Institute of Medical Biology

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.

Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for and proteins and and protein and The is by of of and we of of and that and bodies degraded in inclusion bodies degraded by autophagy. The and we is in degradation of that proteins in cytosolic bodies for bodies and that is for and degradation of bodies by autophagy. Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for and proteins and and protein and The is by of of and we of of and that and bodies degraded in inclusion bodies degraded by autophagy. The and we is in degradation of that proteins in cytosolic bodies for bodies and that is for and degradation of bodies by autophagy. for protein and The is for degradation of and proteins with The and proteins by and by autophagy. of autophagy in of and autophagy is of of with and is to degradation for degradation of of proteins and protein for of and The is is by to cytosolic of to of that is with The is of autophagy and and and The of in autophagy is of autophagy cellular to avoid accumulation of proteins in neurodegenerative in of accumulation of protein aggregates in and show of with of that of autophagy by accumulation of protein aggregates that is basal constitutive autophagy that is in to avoid accumulation of protein aggregates to bodies in to various and to in for polyubiquitinated proteins to degradation inclusion bodies proteins of proteins to of and is of that aggregates that by of aggregates is found in inclusion bodies polyubiquitinated protein neurodegenerative is in protein bodies in in and aggregates in protein of of found in bodies of and bodies in and in aggregates The protein is to and It and we that to proteins and and to and to in found to for we to show that is for degradation of inclusion bodies that inclusion bodies and that is for and to and polyubiquitinated proteins and and and The and and and and and and in in in for and by in in and The and with The for and in with and with and with by and The and to and in of with of with of to and to by The protein in and and and proteins in proteins proteins and with in proteins proteins in with of with of proteins in of with for and with of with proteins in of with of in of for with of with and to with with and proteins with proteins to and by with with of proteins to with of in of for with of with of and to and The with by with in and in with and and for to with with with with and and and with for and in and with of and to and with by protein in we in with to that and with is is of It is is for of we and proteins by with and with and The protein in protein protein of we and The and by protein in protein we proteins and by and protein of and protein in is and we with proteins found to and in to of of to found to and avoid of of of and of we of of of of we of of that in in of and to with to to of by with of in of to of and of is for with for to and of and of of by and of by in and in of of of proteins in The show of and with of of by and of to and of The of of to of of and of and of by and in of by to for with of in The is and to in and of in and of we to we in to and in to in and to is by is by of and is that is of is with that of to found that of of and and and of of and of The of with and that of with in to with with of in to and and is of of found in of proteins to to we and in and to of by with in with and with we we and and with in in of by to and proteins found to to is in in of is for to and proteins with with of and of proteins cellular of and to by with proteins with and in to proteins of and with with in and in and with and of and proteins to bodies in with and and and for of proteins to bodies show that is to in to and that proteins to bodies in of we for to of with in bodies It to and in in aggregates that polyubiquitinated proteins and is of that is with of to to by with to The of of to and of and of is that to with to inclusion bodies to to and we protein to The of is protein with we to with of and that and of that in is to of proteins in to to inclusion bodies and and in we to in of and in of and of in that of of and of proteins of is of that of and degradation of with accumulate with found in with to that of in of that is in in found in and The to of to by The in and and of inclusion bodies and with of to with with and with of degraded we in of in of and in of with and to is and that and to protein of proteins with with for of of The of bodies in for that with to and we for with with and and is for and is in we found and of in of with with with to inclusion in inclusion and with with and of is with for of and in with and that we in and in The to by to in is and in inclusion bodies and in in to and in of autophagy in of of is of of in is autophagy to is of of and of in of to in to of that with with and of bodies is autophagy. with and in for to to and autophagy to by is is of inclusion bodies that degraded by autophagy. It is to bodies and of that with proteins and for in we to bodies of with we that