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Exosomes: Smart nanospheres for drug delivery naturally produced by stem cells
Abstract
Exosomes are the consummate delivery vehicle for "systems therapeutics." Naturally produced by stem cells, the exosome can be considered a smart liposome given its ability to protect, target, and co-deliver a payload of about 20,000 proteins in space and in time to the target of choice. Thus, an emergent, systems level effect is achieved by the multiple molecule types acting together as a collective at the one target. Given that thousands of exosomes are released from one stem cell type in a short duration of time, the emergent, systems level effects occur across many targets, affecting many pathways.
Supplementary resource (1)
... Bacteria send signals throughout the body by releasing extracellular vesicles, which are exosome-like nanoparticles (Maguire, 2016). In somatic cells, the content, including microRNA, and functional characteristics of exosomes on other cells has been shown to be regulated by environmental inputs (Lo Cicero et al., 2015). ...
... Another promising methodology for the production of therapeutics are cell-free expression and purification of proteins (Sullivan et al., 2016) using lysates of bacteria and yeast. However, such approaches may not benefit from the synergistic properties of the systems therapeutic where a broader collection of therapeutic molecules are included (Maguire, 2014), and the packaging of the molecules into exosomes that provide protection and delivery properties to the molecules is present, as well as possible important post-translational modifications made within the exosome (Maguire, 2016). Because bacteria secrete molecules and exosomes, using the secretome of commensal bacteria may provide a much more useful therapeutic. ...
... Bacteria send signals throughout the body by releasing extracellular vesicles, which are exosome-like nanoparticles (Maguire, 2016). In somatic cells, the content, including microRNA, and functional characteristics of exosomes on other cells has been shown to be regulated by environmental inputs (Lo Cicero et al., 2015). ...
... Another promising methodology for the production of therapeutics are cell-free expression and purification of proteins (Sullivan et al., 2016) using lysates of bacteria and yeast. However, such approaches may not benefit from the synergistic properties of the systems therapeutic where a broader collection of therapeutic molecules are included (Maguire, 2014), and the packaging of the molecules into exosomes that provide protection and delivery properties to the molecules is present, as well as possible important post-translational modifications made within the exosome (Maguire, 2016). Because bacteria secrete molecules and exosomes, using the secretome of commensal bacteria may provide a much more useful therapeutic. ...
Here we offer a review of the evidence for a hypothesis that a combination of ingestible probiotics, prebiotics, postbiotics, and amino acids will help ameliorate dysbiosis and degeneration of the gut, and therefore promote restoration of nervous system function in a number of neurological indications.
... The dermal papilla (DP), a cluster of specialized broblasts in the follicle, secretes diffusible proteins packaged into exosomes that regulate the growth and activity of the various cells in the follicle, thereby playing a key role within the follicle in the regulation of hair cycling and growth (Zhou et al, 2018). Extracellular vesicles (EVs), including exosomes, are the carriers for the distribution of morphogens and growth and differentiation factors (Riazifar et al, 2017), and can pack thousands of proteins into one exosome, delivering that collective cargo to one target in the same time and space (Maguire, 2016). The DP is encapsulated by an overlying matrix of epithelial cells, and growth factors from DP are believed to cause epithelial cells to proliferate and differentiate to produce hair shafts during the anagen phase. ...
Introduction: Alopecia is a chronic dermatological disorder affecting millions of people, in which people lose some or all of the hair on their head.
Although alopecia has many forms, all are characterized as a chronic inflammatory disease that affects the hair follicles. Alopecia often has
psychological consequences, including high levels of anxiety and depression.
Case presentation: We report hair regrowth in 12 of 13 patients with alopecia treated with adult stem cell released molecules in combination with
micro needling.
Conclusion: Adult stem cell released molecules in combination with micro needling is an efficacious, safe, and affordable treatment for alopecia
... The dermal papilla (DP), a cluster of specialized broblasts in the follicle, secretes diffusible proteins packaged into exosomes that regulate the growth and activity of the various cells in the follicle, thereby playing a key role within the follicle in the regulation of hair cycling and growth (Zhou et al, 2018). Extracellular vesicles (EVs), including exosomes, are the carriers for the distribution of morphogens and growth and differentiation factors (Riazifar et al, 2017), and can pack thousands of proteins into one exosome, delivering that collective cargo to one target in the same time and space (Maguire, 2016). The DP is encapsulated by an overlying matrix of epithelial cells, and growth factors from DP are believed to cause epithelial cells to proliferate and differentiate to produce hair shafts during the anagen phase. ...
Introduction: Alopecia is a chronic dermatological disorder affecting millions of people, in which people lose some or all of the hair on their head.
Although alopecia has many forms, all are characterized as a chronic inflammatory disease that affects the hair follicles. Alopecia often has
psychological consequences, including high levels of anxiety and depression.
Case presentation: We report hair regrowth in 12 patients with alopecia treated with adult stem cell released molecules in combination with micro
needling.
Conclusion: Adult stem cell released molecules in combination with micro needling is an efficacious, safe, and affordable treatment for alopecia
... Whereas the quiescent state and collagen production in fibroblasts seems to be induced by adipose-derived mesenchymal stem cells [61], leading to scarless wound healing [14], bone marrow derived mesenchymal stem cells (BMSCs) seem to induce proliferation, migration, and chemotaxis [62], and differentiation of resident stem cells to somatic cells [63] by releasing factors including Growth differentiation factor 11 (GDF11) [64]. However, adipose derived mesenchymal stem cells release molecules, packaged in exosomes [65], that have been shown to switch the ratio of collagen I to collagen III from a scarpromoting high ratio to an anti-scaring low ratio, similar to that seen in fetal wound healing [14]. ...
Wounds, aging, and autoimmune conditions of the skin involve a disruption of skin homeostasis, especially a disruption of proteostasis. In this study we used S2RM technology, a proprietary combination of stem cell released molecules from multiple types of skin stem cells, to renormalize homeostasis of the skin, including a renormalization of proteostasis. Dramatic reductions in scarring, pain, redness, and inflammation, more rapid and complete wound healing, and an overall enhancement of the appearance of the skin were achieved in a number of skin conditions. Prevention of radiation dermatitis was achieved by concurrent topical administration of S2RM during radiation treatment. The current study demonstrates that simple topical application of S2RM technology is a powerful means to renormalize homeostasis of the skin and remediate and prevent a number of skin indications.
... Future directions include exosomes being engineered via nanoparticle-based technology to carry speci c cargo (proteins or surface markers) to regulate intercellular communication in a targeted manner. Technology and engineering approaches are necessary to scale technology for clinical feasibility [177,178]. ...
Current treatment strategies for osteoarthritis (OA) are generally palliative, with the goal of alleviating pain and inflammation rather than addressing underlying pathology or slowing OA progression. The presence of clinical symptoms and radiographic changes are indicative of irreversible damage. It is becoming clear that the earlier we intervene, the better the likelihood of preserving cartilage and preventing or delaying end-stage disease. Due to the paucity of effective treatment options for OA, regenerative medicine strategies have recently generated considerable excitement. Therapeutic strategies based on mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (EVs) have rapidly emerged as the most promising approaches for preserving articular cartilage and slowing OA disease progression. Despite the considerable momentum to explore novel regenerative approaches for OA, research is still in the early stages, and paradigms continue to rapidly evolve. This chapter examines current knowledge regarding the biological aspects of MSCs and MSC-derived EVs, their potential mechanisms of action, and promising therapeutic approaches being explored in the context of early OA.
... In addition, as exosomes contain both RNA and DNA (reflecting tumor mutations), the use of a single platform to study both molecular species is a clear advantage for finding rare or not-abundant mutations. Finally, the protein content of a single exosome reaches up to 400 unique proteins [17,18]. Liquid biopsy analytes. ...
Simple Summary
Exosomes are small vesicles of 100 nm in size that are released from every cell constantly. They contain different molecules (DNA, RNA, lipids, metabolites, etc.) that reflect the content of the cell they come from. Exosomes can be found in all biological fluids. In cancer, exosomes are involved in several events such as tumor growth, metastasis, and the immune response, by delivering their cargos to recipient cells. Due to their unique features, exosomes have become promising analytes in the field of liquid biopsy, which searches for biomarkers to manage different steps of the tumor process. We believe that exosomes will become an important tool in liquid biopsy in the near future. In this review we provide an updated literature compilation about exosomes as biomarkers in oncology and discuss their possibilities and limitations.
