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Lung carcinoma is the most common type of cancer and the leading cause of cancer-related death worldwide. Among the numerous therapeutic strategies for the treatment of lung cancer, adeno-associated virus (AAV)-mediated gene transfer has been demonstrated to have the potential to effectively suppress tumor growth or reverse the progression of the disease in a number of preclinical studies. AAV vector has a safety profile; however, the relatively low delivery efficacy to particular subtypes of lung carcinoma has limited its prospective clinical translation. Exosomes are nanosized extracellular vesicles secreted from nearly all known cell types. Exosomes have a membrane-enclosed structure carrying a range of cargo molecules for efficient intercellular transfer of functional entities, thus are considered as a superior vector for drug delivery. In the present study, we developed a novel strategy to produce and purify AAV-containing exosomes (AAVExo) from AAV-packaging HEK 293T cells. The cellular uptake capacity of exosomes assisted and enhanced AAV entry into cells and protected AAV from antibody neutralization, which was a serious challenge for AAV in vivo application. We tested a list of lung cancer cell lines representing non-small-cell lung cancer and small-cell lung cancer and found that AAVExo apparently improved the gene transfer efficiency compared to conventional AAV vector. Our in vitro results were supported in vivo in a lung cancer xenograft rodent model. Additionally, we evaluated the gene delivery efficiency in the presence of neutralizing antibody on lung cancer cells. The results demonstrated that AAVExo-mediated gene transfer was not impacted, while the AAV vectors were significantly blocked by the neutralizing antibody. Taken together, we established an efficient methodology for AAVExo purification, and the purified AAVExo largely enhanced gene delivery to lung cancer cells with remarkable resistance to antibody neutralization.

Extracellular vesicles (EVs) are released by many cell types and distributed within various biofluids. EVs have a lipid membrane‐confined structure that allows for carrying unique molecular information originating from their parent cells. The species and quantity of EV cargo molecules, including nucleic acids, proteins, lipids, and metabolites, may vary largely owing to their parent cell types and the pathophysiologic status. Such heterogeneity in EV populations provides immense challenges to researchers, yet allows for the possibility to prognosticate the pathogenesis of a particular tissue from unique molecular signatures of dispersing EVs within biofluids. However, the inherent nature of EV's small size requires advanced methods for EV purification and evaluation from the complex biofluid. Recently, the interdisciplinary significance of EV research has attracted growing interests, and the EV analytical platforms for their diagnostic prospect have markedly progressed. This review summarizes the recent advances in these EV detection techniques and methods with the intention of translating an EV‐based liquid biopsy into clinical practice. This article aims to present an overview of current EV assessment techniques, with a focus on their progress and limitations, as well as an outlook on the clinical translation of an EV‐based liquid biopsy that may augment current paradigms for the diagnosis, prognosis, and monitoring the response to therapy in a variety of disease settings.

Rationale: Paracrine secretions seem to mediate therapeutic effects of human CD34 + stem cells locally transplanted in patients with myocardial and critical limb ischemia and in animal models. Earlier, we had discovered that paracrine secretion from human CD34 + cells contains proangiogenic, membrane-bound nanovesicles called exosomes (CD34Exo). Objective: Here, we investigated the mechanisms of CD34Exo-mediated ischemic tissue repair and therapeutic angiogenesis by studying their miRNA content and uptake. Methods and Results: When injected into mouse ischemic hindlimb tissue, CD34Exo, but not the CD34Exo-depleted conditioned media, mimicked the beneficial activity of their parent cells by improving ischemic limb perfusion, capillary density, motor function, and their amputation. CD34Exo were found to be enriched with proangiogenic miRNAs such as miR-126-3p. Knocking down miR-126-3p from CD34Exo abolished their angiogenic activity and beneficial function both in vitro and in vivo. Interestingly, injection of CD34Exo increased miR-126-3p levels in mouse ischemic limb but did not affect the endogenous synthesis of miR-126-3p, suggesting a direct transfer of stable and functional exosomal miR-126-3p. miR-126-3p enhanced angiogenesis by suppressing the expression of its known target, SPRED1 , simultaneously modulating the expression of genes involved in angiogenic pathways such as VEGF (vascular endothelial growth factor), ANG1 (angiopoietin 1), ANG2 (angiopoietin 2), MMP9 (matrix metallopeptidase 9), TSP1 (thrombospondin 1), etc. Interestingly, CD34Exo, when treated to ischemic hindlimbs, were most efficiently internalized by endothelial cells relative to smooth muscle cells and fibroblasts, demonstrating a direct role of stem cell–derived exosomes on mouse endothelium at the cellular level. Conclusions: Collectively, our results have demonstrated a novel mechanism by which cell-free CD34Exo mediates ischemic tissue repair via beneficial angiogenesis. Exosome-shuttled proangiogenic miRNAs may signify amplification of stem cell function and may explain the angiogenic and therapeutic benefits associated with CD34 + stem cell therapy.