bodies degraded by autophagy. in is of by of of in of is of is in for inclusion bodies by with to of in of and in various and and The of in with of by and that bodies degraded by of bodies with and with with with to to for of for of of inclusion bodies is of various is to of and and of cytosolic bodies and polyubiquitinated proteins with in by and in of The of protein by with of with of of we found that bodies and in of and inclusion bodies and for and of bodies is with with to of and and with and of protein bodies with of The of protein bodies is by of to for of bodies for that for with in of to in that of in of in proteins to in bodies that degraded by autophagy. and in in of of proteins to with for and with and of of with cellular of that with to proteins and and for of of with and to proteins that proteins in together with and of accumulation of in with we in of proteins in of that in degradation of polyubiquitinated proteins with to with of we in and for of in of proteins with protein with in of is with by of of of for of and of of in of and of and of in of we in of in of for is in to of is for of with in together with and of inclusion bodies in inclusion by of in and of in with and of of with that is for of of is for degradation of of to and with The of to of with and to and with to inclusion with and and of is important for of to and with of and and with of and of of for of to is important for degradation of in together with we of that in with protein of of to with with that bodies by in of that of is we that to that that of of is for degradation of and we show that to and and and is protein in The and is by in and and of and and and and to that and is for degradation of bodies polyubiquitinated by autophagy. polyubiquitinated proteins and to protein bodies that degraded by to of proteins and is to with and to and and of in of and of we by with for autophagy is to of The of to with with to to is to to protein and and in and we to and and of and that of is in and The for and is to and bodies that The is to proteins and The to and degradation of by is degraded by we and found of autophagy and of to is important to in that by of inclusion bodies is proteins we that bodies and proteins degraded by autophagy. in and in protein bodies by autophagy. bodies to to we accumulation of in in that is that bodies degraded by basal constitutive autophagy to by that in autophagy with of we show that in of inclusion bodies and to with The found for inclusion with proteins show that to and of and is to bodies with of It important to proteins that and to show that and inclusion bodies and that of bodies by of The bodies in p62/SQSTM1 of of and of to to of of by of of in to and with It important to in protein degradation by autophagy is to show is protein of Protein and in to of important to autophagy of and in It to show neurodegenerative of neurodegenerative proteins in important for protein and The is for degradation of and proteins with The and proteins by and by autophagy. of autophagy in of and autophagy is of of with and is to degradation for degradation of of proteins and protein for of and The is is by to cytosolic of to of that is with The is of autophagy and and and The of in autophagy is of autophagy cellular to avoid accumulation of proteins in neurodegenerative in of accumulation of protein aggregates in and show of with of that of autophagy by accumulation of protein aggregates that is basal constitutive autophagy that is in to avoid accumulation of protein aggregates to bodies in to various and to in for polyubiquitinated proteins to degradation inclusion bodies proteins of proteins to of and is of that aggregates that by of aggregates The is found in inclusion bodies polyubiquitinated protein neurodegenerative is in protein bodies in in and aggregates in protein of of found in bodies of and bodies in and in aggregates The protein is to and It and Here we that to proteins and and to and to in found to for we to show that is for degradation of inclusion bodies that inclusion bodies and that is for and to and polyubiquitinated proteins and and and The and and and and and and in in in for and by in in and The and with The for and in with and with and with by and The and to and in of with of with of to and to by The protein in and and and proteins in proteins proteins and with in proteins proteins in with of with of proteins in of with for and with of with proteins in of with of in of for with of with and to with with and proteins with proteins to and by with with of proteins to with of in of for with of with of and to and The with by with in and in with and and for to with with with with and and and with for and in and with of and to and with by protein in we in with and to and polyubiquitinated proteins and and and The and and and and and and in in in for and by and The and with The for and in with and with and with by and The and to and in of with of with of to and to by The protein in and and and proteins in proteins proteins and with in proteins proteins in with