Abstract
Among the different components that can be analyzed in liquid biopsy, the utility of exosomes is particularly promising because of their presence in all biological fluids and their potential for multicomponent analyses. Exosomes are extracellular vesicles with an average size of ~100 nm in diameter with an endosomal origin. All eukaryotic cells release exosomes as part of their active physiology. In an oncologic patient, up to 10% of all the circulating exosomes are estimated to be tumor-derived exosomes. Exosome content mirrors the features of its cell of origin in terms of DNA, RNA, lipids, metabolites, and cytosolic/cell-surface proteins. Due to their multifactorial content, exosomes constitute a unique tool to capture the complexity and enormous heterogeneity of cancer in a longitudinal manner. Due to molecular features such as high nucleic acid concentrations and elevated coverage of genomic driver gene sequences, exosomes will probably become the “gold standard” liquid biopsy analyte in the near future.
... For example, studies have shown that exosomes secretion could be obviously increased in tumor cells than that in normal cells. The delivery of exosomes throughout the body can transport mRNAs, miRNAs, and proteins to targeted cells, including tumor cells, and ultimately affect the occurrence, invasion, and metastasis of tumor [28][29][30][31][32]. As the increasing recognition of the potential therapeutics, exosomes will play more and more important roles in clinic [33][34][35][36]. ...
Background: Colorectal cancer (CRC) is one of the most common cancers with high mortality worldwide. Uncontrolled growth is an important hallmark of CRC. However, the mechanisms are poorly understood. Methods: Syntaxin 2 (STX2) expression was analyzed in 160 cases of paraffin-embedded CRC tissue by immunohistochemistry, in 10 cases of fresh CRC tissue by western blot, and in 2 public databases. Gain- and loss-of-function analyses were used to investigate the biological function of STX2 in CRC growth. Exosomes isolation, characterization, Co-immunoprecipitation (Co-IP), flow cytometry and fluorescence were conducted to study the molecular mechanism of STX2 in CRC growth. Results: The expression of STX2 was obviously up-regulated in human CRC tissues. Overexpression of STX2 increased the growth of CRC cells in vitro and in vivo. Downregulation of STX2 repressed the growth of CRC. STX2 modulated exosomes secretion of CRC cells which might correlated with Rab8a expression. The secreted exosomes could be ingested by CRC cells, and ultimately promoted the growth of CRC by arresting the tumor cells at S phase. Conclusions: Our data provide evidence that STX2 promotes CRC growth by increasing exosomes secretion of CRC cells; And the modulation of STX2 in exosomes secretion correlates with Rab8a. Thus, our study identified a new mechanism of STX2 in CRC growth and may provide a possible strategy for CRC therapy.
... The fact that exosomes are available in large quantities in synovial fluid means they can be easily isolated and analysed [129]. The idea that exosome cargo could potentially be manufactured and engineered to carry specific proteins and surface markers, for instance using nanoparticlebased technology, could ultimately allow targeted regulation of intercellular communication [130,131]. For example, exosomes derived from miR92a3p overexpressing tumour MSCs increased chondrogenesis and decreased cartilage damage via interaction with Wnt, a critical protein involved in bone and cartilage development [115]. ...
Osteoarthritis (OA) is the most prevalent of the musculoskeletal conditions and represents a significant public health burden. While degeneration of articular cartilage is a key feature, it is now increasingly recognized as a complex condition affecting the whole joint, with synovial inflammation present in a significant proportion of patients. As a secretory tissue, the OA synovium is a rich source of both soluble inflammatory mediators and extracellular vesicles, including exosomes, which have been implicated in cell-cell communication. Exosome cargo has been found to include proteins, lipids and various RNA subtypes such as mRNA and miRNA, potentially capable of regulating gene expression in target cells and tissues. Profiling of exosome cargo and understanding effects on cartilage could elucidate novel regulatory mechanisms within the joint, providing insight for targeted treatment. The aim of this article is to review current literature on exosome biology, highlighting the relevance and application for OA pathogenesis.
... Important to our development of a systems therapeutic is allowing the stem cells to release their molecules as opposed to using an extraction process thereby allowing the molecules to fully form and fold correctly, and to package into exosomes. The nanosphere exosome will impart protective, targeting, and penetration qualities to the molecules that would not be present in the molecule's native state without packaging into exosomes (Maguire 2016b). ...
Evidence suggests that adult stem cell types and progenitor cells act collectively in a given tissue to maintain and heal organs, such as muscle, through a release of a multitude of molecules packaged into exosomes from the different cell types. Using this principle for the development of bioinspired therapeutics that induces homeostatic renormalization, here we show that the collection of molecules released from four cell types, including mesenchymal stem cells, fibroblast, neural stem cells, and astrocytes, rescues degenerating neurons and cells. Specifically, oxidative stress induced in a human recombinant TDP-43- or FUS‐tGFP U2OS cell line by exposure to sodium arsenite was shown to be significantly reduced by our collection of molecules using in vitro imaging of FUS and TDP-43 stress granules. Further, we also show that the collective secretome rescues cortical neurons from glutamate toxicity as evidenced by increased neurite outgrowth, reduced LDH release, and reduced caspase 3/7 activity. These data are the first in a series supporting the development of stem cell-based exosome systems therapeutics that uses a physiological renormalization strategy to treat neurodegenerative diseases.
... This is evidenced by the high profile individuals who have had such procedures, for joint repair as an example, with little or no benefit. 17 Like therapeutic development with stem cells, as described in the aforementioned section, medical procedures with stem cells currently exist in the "trough of disillusionment". ...
The Gartner curve for regenerative and stem cell therapeutics is currently climbing out of the “trough of disillusionment” and into the “slope of enlightenment”. Understanding that the early years of stem cell therapy relied on the model of embryonic stem cells (ESCs), and then moved into a period of the overhype of induced pluripotent stem cells (iPSCs), instead of using the model of 40 years of success, i.e. adult stem cells used in bone marrow transplants, the field of stem cell therapy has languished for years, trying to move beyond the early and poorly understood success of bone marrow transplants. Recent studies in the lab and clinic show that adult stem cells of various types, and the molecules that they release, avoid the issues associated with ESCs and iPSCs and lead to better therapeutic outcomes and into the slope of enlightenment.
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by bradykinesia, rigidity, and tremor. Considerable progress has been made to understand the exact mechanism leading to this disease. Most of what is known comes from the evidence of PD brains’ autopsies showing a deposition of Lewy bodies—containing a protein called α-synuclein (α-syn)—as the pathological determinant of PD. α-syn predisposes neurons to neurotoxicity and cell death, while the other associated mechanisms are mitochondrial dysfunction and oxidative stress, which are underlying precursors to the death of dopaminergic neurons at the substantia nigra pars compacta leading to disease progression. Several mechanisms have been proposed to unravel the pathological cascade of these diseases; most of them share a particular similarity: cell-to-cell communication through exosomes (EXOs). EXOs are intracellular membrane-based vesicles with diverse compositions involved in biological and pathological processes, which their secretion is driven by the NLR family pyrin domain-containing three proteins (NLRP3) inflammasome. Toxic biological fibrils are transferred to recipient cells, and the disposal of damaged organelles through generating mitochondrial-derived vesicles are suggested mechanisms for developing PD. EXOs carry various biomarkers; thus, they are promising to diagnose different neurological disorders, including neurodegenerative diseases (NDDs). As nanovesicles, the applications of EXOs are not only restricted as diagnostics but also expanded to treat NDDs as therapeutic carriers and nano-scavengers. Herein, the aim is to highlight the potential incrimination of EXOs in the pathological cascade and progression of PD and their role as biomarkers and therapeutic carriers for diagnosing and treating this neuro-debilitating disorder.
Exosome‐based therapy is an emerging novel approach for myocardial infarction (MI) treatment. Exosomes are identified as extracellular vesicles that are produced within multi vesicular bodies in the cells cytosols and then are secreted from the cells. Exosomes are 30–100 nm in diameter that are released from viable cells and are different from other secreted vesicles such as apoptotic bodies and microvesicles in their origin and contents such as RNAs, proteins and nucleic acid. The recent advances in exosomes researches have demonstrated the role of these bio‐nanovesicles in the physiolocal, pathological and molecular aspects of the heart.
