifactorial, as recently summarized, and comprises primarily processes contributing to increased bilirubin load, or diminished bilirubin clearance. 1,2 The former may be the result of factors that augment bilirubin production and the enterohepatic circulation, whereas the latter is primarily the result of immature conjugative capacity, although impaired hepatic uptake or excretion may also play a part. It has been suggested that serum total bilirubin (STB) concentrations that remain within the physiologic range result from equilibrium between bilirubin production and elimination. In contrast, in some neonates, imbalance between these processes may occur, with bilirubin production being relatively higher than conjugation. This imbalance is thought to result in hyperbilirubinemia. 3 Assessment of the role of hemolysis may be accomplished through assessment of endogenous carbon monoxide (CO) production by accurately measuring blood carboxyhemoglobin (COHb), or end tidal CO, both with correction for ambient ...