of with of proteins in of with for and with of with proteins in of with of in of for with of with and to with with and proteins with proteins to and by with with of proteins to with of in of for with of with of and to and The with by with in and in with and and for to with with with with and and and with for and in and with of and to and with by protein in we in with to that and with is is of It is is for of we and proteins by with and with and The protein in protein protein of we and The and by protein in protein we proteins and by and protein of and protein in is and we with proteins found to and in to of of to found to and avoid of of of and of we of of of of we of of that in in of and to with to to of by with of in of to of and of of by and of to and of The of of to of of and of and of by and in of by to for with of in The is and to in and of in and of we to we in to and in to in and to is by is by of and is that is of is with that of to found that of of and and and of of and of The of with and that of with in to with with of in to and and is of of found in of proteins to to we and in and to of by with in with and with we we and and with in in of by to and proteins found to to is in in of is for to and proteins with with of and of proteins cellular of and to by with proteins with and in to proteins of and with with in and in and with and of and proteins to bodies in with and and and for of proteins to bodies show that is to in to and that proteins to bodies in of we for to of with in bodies It to and in in aggregates that polyubiquitinated proteins and is of that is with of to to by with to The of of to and of and of is that to with to inclusion bodies to to and we protein to The of is protein with we to with of and that and of that in is to of proteins in to to inclusion bodies and and in we to in of and in of and of in that of of and of proteins of is of that of and degradation of with accumulate with found in with to that of in of that is in in found in and The to of to by The in and and of inclusion bodies and with of to with with and with of degraded we in of in of and in of with and to is and that and to protein of proteins with with for of of The of bodies in for that with to and we for with with and and is for and is in we found and of in of with with with to inclusion in inclusion and with with and of is with for of and in with and that we in and in The to by to in is and in inclusion bodies and in in to and in of autophagy in of of is of of in is autophagy to is of of and of in of to in to of that with with and of bodies is autophagy. with and in for to to and autophagy to by is is of inclusion bodies that degraded by autophagy. It is to bodies and of that with proteins and for in we to bodies of with we that bodies degraded by autophagy. in is of by of of in of is of is in for inclusion bodies by with to of in of and in various and and The of in with of by and that bodies degraded by of bodies with and with with with to to for of for of of inclusion bodies is of various is to of and and of cytosolic bodies and polyubiquitinated proteins with in by and in of The of protein by with of with of of we found that bodies and in of and inclusion bodies and for and of bodies is with with to of and and with and of protein bodies with of The of protein bodies is by of to for of bodies for that for with in of to in that of in of in proteins to in bodies that degraded by autophagy. and in in of of proteins to with for and with and of of with cellular of that with to proteins and and for of of with and to proteins that proteins in together with and of accumulation of in with we in of proteins in of that in degradation of polyubiquitinated proteins with to with of we in and for of in of proteins with protein with in of is with by of of of for of and of of in of and of and of in of we in of in of for is in to of is for of with in together with and of inclusion bodies in inclusion by of in and of in with and of of with that is for of of is for degradation of of to and with The of to of with and to and with to inclusion with and and of is important for of to and with of and and with of and of of for of to is important for degradation of in together with we of that in with protein of of to with with that bodies by in of that of is we that to that that of of is for degradation of and p62/SQSTM1 to that and with is is of It is is for of we and proteins by with and with and The protein in protein protein of we and The and by protein in protein we proteins and by and protein of and protein in is and we with proteins found to and in to of of to found to and avoid of of of and of we of of of of we of of that in in of and to with to to of by with of in of to of and The is and to in and of in and of we to we in to and in to in and to is by is by of and is that is of is with that of to found that of of and and and of of and of The of with and that of with in to with with of in to and and is of of found in of proteins to to we and in and to of by with in with and with we we and and