The results of In vitro and preclinical models have shown that exosmes from different cardiac cells can improve the cardiac function following MI. For example, mesenchymal stem cells (MSCs) and cardiac progenitor cells(CPCs) containing exosomes can affect proliferation, survival and differentiation of cardiac fibroblasts and cardiomyocytes. Moreover, MSCs and CPCs‐ derived exosomes can enhance the migration of endothelial cells. Exosome‐based therapy approaches augment the cardiac function by multiple means, such as reducing fibrosis, stimulation of vascular angiogenesis and proliferation of cardiomyocytes that result in replacing damaged heart tissue with newly generated functionall myocytes. This review article aims to briefly discuss the recent advancements in the role of secreted exosomes in myocardial repair, by focusing on cardiac cells‐ derived exosomes.
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Owing to the role of exosome as a cargo for intercellular communication, especially in cancer metastasis, the evidence has been consistently accumulated that exosomes can be used as a non-invasive indicator of cancer. Consequently, several studies applying exosome have been proposed for cancer diagnostic methods such as ELISA assay. However, it has been still challenging to get reliable results due to the requirement of a labeling process and high concentration of exosome. Here, we demonstrate a label-free and highly sensitive classification method of exosome by combining surface enhanced Raman scattering (SERS) and statistical pattern analysis. Unlike the conventional method to read different peak positions and amplitudes of spectrum, whole SERS spectra of exosomes were analyzed by principal component analysis (PCA). By employing this pattern analysis, lung cancer cell derived exosomes were clearly distinguished from normal cell derived exosomes by 95.3% sensitivity and 97.3% specificity. Moreover, by analyzing the PCA result, we could suggest that this difference was induced by 11 different points in SERS signals from lung cancer cell derived exosomes. This result paved the way of new real-time diagnosis and classification of lung cancer by exosome as a cancer marker.
Exosomes are membrane vesicles with a diameter of 40–100 nm that are secreted by many cell types into the extracellular milieu. Exosomes are found in cell culture supernatants and in different biological fluids and are known to be secreted by most cell types under normal and pathological conditions. Considerable research is focusing on the exploitation of exosomes in biological fluids for biomarkers in the diagnosis of disease. More recently, exosomes are being exploited for their therapeutic potential. Exosomes derived from dendritic cells, tumor cells, and malignant effusions demonstrate immunomodulatory functions and are able to present antigens to T-cells and stimulate antigen-specific T-cell responses. Exosomes have also been examined for their therapeutic potential in the treatment of infections such as toxoplasmosis, diphtheria, tuberculosis and atypical severe acute respiratory syndrome as well as autoimmune diseases. Attempts to find practical applications for exosomes continue to expand with the role of exosomes as a drug delivery system for the treatment of autoimmune/inflammatory diseases and cancers.
We sought to evaluate the effects of adipose-derived mesenchymal stem cells (ADMSCs) exosomes on hepatocellular carcinoma (HCC) in rats using apparent diffusion coefficient (ADC), natural killer T-cell (NKT-cell) responses, and histopathological features. ADMSC-derived exosomes appeared as nanoparticles (30–90 nm) on electron microscopy and were positive for CD63, tumor susceptibility gene-101, and
β
-catenin on western blotting. The control (
n
=
8
) and exosome-treated (
n
=
8
) rats with N1S1-induced HCC underwent baseline and posttreatment day 10 and day 20 magnetic resonance imaging and measurement of ADC. Magnetic resonance imaging showed rapidly enlarged HCCs with low ADCs in the controls. The exosome-treated rats showed partial but nonsignificant tumor reduction, and significant ADC and ADC ratio increases on day 10. On day 20, the exosome-treated rats harbored significantly smaller tumors and volume ratios, higher ADC and ADC ratios, more circulating and intratumoral NKT-cells, and low-grade HCC (
P
<
0.05
for all comparisons) compared to the controls. The ADC and volume ratios exhibited significant inverse correlations (
P
<
0.001
,
R
2
=
0.679
). ADMSC-derived exosomes promoted NKT-cell antitumor responses in rats, thereby facilitating HCC suppression, early ADC increase, and low-grade tumor differentiation. ADC may be an early biomarker of treatment response.
Cardiovascular diseases (CVDs) are still a major cause of people deaths worldwide, and mesenchymal stem cells (MSCs) transplantation holds great promise due to its capacity to differentiate into cardiovascular cells and secrete protective cytokines, which presents an important mechanism of MSCs therapy for CVDs. Although the capability of MSCs to differentiate into cardiomyocytes (CMCs), endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) has been well recognized in massive previous experiments both in vitro and in vivo, low survival rate of transplanted MSCs in recipient hearts suggests that therapeutic effects of MSCs transplantation might be also correlated with other underlying mechanisms. Notably, recent studies uncovered that MSCs were able to secret cholesterol-rich, phospholipid exosomes which were enriched with microRNAs (miRNAs). The released exosomes from MSCs acted on hearts and vessels, and then exerted anti-apoptosis, cardiac regeneration, anti-cardiac remodeling, anti-inflammatory effects, neovascularization and anti-vascular remodeling, which are considered as novel molecular mechanisms of therapeutic potential of MSCs transplantation. Here we summarized recent advances about the role of exosomes in MSCs therapy for CVDs, and discussed exosomes as a novel approach in the treatment of CVDs in the future.
Stem cell released molecules (SRM) production employs a proprietary manufacturing process that allows the collection of a wide variety of soluble and properly folded proteins and signaling molecules with complete post-translational modifications and exosome packaging from multiple stem cell types important to immune modulation, and tissue repair and regeneration. The advantages of the process are in the production of biologically active proteins, exosomes, and signaling molecules, developing a "systems therapeutic" yielding a combination of many molecules, that act at multiple targets, resulting in a synergistic therapeutic with emergent therapeutic value. Furthermore, the production of the SRM (stem cell released molecules) does not require downstream solubilization, refolding, or other processes. Additionally, the process offers reduced purification requirements and lower production costs than other pharmacological and biological processes. The production of a "systems therapeutic" with a multitude of molecules represents a multi-targeted, systems biology approach to designing and manufacturing therapeutics, including therapeutics designed to work alone, or augment scaffolding-based and/or cell-based regenerative medicine.
The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migration and axon guidance in a wide range of organisms. During development, the Hh morphogen directs tissue patterning according to a concentration gradient. Lipid modifications on Hh are needed to achieve graded distribution, leading to debate about how Hh is transported to target cells despite being membrane-tethered. Cytonemes in the region of Hh signalling have been shown to be essential for gradient formation, but the carrier of the morphogen is yet to be defined. Here we show that Hh and its co-receptor Ihog are in exovesicles transported via cytonemes. These exovesicles present protein markers and other features of exosomes. Moreover, the cell machinery for exosome formation is necessary for normal Hh secretion and graded signalling. We propose Hh transport via exosomes along cytonemes as a significant mechanism for the restricted distribution of a lipid-modified morphogen.
Introduction
Osteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in communication among joint tissue cells.
Methods
Human synovial fibroblasts (SFB) and articular chondrocytes were obtained from normal knee joints. Exosomes isolated from conditioned medium of SFB were analyzed for size, numbers, markers and function. Normal articular chondrocytes were treated with exosomes from SFB, and Interleukin-1β (IL-1β) stimulated SFB. OA-related genes expression was quantified using real-time PCR. To analyze exosome effects on cartilage tissue, we performed glycosaminoglycan release assay. Angiogenic activity of these exosomes was tested in migration and tube formation assays. Cytokines and miRNAs in exosomes were analyzed by Bio-Plex multiplex assay and NanoString analysis.
Results
Exosomes from IL-1β stimulated SFB significantly up-regulated MMP-13 and ADAMTS-5 expression in articular chondrocytes, and down-regulated COL2A1 and ACAN compared with SFB derived exosomes. Migration and tube formation activity were significantly higher in human umbilical vein endothelial cells (HUVECs) treated with the exosomes from IL-1β stimulated SFB, which also induced significantly more proteoglycan release from cartilage explants. Inflammatory cytokines, IL-6, MMP-3 and VEGF in exosomes were only detectable at low level. IL-1β, TNFα MMP-9 and MMP-13 were not detectable in exosomes. NanoString analysis showed that levels of 50 miRNAs were differentially expressed in exosomes from IL-1β stimulated SFB compared to non-stimulated SFB.
Conclusions
Exosomes from IL-1β stimulated SFB induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints.
The standard drug development model uses reductionist approaches to discover small molecules targeting one pathway. Although systems biology analyzes multiple pathways, the approach is often used to develop a small molecule interacting at only one pathway in the system. Similar to that in physics where a departure from the old reductionist "Copenhagen View" of quantum physics to a new and predictive systems based, collective model has emerged yielding new breakthroughs such as the LASER, a new model is emerging in biology where systems biology is used to develop a new technology acting at multiple pathways called "systems therapeutics."
Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. Although their potential as cancer biomarkers has been promising, the identification and quantification of EVs in clinical samples remains challenging. Here we describe a sensitive and rapid analytical technique for profiling circulating EVs directly from blood samples of patients with colorectal cancer. EVs are captured by two types of antibodies and are detected by photosensitizer-beads, which enables us to detect cancer-derived EVs without a purification step. We also show that circulating EVs can be used for detection of colorectal cancer using the antigen CD147, which is embedded in cancer-linked EVs. This work describes a new liquid biopsy technique to sensitively detect disease-specific circulating EVs and provides perspectives in translational medicine from the standpoint of diagnosis and therapy.
As an example of the burgeoning importance of stem cell therapy, this past month the California Institute for Regenerative Medicine (CIRM) has approved $70 million to create a new network of stem cell clinical trial centers. Much work in the last decade has been devoted to developing the use of autologous and allogeneic adult stem cell transplants to treat a number of conditions, including heart attack, dementia, wounds, and immune system-related diseases. The standard model teaches us that adult stem cells exists throughout most of the body and provide a means to regenerate and repair most tissues through replication and differentiation. Although we have often witnessed the medical cart placed in front of the scientific horse in the development of stem cell therapies outside of academic circles, great strides have been made, such as the use of purified stem cells(1) instead of whole bone marrow transplants in cancer patients, where physicians avoid re-injecting the patients with their own cancer cells.(2) We most often think of stem cell therapy acting to regenerate tissue through replication and then differentiation, but recent studies point to the dramatic effects adult stem cells exert in the repair of various tissues through the release of paracrine and autocrine substances, and not simply through differentiation. Indeed, up to 80% of the therapeutic effect of adult stem cells has been shown to be through paracrine mediated actions.(3) That is, the collected types of molecules released by the stem cells, called the secretome, or stem cell released molecules (SRM), number in the 100s, including proteins, microRNA, growth factors, antioxidants, proteasomes, and exosomes, and target a multitude of biological pathways through paracrine actions. The composition of the different molecule types in SRM is state dependent, and varies with cell type and conditions such as age and environment.
Exosomes are small membrane vesicles released by a variety of cell types. Exosomes contain genetic materials, such as mRNAs and microRNAs (miRNAs), implying that they may play a pivotal role in cell-to-cell communication. Mesenchymal stem cells (MSCs), which potentially differentiate into multiple cell types, can migrate to the tumor sites and have been reported to exert complex effects on tumor progression. To elucidate the role of MSCs within the tumor microenvironment, previous studies have suggested various mechanisms such as immune modulation and secreted factors of MSCs. However, the paracrine effects of MSC-derived exosomes on the tumor microenvironment remain to be explored. The hypothesis of this study was that MSC-derived exosomes might reprogram tumor behavior by transferring their molecular contents. To test this hypothesis, exosomes from MSCs were isolated and characterized. MSC-derived exosomes exhibited different protein and RNA profiles compared with their donor cells and these vesicles could be internalized by breast cancer cells. The results demonstrated that MSC-derived exosomes significantly down-regulated the expression of vascular endothelial growth factor (VEGF) in tumor cells, which lead to inhibition of angiogenesis in vitro and in vivo. Additionally, miR-16, a miRNA known to target VEGF, was enriched in MSC-derived exosomes and it was partially responsible for the anti-angiogenic effect of MSC-derived exosomes. The collective results suggest that MSC-derived exosomes may serve as a significant mediator of cell-to-cell communication within the tumor microenvironment and suppress angiogenesis by transferring anti-angiogenic molecules.
Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, the complexity of body fluids often hampers biomarker discovery. An attractive alternative approach is the isolation of small vesicles, i.e. exosomes, ∼100 nm, which contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific biomarker discovery.
Exosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. After tryptic digestion, proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode. Accurate Mass and Time (AMT) tag approach was employed for peptide identification and quantitation. Candidate biomarkers were validated by Western blotting and Immunohistochemistry.
Proteomic characterization resulted in the identification of 248, 233, 169, and 216 proteins by at least 2 peptides in exosomes from PNT2C2, RWPE-1, PC346C, and VCaP, respectively. Statistical analyses revealed 52 proteins differently abundant between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes.
Identification of exosomal proteins using high performance LC-FTMS resulted in the discovery of PDCD6IP, FASN, XPO1 and ENO1 as new candidate biomarkers for prostate cancer.
The antibacterial activities of lipids have been known for more than a century. With the advent of antibiotics in the 1940s and 50s the interest in fatty acids and other lipids as antibacterial agents diminished but in the 1970s there was a renewed interest in antimicrobial lipids, particularly milk lipids. Fatty acids and monoglycerides released by hydrolysis of milk fats were found to inactivate enveloped viruses and the stomach contents of neonates became microbicidal one hour after feeding of breast milk. Fatty acids and monoglycerides inactivate all enveloped viruses which have been studied. They kill Gram positive and Gram negative bacteria, but the susceptibility of bacteria varies. Long-chain unsaturated fatty acids and medium-chain saturated fatty acids and monoglycerides thereof are most active. A potential use of these lipids in prevention and treatment of skin infections, sexually transmitted infections, respiratory infections, oral infections and foodborne infections is discussed.
Mesenchymal stem cell (MSC) was previously shown to secrete lipid vesicles that when purified by high performance liquid chromatography as a population of homogenously sized particles with a hydrodynamic radius of 55-65 nm reduce infarct size in a mouse model of myocardial ischemia/reperfusion injury. As these vesicles exhibit many biophysical and biochemical properties of exosomes, they were identified as exosomes. Here we investigated if these lipid vesicles were indeed exosomes that have an endosomal biogenesis.
In most cells, endocytosis is thought to occur at specialized microdomains known as lipid rafts. To demonstrate an endosomal origin for MSC exosomes, MSCs were pulsed with ligands e.g. transferrin (Tfs) and Cholera Toxin B (CTB) that bind receptors in lipid rafts. The endocytosed ligands were then chased to determine if they were incorporated into the exosomes.
A fraction of exogenous Tfs was found to recycle into MSC exosomes. When MSCs were pulsed with labelled Tfs in the presence of chlorpromazine, an inhibitor of clathrin-mediated endocytosis, Tf incorporation in CD81-immunoprecipitate was reduced during the chase. CTB which binds GM1 gangliosides that are enriched in lipid rafts extracted exosome-associated proteins, CD81, CD9, Alix and Tsg101 from MSC-conditioned medium. Exogenous CTBs were pulse-chased into secreted vesicles. Extraction of Tf- or CTB-binding vesicles in an exosome preparation mutually depleted each other. Inhibition of sphingomyelinases reduced CTB-binding vesicles.
Together, our data demonstrated that MSC exosomes are derived from endocytosed lipid rafts and that their protein cargo includes exosome-associated proteins CD81, CD9, Alix and Tsg101.
Systems biology is a recent addition to the necessary but insufficient reductionist approach used in biological research. Systems biology is focused on understanding living things as a function of their various interactions at multiple levels: not simply as a sum of all their individual parts at any one level. This integrative approach yields predictive models of the normal state, the disease state and therapeutic actions. Although molecular biology has collected an enormous amount of information, including the sequencing of the entire human genome in the year 2000, few real-world applications have resulted from this molecular approach. The pharmaceutical industry's R&D expenditure has increased substantially since 2000, but the number of approved therapeutics has dropped simultaneously, due in part to over-reliance on reductionist genomic, and not systems, approaches. Instead of using reductionist genomics approaches alone, genomics should be incorporated into a multi-level systems biology approach to develop diagnostics and therapeutics.
Exosomes are nanosized vesicles of endocytic origin that are released into the extracellular environment by many different cells. It has been shown that exosomes from various cellular origins contain a substantial amount of RNA (mainly mRNA and microRNA). More importantly, exosomes are capable of delivering their RNA content to target cells, which is a novel way of cell-to-cell communication. The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34(+) progenitor cells.