with in in of by to and proteins found to to is in in of is for to and proteins of and proteins to bodies in with and and and for of proteins to bodies show that is to in to and that proteins to bodies in of we for to of with in bodies It to and in in aggregates that polyubiquitinated proteins and is of that is with of to to by with to The of of to and of and of is that to with to inclusion bodies to to and we protein to The of is protein with we to with of and that and of that in is to of proteins in to to inclusion bodies and and in we to in of and in of and of in that of of and of proteins of is of that of and degradation of with accumulate with found in with to that of in of that is in in found in and The to of to by The in and and of inclusion bodies and with of to with with and with of degraded we in of in of and in of with and to is and that and that with to and we for with with and and is for and is in we found and of in of with with with that we in and in The to by to in is and in inclusion bodies and in in to and in of autophagy in of of is of of in is autophagy to is of of and of in of to in to of that with with and by is is of inclusion bodies that degraded by autophagy. It is to bodies and of that with proteins and for in we to bodies of with we that bodies degraded by autophagy. in is of by of of in of is of is in for inclusion bodies by with to of in of and in various and and The of in with of by and that bodies degraded by autophagy. for of of inclusion bodies is of various is to of and and of cytosolic bodies and polyubiquitinated proteins with in by and in of The of protein by with of with of of we found that bodies and in of and for of of with and to proteins that proteins in together with and of accumulation of in with we in of proteins in of that in degradation of polyubiquitinated proteins with to with of we in and for of in of proteins with protein with in of is with by of of The of for of and of of in of and of and of in of we in of in of for is in to of is for of with in together with and of inclusion bodies in inclusion by of in and of in with and of of with that is for of is important for degradation of in together with we of that in with protein of of to with with that bodies by in of that of is we that to that that of of is for degradation of and we show that to and and and is protein in The and is by in and and of and and and and to that and is for degradation of bodies polyubiquitinated by autophagy. polyubiquitinated proteins and to protein bodies that degraded by to of proteins and is to with and to and and of in of and of we by with for autophagy is to of The of to with with to to is to to protein and and in and we to and and of and that of is in and The for and is to and bodies that The is to proteins and The to and degradation of by is degraded by we and found of autophagy and of to is important to in that by of inclusion bodies is proteins we that bodies and proteins degraded by autophagy. in and in protein bodies by autophagy. bodies to to we accumulation of in in that is that bodies degraded by basal constitutive autophagy to by that in autophagy with of we show that in of inclusion bodies and to with The found for inclusion with proteins show that to and of and is to bodies with of It important to proteins that and to show that and inclusion bodies and that of bodies by of The bodies in p62/SQSTM1 of of and of to to of of by of of in to and with It important to in protein degradation by autophagy is to show is protein of Protein and in to of important to autophagy of and in It to show neurodegenerative of neurodegenerative proteins in important we show that to and and and is protein in The and is by in and and of and and and and to that and is for degradation of bodies polyubiquitinated by autophagy. polyubiquitinated proteins and to protein bodies that degraded by autophagy. to of proteins and is to with and to and and of in of and of we by with for autophagy is to of The of to with with to to is to to protein and and in and we to and and of and that of is in and The for and is to and bodies that The is to proteins and The to and degradation of by is degraded by we and found of autophagy and of to is important to in that by of inclusion bodies is proteins we that bodies and proteins degraded by autophagy. in and in protein bodies by autophagy. bodies to to we accumulation of in in that is that bodies degraded by basal constitutive autophagy to by that in autophagy with of we show that in of inclusion bodies and to with The found for inclusion with proteins show that to and of and is to bodies with of It important to proteins that and to show that and inclusion bodies and that of bodies by of The bodies in p62/SQSTM1 of of and of to to of of by of of in to and with It important to in protein degradation by autophagy is to show is protein of Protein and in to of important to autophagy of and in It to show neurodegenerative of neurodegenerative proteins in important to for of to for of and for for with to for of and for of with with