The mRNA and microRNA content of exosomes from a human mast cell line, HMC-1, was analysed by using microarray technology. Co-culture experiments followed by flow cytometry analysis and confocal microscopy as well as radioactive labeling experiments were performed to examine the uptake of these exosomes and the shuttle of the RNA to other mast cells and CD34(+) progenitor cells.
In this study, we show that human mast cells release RNA-containing exosomes, with the capacity to shuttle RNA between cells. Interestingly, by using microRNA microarray analysis, 116 microRNAs could be identified in the exosomes and 134 microRNAs in the donor mast cells. Furthermore, DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. In addition, transfer experiments revealed that exosomes can shuttle RNA between human mast cells and to CD34(+) hematopoietic progenitor cells.
These findings suggest that exosomal shuttle RNA (esRNA) can play a role in the communication between cells, including mast cells and CD34(+) progenitor cells, implying a role in cells maturation process.
The potential of stem cell (SC) therapies for eye diseases is well-recognized. However, the results remain only encouraging as little is known about the mechanisms responsible for eye renewal, regeneration and/or repair. Therefore, it is critical to gain knowledge about the specific tissue environment (niches) where the stem/progenitor cells reside in eye. A new type of interstitial cell-telocyte (TC) (www.telocytes.com) was recently identified by electron microscopy (EM). TCs have very long (tens of micrometres) and thin (below 200 nm) prolongations named telopodes (Tp) that form heterocellular networks in which SCs are embedded. We found TCs by EM and electron tomography in sclera, limbus and uvea of the mouse eye. Furthermore, EM showed that SCs were present in the anterior layer of the iris and limbus. Adhaerens and gap junctions were found to connect TCs within a network in uvea and sclera. Nanocontacts (electron-dense structures) were observed between TCs and other cells: SCs, melanocytes, nerve endings and macrophages. These intercellular 'feet' bridged the intercellular clefts (about 10 nm wide). Moreover, exosomes (extracellular vesicles with a diameter up to 100 nm) were delivered by TCs to other cells of the iris stroma. The ultrastructural nanocontacts of TCs with SCs and the TCs paracrine influence via exosomes in the epithelial and stromal SC niches suggest an important participation of TCs in eye regeneration.
Here, for the first time, we test a novel hypothesis that systemic treatment of stroke with exosomes derived from multipotent mesenchymal stromal cells (MSCs) promote neurovascular remodeling and functional recovery after stroke in rats. Adult male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAo) followed by tail vein injection of 100 μg protein from MSC exosome precipitates or an equal volume of vehicle phosphate-buffered saline (PBS) (n=6/group) 24 hours later. Animals were killed at 28 days after stroke and histopathology and immunohistochemistry were employed to identify neurite remodeling, neurogenesis, and angiogenesis. Systemic administration of MSC-generated exosomes significantly improved functional recovery in stroke rats compared with PBS-treated controls. Axonal density and synaptophysin-positive areas were significantly increased along the ischemic boundary zone of the cortex and striatum in MCAo rats treated with exosomes compared with PBS control. Exosome treatment significantly increased the number of newly formed doublecortin (a marker of neuroblasts) and von Willebrand factor (a marker of endothelial cells) cells. Our results suggest that intravenous administration of cell-free MSC-generated exosomes post stroke improves functional recovery and enhances neurite remodeling, neurogenesis, and angiogenesis and represents a novel treatment for stroke.Journal of Cerebral Blood Flow & Metabolism advance online publication, 21 August 2013; doi:10.1038/jcbfm.2013.152.
Despite the therapeutic potential of nucleic acid drugs, their clinical application has been limited in part by a lack of appropriate delivery systems. Exosomes or microvesicles are small endosomally derived vesicles that are secreted by a variety of cell types and tissues. Here, we show that exosomes can efficiently deliver microRNA (miRNA) to epidermal growth factor receptor (EGFR)-expressing breast cancer cells. Targeting was achieved by engineering the donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the GE11 peptide. Intravenously injected exosomes delivered let-7a miRNA to EGFR-expressing xenograft breast cancer tissue in RAG2(-/-) mice. Our results suggest that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.Molecular Therapy (2012); doi:10.1038/mt.2012.180.
Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56(+) and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.
Conventionally, cells described in the stroma of the intestinal wall are fibroblasts/fibrocytes, mast cells, plasma cells, eosinophils, macrophages and, interstitial cells of Cajal (ICCs), the latter being considered as the pacemakers of gastrointestinal rhythmicity. Recently, a new type of stromal cell called telocyte (TCs) was found in various cavitary and non-cavitary organs (www.telocytes.com). We show here direct electron microscopical evidence for the presence of TCs in the lamina propria of rat jejunum just beneath the epithelial layer of the mucosal crypts and in between the smooth muscle cells (SMCs) of muscularis mucosae. TCs are characterized by: several very long (tens to hundreds of µm) prolongations called telopodes (Tps). Tps (with caliber below the resolving power of light microscopy) display podomeres (thin segments ≤ 0.2 µm) and podoms (dilations accommodating caveolae, mitochondria, and endoplasmic reticulum). Tps present dichotomous branching and form a three dimensional network close to immune cells, SMCs or nerve bundles. TCs could play a role in intercellular signaling and control of local tissue homeostasis.
S ummary
Surface characteristics and wettability of the leaves of six plant species have been examined in relation to their susceptibility to damage from simulated acid rain. The species examined differed in the type and extent of epicuticular wax deposits, surface topography of the cuticle, trichome type and distribution, and epidermal cell shape. Leaf wettability, as measured by either water‐holding capacity or droplet contact angle, was significantly different among species, and is highly correlated with previous reports of damage from simulated acid rain. The leaves of Platanus occidentalis L. were the most wettable of the species examined and are reported to be damaged the most by repeated applications of simulated acid rain. Mature leaves of hiriodendron tulipifera L., with high contact angles and very low water‐holding capacity, are the least damaged according to published reports. Leaf water‐holding capacity and surface‐droplet contact angle may be useful first indicators of resistance to acid rain, and should be included m future studies of foliar effects of acid rain.
Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics
is to deliver nucleic acids across the cell’s plasma membrane. This study demonstrates that exosome vesicles derived from
humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic
origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies,
an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes
(exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells.
The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing
of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous
nucleic acids such as therapeutic siRNAs.
Exosomes are small membranous vesicles secreted by a number of cell types including neurons and can be isolated from conditioned cell media or bodily fluids such as urine and plasma. Exosome biogenesis involves the inward budding of endosomes to form multivesicular bodies (MVB). When fused with the plasma membrane, the MVB releases the vesicles into the extracellular environment as exosomes. Proposed functions of these vesicles include roles in cell-cell signaling, removal of unwanted proteins, and the transfer of pathogens between cells. One such pathogen which exploits this pathway is the prion, the infectious particle responsible for the transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) of humans or bovine spongiform encephalopathy (BSE) of cattle. Similarly, exosomes are also involved in the processing of the amyloid precursor protein (APP) which is associated with Alzheimer's disease. Exosomes have been shown to contain full-length APP and several distinct proteolytically cleaved products of APP, including Aβ. In addition, these fragments can be modulated using inhibitors of the proteases involved in APP cleavage. These observations provide further evidence for a novel pathway in which PrP and APP fragments are released from cells. Other proteins such as superoxide dismutase I and alpha-synuclein (involved in amyotrophic lateral sclerosis and Parkinson's disease, respectively) are also found associated with exosomes. This review will focus on the role of exosomes in neurodegenerative disorders and discuss the potential of these vesicles for the spread of neurotoxicity, therapeutics, and diagnostics for these diseases.
In recent years, a variety of low molecular weight antibiotics have been isolated from diverse animal species. These agents, which include peptides, lipids, and alkaloids, exhibit antibiotic activity against environmental microbes and are thought to play a role in innate immunity. We report here the discovery of a broad-spectrum steroidal antibiotic isolated from tissues of the dogfish shark Squalus acanthias. This water-soluble antibiotic, which we have named squalamine, exhibits potent bactericidal activity against both Gram-negative and Gram-positive bacteria. In addition, squalamine is fungicidal and induces osmotic lysis of protozoa. The chemical structure of the antibiotic 3 beta-N-1-(N-[3-(4-aminobutyl)]- 1,3-diaminopropane)-7 alpha,24 zeta-dihydroxy-5 alpha-cholestane 24-sulfate has been determined by fast atom bombardment mass spectroscopy and NMR. Squalamine is a cationic steroid characterized by a condensation of an anionic bile salt intermediate with spermidine. The discovery of squalamine in the shark implicates a steroid as a potential host-defense agent in vertebrates and provides insights into the chemical design of a family of broad-spectrum antibiotics.