We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment.

Mounting evidence suggests that autophagy is a more selective process than originally anticipated. The discovery and characterization of autophagic adapters, like p62 and NBR1, has provided mechanistic insight into this process. p62 and NBR1 are both selectively degraded by autophagy and able to act as cargo receptors for degradation of ubiquitinated substrates. A direct interaction between these autophagic adapters and the autophagosomal marker protein LC3, mediated by a so-called LIR (LC3-interacting region) motif, their inherent ability to polymerize or aggregate as well as their ability to specifically recognize substrates are required for efficient selective autophagy. These three required features of autophagic cargo receptors are evolutionarily conserved and also employed in the yeast cytoplasm-to-vacuole targeting (Cvt) pathway and in the degradation of P granules in C. elegans. Here, we review the mechanistic basis of selective autophagy in mammalian cells discussing the degradation of misfolded proteins, p62 bodies, aggresomes, mitochondria and invading bacteria. The emerging picture of selective autophagy affecting the regulation of cell signaling with consequences for oxidative stress responses, tumorigenesis and innate immunity is also addressed.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.

Melanoma, a skin cancer associated with high mortality rates, is highly radio- and chemotherapy resistant but can also be very immunogenic. These circumstances have led to a recent surge in research into therapies aiming to boost anti-tumor immune responses in cancer patients. Among these immunotherapies, neutralizing antibodies targeting the immune checkpoints T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) are being hailed as particularly successful. These antibodies have resulted in dramatic improvements in disease outcome and are now clinically approved in many countries. However, the majority of advanced stage melanoma patients do not respond or will relapse, and the hunt for the "magic bullet" to treat the disease continues. This review examines the mechanisms of action and the limitations of anti-PD-1/PD-L1 and anti-CTLA-4 antibodies which are the two types of checkpoint inhibitors currently available to patients and further explores the future avenues of their use in melanoma and other cancers.

Chronic wounds are a health problem that have devastating consequences for patients and contribute major costs to healthcare systems and societies. To understand the magnitude of this health issue, a systematic review was undertaken. Searches were conducted in MEDLINE, EMBASE, EBM Reviews and Cochrane library, CINAHL, EBSCO, PsycINFO, and Global Health databases for articles published between 2000 and 2015. Included publications had to target adults (≥18 years of age), state wound chronicity (≥3 weeks) and/or label the wounds as chronic, complex, hard-to-heal, or having led to an amputation. The review excluded studies that did not present data on generic health-related quality of life and/or cost data, case studies, randomized controlled trials, economic modeling studies, abstracts, and editorials. Extracted data were summarized into a narrative synthesis, and for a few articles using the same health-related quality of life instrument, average estimates with 95% confidence intervals were calculated. Thirty articles met the inclusion criteria. Findings revealed that health-related quality of life was lowest for physical pathologies, and based on average estimates were scores most inferior in the domain physical role for both patients with chronic wounds and for those with wound-related amputations. The cost burden was mainly attributed to amputations for patients also comorbid with diabetes, where the cost for hospitalization ranged from US$12,851 to US$16,267 (median) for this patient group. Patients with chronic wounds have poor health-related quality of life in general and wound-related costs are substantial. Development and implementation of wound management strategies that focus on increasing health-related quality of life and effectively reduce costs for this patient group are urgently needed.

Mesenchymal stem cells (MSC) hold great potential for regenerative medicine because of their ability for self-renewal and differentiation into tissue-specific cells such as osteoblasts, chondrocytes, and adipocytes. MSCs orchestrate tissue development, maintenance and repair, and are useful for musculoskeletal regenerative therapies to treat age-related orthopedic degenerative diseases and other clinical conditions. Importantly, MSCs produce secretory factors that play critical roles in tissue repair that support both engraftment and trophic functions (autocrine and paracrine). The development of uniform protocols for both preparation and characterization of MSCs, including standardized functional assays for evaluation of their biological potential, are critical factors contributing to their clinical utility. Quality control and release criteria for MSCs should include cell surface markers, differentiation potential, and other essential cell parameters. For example, cell surface marker profiles (surfactome), bone-forming capacities in ectopic and orthotopic models, as well as cell size and granularity, telomere length, senescence status, trophic factor secretion (secretome), and immunomodulation, should be thoroughly assessed to predict MSC utility for regenerative medicine. We propose that these and other functionalities of MSCs should be characterized prior to use in clinical applications as part of comprehensive and uniform guidelines and release criteria for their clinical-grade production to achieve predictably favorable treatment outcomes for stem cell therapy. Stem Cells Translational Medicine 2017;6:2173-2185.

Methylation of DNA is an essential epigenetic control mechanism in mammals. During embryonic development, cells are directed toward their future lineages, and DNA methylation poses a fundamental epigenetic barrier that guides and restricts differentiation and prevents regression into an undifferentiated state. DNA methylation also plays an important role in sex chromosome dosage compensation, the repression of retrotransposons that threaten genome integrity, the maintenance of genome stability, and the coordinated expression of imprinted genes. However, DNA methylation marks must be globally removed to allow for sexual reproduction and the adoption of the specialized, hypomethylated epigenome of the primordial germ cell and the preimplantation embryo. Recent technological advances in genome-wide DNA methylation analysis and the functional description of novel enzymatic DNA demethylation pathways have provided significant insights into the molecular processes that prepare the mammalian embryo for normal development.

Mesenchymal stem cells (MSCs) have been shown to secrete exosomes that are cardioprotective. Here, we demonstrated that MSC exosome, a secreted membrane vesicle, is immunologically active. MSC exosomes induced polymyxin-resistant, MYD88-dependent secreted embryonic alkaline phosphatase (SEAP) expression in a THP1-Xblue, a THP-1 reporter cell line with an NFκB-SEAP reporter gene. In contrast to lipopolysaccharide, they induced high levels of anti-inflammatory IL10 and TGFβ1 transcript at 3 and 72 h, and much attenuated levels of pro-inflammatory IL1B, IL6, TNFA and IL12P40 transcript at 3-h. The 3-h but not 72-h induction of cytokine transcript was abrogated by MyD88 deficiency. Primary human and mouse monocytes exhibited a similar exosome-induced cytokine transcript profile. Exosome-treated THP-1 but not MyD88-deficient THP-1 cells polarized activated CD4(+) T cells to CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) at a ratio of one exosome-treated THP-1 cell to 1,000 CD4(+) T cells. Infusion of MSC exosomes enhanced the survival of allogenic skin graft in mice and increased Tregs.

Intercellular exchange of protein and RNA-containing microparticles is an increasingly important mode of cell-cell communication. Here we investigate if mesenchymal stem cells (MSCs) known for secreting therapeutic paracrine factors also secrete RNA-containing microparticles. We observed that human embryonic stem cell (hESC)-derived MSC conditioned medium contained small RNAs (less than 300 nt) encapsulated in cholesterol-rich phospholipid vesicles as evidenced by their RNase sensitivity only in the presence of a sodium dodecyl sulfate-based cell lysis buffer, phospholipase A2 and a chelator of cholesterol, cyclodextrin and the restoration of their lower than expected density by detergent or phospholipase A2 treatment. MicroRNAs (miRNAs) such as hsa-let-7b and hsa-let-7g were present in a high precursor (pre)- to mature miRNA ratio by microarray analysis and quantitative reverse transcription-polymerase chain reaction. The pre-miRNAs were cleaved to mature miRNA by RNase III in vitro. High performance liquid chromatography-purified RNA-containing vesicles have a hydrodynamic radius of 55-65 nm and were readily taken up by H9C2 cardiomyocytes. This study suggests that MSCs could facilitate miRNA-mediated intercellular communication by secreting microparticles enriched for pre-miRNA.

OBJECTIVE: Clinical and animal studies have demonstrated the efficacy of mesenchymal stem cell (MSC) therapies in cartilage repair. As the efficacy of many MSC-based therapies has been attributed to paracrine secretion, particularly extracellular vesicles/exosomes, we determine here if weekly intra-articular injections of human embryonic MSC-derived exosomes would repair and regenerate osteochondral defects in a rat model. METHODS: In this study, osteochondral defects were created on the trochlear grooves of both distal femurs in 12 adult rats. In each animal, one defect was treated with 100 μg exosomes and the contralateral defect treated with phosphate buffered saline (PBS). Intra-articular injections of exosomes or PBS were administered after surgery and thereafter weekly for a period of 12 weeks. Three unoperated age-matched animals served as native controls. Analyses were performed by histology, immunohistochemistry, and scoring at 6 and 12 weeks after surgery. RESULTS: Generally, exosome-treated defects showed enhanced gross appearance and improved histological scores than the contralateral PBS-treated defects. By 12 weeks, exosome-treated defects displayed complete restoration of cartilage and subchondral bone with characteristic features including a hyaline cartilage with good surface regularity, complete bonding to adjacent cartilage, and extracellular matrix deposition that closely resemble that of age-matched unoperated control. In contrast, there were only fibrous repair tissues found in the contralateral PBS-treated defects. CONCLUSION: This study demonstrates for the first time the efficacy of human embryonic MSC exosomes in cartilage repair, and the utility of MSC exosomes as a ready-to-use and 'cell-free' therapeutic alternative to cell-based MSC therapy.