Cancer biomarkers are invaluable tools for cancer detection, prognosis, and treatment. Recently, microvesicles have appeared as a novel source for cancer biomarkers. We present here the results from a proteomic analysis of microvesicles released to the extracellular environment by the metastatic prostate cancer cell line PC-3. Using nanocapillary liquid chromatography-tandem mass spectrometry 266 proteins were identified with two or more peptide sequences. Further analysis showed that 16% of the proteins were classified as extracellular and that intracellular proteins were annotated in a variety of locations. Concerning biological processes, the proteins found in PC-3 cell-released microvesicles are mainly involved in transport, cell organization and biogenesis, metabolic process, response to stimulus, and regulation of biological processes. Several of the proteins identified (tetraspanins, annexins, Rab proteins, integrins, heat shock proteins, cytoskeletal proteins, 14-3-3 proteins) have previously been found in microvesicles isolated from other sources. However, some of the proteins seem to be more specific to the vesicular population released by the metastatic prostate cancer PC-3 cell line. Among these proteins are the tetraspanin protein CD151 and the glycoprotein CUB domain-containing protein 1. Interestingly, our results show these proteins are promising biomarkers for prostate cancer and therefore candidates for clinical validation studies in biological fluids.
The behavior of each of a series of proteins during chromatography on columns of Sephadex G-200 may be correlated with the Stokes radius of the protein, but does not correlate with molecular weight. Proteins with Stokes radii as high as 107 Å or molecular weights as high as 1 300 000 may be characterized by the use of such columns. The Sephadex data are used in a critique of earlier mathematical treatments of the phenomenon known as “gel filtration”.With a Stokes radius measured by the chromatographic method and a sedimentation coefficient determined by density gradient centrifugation, reasonable estimates for both the molecular weight and the frictional ratio (f/f0) of a macromolecule are available. Since both of these methods are applicable to proteins present in mixtures, valuable information concerning the molecular weights and shapes of proteins may be obtained in anticipation of the achievement of high degrees of purity. The determination of the molecular weight and the f/f0 for each of several enzymes in unfractionated extracts of Salmonella typhimurium and Neurospora crassa illustrates this application.
Diseases such as tuberculosis, hepatitis, and HIV/AIDS are caused by intracellular pathogens and are a major burden to the global medical community. Conventional treatments for these diseases typically consist of long-term therapy with a combination of drugs, which may lead to side effects and contribute to low patient compliance. The pathogens reside within intracellular compartments of the cell, which provide additional barriers to effective treatment. Therefore, there is a need for improved and more effective therapies for such intracellular diseases. This review will summarize, for the first time, the intracellular compartments in which pathogens can reside and discuss how nanomedicine has the potential to improve intracellular disease therapy by offering properties such as targeting, sustained drug release, and drug delivery to the pathogen's intracellular location. The characteristics of nanomedicine may prove advantageous in developing improved or alternative therapies for intracellular diseases.
We previously reported the purification, culture-expansion, and osteogenic differentiation potential of mesenchymal progenitor cells (MPCs) derived from human bone marrow. As a first step to establishing the phenotypic characteristics of MPCs, we reported on the identification of unique cell surface proteins which were detected with monoclonal antibodies. In this study, the phenotypic characterization of human marrow-derived MPCs is further established through the identification of a cytokine expression profile under standardized growth medium conditions and in the presence of regulators of the osteogenic and stromal cell lineages, dexamethasone and interleukin-1α (IL-1α), respectively. Constitutively expressed cytokines in this growth phase include G-CSF, SCF, LIF, M-CSF, IL-6, and IL-11, while GM-CSF, IL-3, TGF-β2, and OSM were not detected in the growth medium. Exposure of cells in growth medium to dexamethasone resulted in a decrease in the expression of LIF, IL-6, and IL-11. These cytokines have been reported to exert influence on the differentiation of cells derived from the bone marrow stroma through target cell receptors that utilize gp130-associated signal transduction pathways. Dexamethasone had no effect on the other cytokines expressed under growth medium conditions and was not observed to increase the expression of any of the cytokines measured in this study. In contrast, IL-1α increased the expression of G-CSF, M-CSF, LIF, IL-6, and IL-11 and induced the expression of GM-CSF. IL-1α had no effect on SCF expression and was not observed to decrease the production of any of the cytokines assayed. These data indicate that MPCs exhibit a distinct cytokine expression profile. We interpret this cytokine profile to suggest that MPCs serve specific supportive functions in the microenvironment of bone marrow. MPCs provide inductive and regulatory information which are consistent with the ability to support hematopoiesis, and also supply autocrine, paracrine, and juxtacrine factors that influence the cells of the marrow microenvironment itself. In addition, the cytokine profiles expressed by MPCs, in response to dexamethasone and IL-1α, identify specific cytokines whose levels of expression change as MPCs differentiate or modulate their phenotype during osteogenic or stromagenic lineage entrance/progression. © 1996 Wiley-Liss, Inc.
Microtubules are long, proteinaceous filaments that perform structural functions in eukaryotic cells by defining cellular shape and serving as tracks for intracellular motor proteins. We report the first accurate measurements of the flexural rigidity of microtubules. By analyzing the thermally driven fluctuations in their shape, we estimated the mean flexural rigidity of taxol-stabilized microtubules to be 2.2 x 10(-23) Nm2 (with 6.4% uncertainty) for seven unlabeled microtubules and 2.1 x 10(-23) Nm2 (with 4.7% uncertainty) for eight rhodamine-labeled microtubules. These values are similar to earlier, less precise estimates of microtubule bending stiffness obtained by modeling flagellar motion. A similar analysis on seven rhodamine-phalloidin-labeled actin filaments gave a flexural rigidity of 7.3 x 10(-26) Nm2 (with 6% uncertainty), consistent with previously reported results. The flexural rigidity of these microtubules corresponds to a persistence length of 5,200 microns showing that a microtubule is rigid over cellular dimensions. By contrast, the persistence length of an actin filament is only approximately 17.7 microns, perhaps explaining why actin filaments within cells are usually cross-linked into bundles. The greater flexural rigidity of a microtubule compared to an actin filament mainly derives from the former's larger cross-section. If tubulin were homogeneous and isotropic, then the microtubule's Young's modulus would be approximately 1.2 GPa, similar to Plexiglas and rigid plastics. Microtubules are expected to be almost inextensible: the compliance of cells is due primarily to filament bending or sliding between filaments rather than the stretching of the filaments themselves.
Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N-terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Object:
Transplanted multipotent mesenchymal stromal cells (MSCs) improve functional recovery in rats after traumatic brain injury (TBI). In this study the authors tested a novel hypothesis that systemic administration of cell-free exosomes generated from MSCs promotes functional recovery and neurovascular remodeling in rats after TBI.
Methods:
Two groups of 8 Wistar rats were subjected to TBI, followed 24 hours later by tail vein injection of 100 μg protein of exosomes derived from MSCs or an equal volume of vehicle (phosphate-buffered saline). A third group of 8 rats was used as sham-injured, sham-treated controls. To evaluate cognitive and sensorimotor functional recovery, the modified Morris water maze, modified Neurological Severity Score, and foot-fault tests were performed. Animals were killed at 35 days after TBI. Histopathological and immunohistochemical analyses were performed for measurements of lesion volume, neurovascular remodeling (angiogenesis and neurogenesis), and neuroinflammation.
Results:
Compared with the saline-treated group, exosome-treated rats with TBI showed significant improvement in spatial learning at 34-35 days as measured by the modified Morris water maze test (p < 0.05), and sensorimotor functional recovery (i.e., reduced neurological deficits and foot-fault frequency) was observed at 14-35 days postinjury (p < 0.05). Exosome treatment significantly increased the number of newly generated endothelial cells in the lesion boundary zone and dentate gyrus and significantly increased the number of newly formed immature and mature neurons in the dentate gyrus as well as reducing neuroinflammation.
Conclusions:
The authors demonstrate for the first time that MSC-generated exosomes effectively improve functional recovery, at least in part, by promoting endogenous angiogenesis and neurogenesis and by reducing inflammation in rats after TBI. Thus, MSC-generated exosomes may provide a novel cell-free therapy for TBI and possibly for other neurological diseases.