ABSTRACT Small extracellular vesicles (sEVs) from mesenchymal stromal/stem cells (MSCs) are transiting rapidly towards clinical applications. However, discrepancies and controversies about the biology, functions, and potency of MSC‐sEVs have arisen due to several factors: the diversity of MSCs and their preparation; various methods of sEV production and separation; a lack of standardized quality assurance assays; and limited reproducibility of in vitro and in vivo functional assays. To address these issues, members of four societies (SOCRATES, ISEV, ISCT and ISBT) propose specific harmonization criteria for MSC‐sEVs to facilitate data sharing and comparison, which should help to advance the field towards clinical applications. Specifically, MSC‐sEVs should be defined by quantifiable metrics to identify the cellular origin of the sEVs in a preparation, presence of lipid‐membrane vesicles, and the degree of physical and biochemical integrity of the vesicles. For practical purposes, new MSC‐sEV preparations might also be measured against a well‐characterized MSC‐sEV biological reference. The ultimate goal of developing these metrics is to map aspects of MSC‐sEV biology and therapeutic potency onto quantifiable features of each preparation.

BACKGROUND: Aging and aging-related disorders impair the survival and differentiation potential of bone marrow mesenchymal stem cells (MSCs) and limit their therapeutic efficacy. Induced pluripotent stem cells (iPSCs) may provide an alternative source of functional MSCs for tissue repair. This study aimed to generate and characterize human iPSC-derived MSCs and to investigate their biological function for the treatment of limb ischemia. METHODS AND RESULTS: Human iPSCs were induced to MSC differentiation with a clinically compliant protocol. Three monoclonal, karyotypically stable, and functional MSC-like cultures were successfully isolated using a combination of CD24(-) and CD105(+) sorting. They did not express pluripotent-associated markers but displayed MSC surface antigens and differentiated into adipocytes, osteocytes, and chondrocytes. Transplanting iPSC-MSCs into mice significantly attenuated severe hind-limb ischemia and promoted vascular and muscle regeneration. The benefits of iPSC-MSCs on limb ischemia were superior to those of adult bone marrow MSCs. The greater potential of iPSC-MSCs may be attributable to their superior survival and engraftment after transplantation to induce vascular and muscle regeneration via direct de novo differentiation and paracrine mechanisms. CONCLUSIONS: Functional MSCs can be clonally generated, beginning at a single-cell level, from human iPSCs. Patient-specific iPSC-MSCs can be prepared as an "off-the-shelf" format for the treatment of tissue ischemia.

Defects in nuclear morphology often correlate with the onset of disease, including cancer, progeria, cardiomyopathy, and muscular dystrophy. However, the mechanism by which a cell controls its nuclear shape is unknown. Here, we use adhesive micropatterned surfaces to control the overall shape of fibroblasts and find that the shape of the nucleus is tightly regulated by the underlying cell adhesion geometry. We found that this regulation occurs through a dome-like actin cap that covers the top of the nucleus. This cap is composed of contractile actin filament bundles containing phosphorylated myosin, which form a highly organized, dynamic, and oriented structure in a wide variety of cells. The perinuclear actin cap is specifically disorganized or eliminated by inhibition of actomyosin contractility and rupture of the LINC complexes, which connect the nucleus to the actin cap. The organization of this actin cap and its nuclear shape-determining function are disrupted in cells from mouse models of accelerated aging (progeria) and muscular dystrophy with distorted nuclei caused by alterations of A-type lamins. These results highlight the interplay between cell shape, nuclear shape, and cell adhesion mediated by the perinuclear actin cap.

Under hypoxia, tumor cells produce a secretion that modulates their microenvironment to facilitate tumor angiogenesis and metastasis. Here, we observed that hypoxic or reoxygenated A431 carcinoma cells exhibited enhanced angiogenic and metastatic potential such as reduced cell-cell and cell-extracellular matrix adhesion, increased invasiveness, and production of a secretion with increased chorioallantoic membrane angiogenic activity. Consistent with these observations, quantitative proteomics revealed that under hypoxia the tumor cells secreted proteins involved in angiogenesis, focal adhesion, extracellular matrix-receptor interaction, and immune cell recruitment. Unexpectedly, the secreted proteins were predominantly cytoplasmic and membrane proteins. Ultracentrifugation at 100,000 x g precipitated 54% of the secreted proteins and enriched for many exosome-associated proteins such as the tetraspanins and Alix and also proteins with the potential to facilitate angiogenesis and metastasis. Two tetraspanins, CD9 and CD81, co-immunoprecipitated. Together, these data suggested that tumor cells secrete proteins and exosomes with the potential to modulate their microenvironment and facilitate angiogenesis and metastasis.