The National Institutes of Health (NIH) closure of the agency's Center for Regenerative Medicine (CRM), which focused on therapeutic development of induced pluripotent stem cells (iPS), was caused by the lack of progress in practical development of the iPSs for use in human therapies. As the NIH evaluates priorities in future stem cell therapeutic development, adult stem cell processes in the human body need to be prioritized for a number of key reasons, including (1) adult stem cells release many types of molecules that provide much of the therapeutic benefit of stem cells and (2) adult stem cells and somatic cells exist in a state of dynamic transition between different potency levels and can be naturally driven by the microenvironment to a state of pluripotency. Thus, the study and development of adult stems for therapeutic use can include naturally induced pluripotent stem cells (NiPSs) that lack the problematic genetic and epigenetic reprogramming errors found in iPSs.
In response to stress, the heart undergoes extensive cardiac remodeling that results in cardiac fibrosis and pathological growth of cardiomyocytes (hypertrophy), which contribute to heart failure. Alterations in microRNA (miRNA) levels are associated with dysfunctional gene expression profiles associated with many cardiovascular disease conditions; however, miRNAs have emerged recently as paracrine signaling mediators. Thus, we investigated a potential paracrine miRNA crosstalk between cardiac fibroblasts and cardiomyocytes and found that cardiac fibroblasts secrete miRNA-enriched exosomes. Surprisingly, evaluation of the miRNA content of cardiac fibroblast-derived exosomes revealed a relatively high abundance of many miRNA passenger strands ("star" miRNAs), which normally undergo intracellular degradation. Using confocal imaging and coculture assays, we identified fibroblast exosomal-derived miR-21_3p (miR-21*) as a potent paracrine-acting RNA molecule that induces cardiomyocyte hypertrophy. Proteome profiling identified sorbin and SH3 domain-containing protein 2 (SORBS2) and PDZ and LIM domain 5 (PDLIM5) as miR-21* targets, and silencing SORBS2 or PDLIM5 in cardiomyocytes induced hypertrophy. Pharmacological inhibition of miR-21* in a mouse model of Ang II-induced cardiac hypertrophy attenuated pathology. These findings demonstrate that cardiac fibroblasts secrete star miRNA-enriched exosomes and identify fibroblast-derived miR-21* as a paracrine signaling mediator of cardiomyocyte hypertrophy that has potential as a therapeutic target.
The study of biological form and how it arises is the domain of the developmental biologists; but once the form is achieved, the organ poses a fascinating conundrum for all the life scientists: how are form and function maintained in adult organs throughout most of the life of the organism? That they do appears to contradict the inherently plastic nature of organogenesis during development. How do cells with the same genetic information arrive at, and maintain such different architectures and functions, and how do they keep remembering that they are different from each other? It is now clear that narratives based solely on genes and an irreversible regulatory dynamics cannot answer these questions satisfactorily, and the concept of microenvironmental signaling needs to be added to the equation. During development, cells rearrange and differentiate in response to diffusive morphogens, juxtacrine signals, and the extracellular matrix ( ECM ). These components, which constitute the modular microenvironment, are sensitive to cues from other tissues and organs of the developing embryo as well as from the external macroenvironment. On the other hand, once the organ is formed, these modular constituents integrate and constrain the organ architecture, which ensures structural and functional homeostasis and therefore, organ specificity. We argue here that a corollary of the above is that once the organ architecture is compromised in adults by mutations or by changes in the microenvironment such as aging or inflammation, that organ becomes subjected to the developmental and embryonic circuits in search of a new identity. But since the microenvironment is no longer embryonic, the confusion leads to cancer: hence as we have argued, tumors become new evolutionary organs perhaps in search of an elusive homeostasis. WIREs Dev Biol 2014, 3:147–163. doi: 10.1002/wdev.130
This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Environmental Control of Stem Cells
Gene Expression and Transcriptional Hierarchies > Cellular Differentiation
Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms
Comparative Development and Evolution > Regulation of Organ Diversity
We are told that our present understanding of physical law was ushered
in by the Quantum Revolution, which began around 1900 and was brought to
fruition around 1930 with the formulation of modern Quantum Mechanics.
The photon" was supposed to be the centerpiece of this revolution,
conveying much of its conceptual avor. What happened during that period
was a rather violent redirection of the prevailing world view in and
around physics|a process that has still not settled. In this paper I
critically review the evolution of the concepts involved, from the time
of Maxwell up to the present day. At any given time, discussions in and
around any given topic take place using a language that presupposes a
world view or zeitgeist. The world view itself limits what ideas are
expressible. We are all prisoners of the language we have created to
develop our understanding to its present state. Thus the very concepts
and ways of thinking that have led to progress in the past are often the
source of blind spots that prevent progress into the future. The most
insidious property of the world view at any point in time is that it
involves assumptions that are not stated. In what follows we will have a
number of occasions to point out the assumptions in the current world
view, and to develop a new world view based on a quite di erent set of
assumptions.
Exosomes are small 50-100nm sized extracellular vesicles released from normal and tumour cells and are a source of a new intercellular communication pathway. Tumour exosomes promote tumour growth and progression. What regulates the release and homoeostatic levels of exosomes, in cancer, in body fluids remains undefined.
We utilised a human mammary epithelial cell line (HMEC B42) and a breast cancer cell line derived from it (B42 clone 16) to investigate exosome production and regulation. Exosome numbers were quantified using a Nanosight LM10 and measured in culture supernatants in the absence and presence of exosomes in the medium. Concentrated suspensions of exosomes from the normal mammary epithelial cells, the breast cancer cells and bladder cancer cells were used. The interaction of exosomes with tumour cells was also investigated using fluorescently labelled exosomes.
Exosome release from normal human mammary epithelial cells and breast cancer cells is regulated by the presence of exosomes, derived from their own cells, in the extracellular environment of the cells. Exosomes from normal mammary epithelial cells also inhibit exosome secretion by breast cancer cells, which occurs in a tissue specific manner. Labelled exosomes from mammary epithelial cells are internalised into the tumour cells implicating a dynamic equilibrium and suggesting a mechanism for feedback control.
These data suggest a previously unknown novel feedback regulatory mechanism for controlling exosome release, which may highlight a new therapeutic approach to controlling the deleterious effects of tumour exosomes. This regulatory mechanism is likely to be generic to other tumours.
Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness.
Liposomes are spherical-enclosed membrane vesicles mainly constructed with lipids. Lipid nanoparticles are loaded with therapeutics and may not contain an enclosed bilayer. The majority of those clinically approved have diameters of 50-300 nm. The growing interest in nanomedicine has fueled lipid-drug and lipid-protein studies, which provide a foundation for developing lipid particles that improve drug potency and reduce off-target effects. Integrating advances in lipid membrane research has enabled therapeutic development. At present, about 600 clinical trials involve lipid particle drug delivery systems. Greater understanding of pharmacokinetics, biodistribution, and disposition of lipid-drug particles facilitated particle surface hydration technology (with polyethylene glycol) to reduce rapid clearance and provide sufficient blood circulation time for drug to reach target tissues and cells. Surface hydration enabled the liposome-encapsulated cancer drug doxorubicin (Doxil) to gain clinical approval in 1995. Fifteen lipidic therapeutics are now clinically approved. Although much research involves attaching lipid particles to ligands selective for occult cells and tissues, preparation procedures are often complex and pose scale-up challenges. With emerging knowledge in drug target and lipid-drug distribution in the body, a systems approach that integrates knowledge to design and scale lipid-drug particles may further advance translation of these systems to improve therapeutic safety and efficacy. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:29-52, 2014.
Defective RNAs and proteins are swiftly degraded by cellular quality control mechanisms. A large fraction of their degradation is mediated by the exosome and the proteasome. These complexes have a similar architectural framework based on cylindrical, hollow structures that are conserved from bacteria and archaea to eukaryotes. Mechanistic similarities have also been identified for how RNAs and proteins are channelled into these structures and prepared for degradation. Insights gained from studies of the proteasome should now set the stage for elucidating the regulation, assembly and small-molecule inhibition of the exosome.