The identification of schizophrenia's negative symptoms dates back to the earliest descriptions of Kraepelin and Bleuler, who each highlighted the central role of avolition in the phenomenology and course of this illness. Since, there have been numerous advances in our understanding of schizophrenia, and the present review tracks the changes that have taken place in our understanding of negative symptoms, their description and measurement. That these symptoms represent a distinct domain of the illness is discussed in the context of their ties to other symptoms and functional outcome. The underlying structure of the negative symptom construct is explored, including several lines of investigation that point towards diminished expression and amotivation as key underlying subdomains. We also discuss findings of intact emotional experience and consummatory pleasure in individuals with schizophrenia, calling into question the presence of anhedonia in this illness. We conclude with a reconceptualization of the negative symptoms, suggesting amotivation (ie, avolition) represents the critical component, particularly in regard to functional outcome. Further exploration and clarification of this core deficit will ultimately enhance our neurobiological understanding of schizophrenia, as well as strategies that may improve outcome.

Cardiovascular disease is a major target for many experimental stem cell-based therapies and mesenchymal stem cells (MSCs) are widely used in these therapies. Transplantation of MSCs to treat cardiac disease has always been predicated on the hypothesis that these cells would engraft, differentiate and replace damaged cardiac tissues. However, experimental or clinical observations so far have failed to demonstrate a therapeutically relevant level of transplanted MSC engraftment or differentiation. Instead, they indicate that transplanted MSCs secrete factors to reduce tissue injury and/or enhance tissue repair. Here we review the evidences supporting this hypothesis including the recent identification of exosome as a therapeutic agent in MSC secretion. In particular, we will discuss the potential and practicality of using this relatively novel entity as a therapeutic modality for the treatment of cardiac disease, particularly acute myocardial infarction.

INTRODUCTION: Mesenchymal stem cell-conditioned medium (MSC-CM) has been shown to have protective effects against various cellular-injury models. This mechanism of protection, however, has yet to be elucidated. Recently, exosomes were identified as the active component in MSC-CM. The aim of this study is to investigate the effect of MSC-derived exosomes in an established carbon tetrachloride (CCl4)-induced liver injury mouse model. This potential effect is then validated by using in vitro xenobiotic-induced liver-injury assays: (1) acetaminophen (APAP)- and (2) hydrogen peroxide (H2O2)-induced liver injury. METHODS: The exosomes were introduced concurrent with CCl4 into a mouse model through different routes of administration. Biochemical analysis was performed based on the blood and liver tissues. Subsequently the exosomes were treated in APAP and H2O2-toxicants with in vitro models. Cell viability was measured, and biomarkers indicative of regenerative and oxidative biochemical responses were determined to probe the mechanism of any hepatoprotective activity observed. RESULTS: In contrast to mice treated with phosphate-buffered saline, CCl4 injury in mice was attenuated by concurrent-treatment exosomes, and characterized by an increase in hepatocyte proliferation, as demonstrated with proliferating cell nuclear antigen (PCNA) elevation. Significantly higher cell viability was demonstrated in the exosomes-treated group compared with the non-exosome-treated group in both injury models. The higher survival rate was associated with upregulation of the priming-phase genes during liver regeneration, which subsequently led to higher expression of proliferation proteins (PCNA and cyclin D1) in the exosomes-treated group. Exosomes also inhibited the APAP- and H2O2-induced hepatocytes apoptosis through upregulation of Bcl-xL protein expression. However, exosomes do not mitigate hepatocyte injury via modulation of oxidative stress. CONCLUSIONS: In summary, these results suggest that MSC-derived exosomes can elicit hepatoprotective effects against toxicants-induced injury, mainly through activation of proliferative and regenerative responses.