Over the last decade there has been increasing research interest in urinary exosomes and their relationship with kidney physiology and disease. Protocols for isolating urinary exosomes have been refined and the exosomal proteome has been extensively catalogued and reported to contain proteins from the kidney's glomerulus and all sections of the nephron. In animal and human biomarker discovery studies, this proteome changes to reflect the underlying pathophysiology of certain kidney diseases. In addition to proteins, exosomes from urine have been demonstrated to contain RNA species, another new reservoir for biomarker discovery. Exosomes have the capacity to shuttle their cargo between kidney cells and change the recipient cell's proteome and function, and may represent a mechanism for cell-to-cell signaling along the nephron. Significant challenges remain; methods for urinary exosome collection need optimization if 'real-life' clinical utility is to be achieved, consensus is needed regarding normalization of changes in exosomal protein and RNA, larger scale exosome biomarker validation studies remain to be performed, and whether exosomes signal between cells in vivo remains an intriguing, but untested, hypothesis.
Free energy was measured for the surface of regular aligned closest hexagonal packed −CF3 groups. n-Perfluoroeicosane was vapor deposited onto glass, which gave epitaxially grown single-like crystallites with their molecular axes perpendicular to the glass surface. The dynamic contact angle of water on its surface was 119°, which corresponds to a surface free energy of 6.7 mJ/m2. This value is considered to be the lowest surface free energy of any solid, based on the hexagonal closed alignment of −CF3 groups on the surface.
Telocytes (TCs), a novel type of interstitial cells, were recently described in the interstitial space of tissues (www.telocytes.com). TCs have several very long, moniliform extensions, namely telopodes (Tps). However, TCs functional role(s) is not yet understood. Successive photomicrographs of ultrathin sections were concatenated in order to capture the entire length of Tps which usually measure tens to hundreds of μm. Besides the podoms (dilations) and podomers (thin segments), ultrastructural features of Tps include the dichotomous branching and establishing homo- and heterocellular contacts. Tps make a labyrinthine system by 3D convolution and overlapping, their number being roughly estimated at approximately 20 per 1000 μm(2) . Moreover, the presence of extracellular vesicles (shedding vesicles/exosomes) along the Tps suggests an active intercellular signaling (micro- and macromolecules), with possible significance in regulating uterine contractility. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.
Although mesenchymal stem cells (MSCs) have been increasingly trialed to treat a variety of diseases, the underlying mechanisms remain still elusive. In this study, human umbilical cord (UC)-derived MSCs were stimulated by hypoxia, and the membrane microvesicles (MVs) in the supernatants were collected by ultracentrifugation, observed under an electron microscope, and the origin was identified with the flow cytometric technique. The results showed that upon hypoxic stimulus, MSCs released a large quantity of MVs of ∼100 nm in diameter. The MVs were phenotypically similar to the parent MSCs, except that the majority of them were negative for the receptor of platelet-derived growth factor. DiI-labeling assay revealed that MSC-MVs could be internalized into human UC endothelial cells (UC-ECs) within 8 h after they were added into the culture medium. Carboxyfluorescein succinimidyl ester-labeling technique and MTT test showed that MSC-MVs promoted the proliferation of UC-ECs in a dose-dependent manner. Further, MVs could enhance in vitro capillary network formation of UC-ECs in a Matrigel matrix. In a rat hindlimb ischemia model, both MSCs and MSC-MVs were shown to improve significantly the blood flow recovery compared with the control medium (P<0.0001), as assessed by laser Doppler imaging analysis. These data indicate that MV releasing is one of the major mechanisms underlying the effectiveness of MSC therapy by promoting angiogenesis.
We discuss dynamic hydrophobicity from the perspective of the force required to move a water droplet on a surface and argue that the structure of the three-phase contact line is important. We studied the wettability of a series of silicon surfaces that were prepared by photolithography and hydrophobized using silanization reagents. Hydrocarbon, siloxane, and fluorocarbon surfaces were prepared. The surfaces contain posts of different sizes, shapes, and separations. Surfaces containing square posts with X-Y dimensions of 32 µm and less exhibited ultrahydrophobic behavior with high advancing and receding water contact angles. Water droplets moved very easily on these surfaces and rolled off of slightly tilted surfaces. Contact angles were independent of the post height from 20 to 140 µm and independent of surface chemistry. Water droplets were pinned on surfaces containing square posts with larger dimensions. Increasing the distance between posts and changing the shape of the posts from square to staggered rhombus, star, or indented square caused increases in receding contact angles. We ascribe these contact angle increases to decreases in the contact length and increases in tortuosity of the three-phase contact line. The maximum length scale of roughness that imparts ultrahydrophobicity is ∼32 µm.
Exosomes have a growing inventory of functions, but the mechanism of protein sorting into exosomes has been unclear. Now, a signal sequence first described in viral budding provides just such a cargo sorting mechanism, revealing closer-than-expected parallelism between exosome biogenesis and the ESCRT-dependent endolysosomal pathway.
Chronic myeloid leukemia (CML) is caused by the malignant transformation of hematopoietic stem cells in leukemic stem cells. From the introduction of the anti-cancer drug imatinib, the therapy of CML has been positively transformed. However, following treatment most patients display a residual CML disease attributed to the presence of quiescent leukemic stem cells intrinsically resistant to imatinib. Considering that the later cancer cells lose their chemoresistance in vitro, it appears that the stromal microenvironment plays a crucial role in CML-affected cell chemoresistance. In the present review, we summarize and discuss the recent findings on signaling pathways through which stromal cells sustain CML leukemogenesis, as well as leukemic stem cell maintenance and chemoresistance.
Leukotrienes (LTs) are potent pro-inflammatory mediators involved in asthma. Exosomes, nanosized vesicles released from various cells, can stimulate or down-regulate immune responses, depending on the state and nature of the originating cell. We have recently shown an altered exosome profile in bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis, but their role in asthma is unknown. Our aims were to investigate whether exosomes from BALF have LT biosynthetic capacity and to explore phenotypic and functional characteristics of BALF exosomes in asthma.
Bronchoalveolar lavage fluid exosomes were collected from healthy individuals (n = 13) and patients with mild allergic asthma to birch pollen (n = 12) before and after birch allergen provocation. Exosomes were characterized by flow cytometry and Western blot. Their capacity to induce IL-8 and LT production in the human bronchial epithelial cell (BEC) line 16HB14o- was measured by ELISA and reverse-phase HPLC, respectively.
Compared to BALF exosomes from healthy individuals, BALF exosomes from asthmatics displayed higher levels of exosome-associated markers, such as the tetraspanins CD63 and CD81 and the scavenger receptor CD36. No major differences were observed between BALF exosomes from before and after allergen provocation. Furthermore, we show that BALF exosomes contain enzymes for LT biosynthesis. The effect of exosomes to promote LTC(4) and IL-8 release in BEC was significantly increased for exosomes from asthmatics, and the CysLT(1) receptor antagonist Montelukast reduced exosome-induced IL-8 secretion.
Bronchoalveolar lavage fluid exosomes from asthmatic and healthy individuals exhibit distinct phenotypes and functions. BALF exosomes from asthmatics might contribute to subclinical inflammation by increasing cytokine and LTC(4) generation in airway epithelium.
Exosomes are bioactive vesicles derived from the cell's endosomal membrane system and secreted into surrounding body fluids. Exosomes contain cell and cell-state specific cargos of protein, mRNA and miRNA. Exosome formation, cargo content, and delivery to surrounding cells is of immense biological interest considering the role that exosomes are believed to play in various pathological conditions. They aid antigen presentation by immune cells and can exhibit either anti-inflammatory or pro-inflammatory properties depending on the parent antigen-presenting cell's conditioning. Viruses can hijack a host cell's exosomal machinery to evade host defense systems aiding in the trans-infection of viruses. Tumor derived exosomes may help establish an oncogenic niche systemically via delivery of protein, mRNA, and miRNA that can aid angiogenesis, cell proliferation, and cell survival. Exosomes have also been implicated in the spread of neurodegenerative diseases. Studies have shown that exosomes are selectively taken up by cells distal from their release. They can reprogram the recipient cells due to their active molecular cargo. Cell-lineage and state-specific exosomes imply that they may therefore harbor body fluid-based biomarkers of unparalleled accuracy, particularly for tissues that are difficult or impossible to access. Exosome-specific membrane proteins provide markers enabling exosome identity and selection, while cell type and cell condition-specific protein, mRNA and miRNA cargo provide a rich potential source of biomarkers. This review serves to provide an overview of the current state of the science in the burgeoning field of exosome biology.