Rationale : Studies have demonstrated that exosomes can repair cardiac tissue post–myocardial infarction and recapitulate the benefits of cellular therapy. Objective : We evaluated the role of donor age and hypoxia of human pediatric cardiac progenitor cell (CPC)–derived exosomes in a rat model of ischemia–reperfusion injury. Methods and Results : Human CPCs from the right atrial appendages from children of different ages undergoing cardiac surgery for congenital heart defects were isolated and cultured under hypoxic or normoxic conditions. Exosomes were isolated from the culture-conditioned media and delivered to athymic rats after ischemia–reperfusion injury. Echocardiography at day 3 post–myocardial infarction suggested statistically improved function in neonatal hypoxic and neonatal normoxic groups compared with saline-treated controls. At 28 days post–myocardial infarction, exosomes derived from neonatal normoxia, neonatal hypoxia, infant hypoxia, and child hypoxia significantly improved cardiac function compared with those from saline-treated controls. Staining showed decreased fibrosis and improved angiogenesis in hypoxic groups compared with controls. Finally, using sequencing data, a computational model was generated to link microRNA levels to specific outcomes. Conclusions : CPC exosomes derived from neonates improved cardiac function independent of culture oxygen levels, whereas CPC exosomes from older children were not reparative unless subjected to hypoxic conditions. Cardiac functional improvements were associated with increased angiogenesis, reduced fibrosis, and improved hypertrophy, resulting in improved cardiac function; however, mechanisms for normoxic neonatal CPC exosomes improved function independent of those mechanisms. This is the first study of its kind demonstrating that donor age and oxygen content in the microenvironment significantly alter the efficacy of human CPC-derived exosomes.

It is known that culture media (CM) promotes cellular growth, adhesion, and protects explanted primary brain cells from in vitro stresses. The fetal bovine serum (FBS) supplement used in most CM, however, contains significant quantities of extracellular vesicles (EVs) that confound quantitative and qualitative analyses from the EVs produced by the cultured cells. We quantitatively tested the ability of common FBS EV-depletion protocols to remove exogenous EVs from FBS-supplemented CM and evaluated the influence such methods have on primary astrocyte culture growth and viability. We assessed two methodologies utilized for FBS EV removal prior to adding to CM: (1) an 18-h ultracentrifugation (UC); and (2) a commercial EV-depleted FBS (Exo-FBS™). Our analysis demonstrated that Exo-FBS™ CM provided the largest depletion (75%) of total FBS EVs, while still providing 6.92 × 109 ± 1.39 × 108 EVs/mL. In addition, both UC and Exo-FBS™ CM resulted in poor primary astrocyte cell growth and viability in culture. The two common FBS EV-depletion methods investigated, therefore, not only contaminate in vitro primary cell-derived EV analyses, but also provide a suboptimal environment for primary astrocyte cell growth and viability. It appears likely that future CM optimization, using a serum-free alternative, might be required to advance analyses of cell-specific EVs isolated in vitro.

Extracellularly secreted membrane vesicles (EMVs) including exosomes were first discovered 30 years ago and were considered to be garbage bags comprised of unwanted cellular components [(1)][1]. Exosomes are relatively homogenous in size (about 30 to 100 nm) and are different from other secreted

Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs.

Exosomes are cell-derived nanovesicles that carry and shuttle microRNAs (miRNAs) to mediate cell-cell communication. Vast majority of cell types including cardiac myocytes and progenitors actively secrete exosomes, whose miRNA contents are altered after physiological or pathological changes such as myocardial ischemia (MI). In this new study, we have discovered that chemical modification to mRNAs is a novel regulator of ischemia-induced gene expression changes in the heart. We hypothesized that the benefits of human CD34 + stem cell-derived exosomes (CD34exo) are mediated by mRNA modifications in the target cells via miRNA delivery. MiRNA profiling and bioinformatic analysis identified that CD34exo is selectively enriched with a number of miRNAs that directly target genes implicated in regulation of mRNA modifications. Interestingly, under myocardial ischemia, there was a significant increase in mRNA modifications in the mouse heart, which was decreased by about 70% with CD34exo-treatment. In line with the in vivo MI data, in vitro hypoxic stimulation in neonatal / adult rodent myocytes and non-myocytes increased mRNA modifications and controls known regulators of those mRNA modifications. Loss-of-function studies for regulators of mRNA modifications attenuated hypoxia-induced changes to epitranscriptome indicating important roles for these molecules under stress conditions. Finally, using gain-of-function and loss-of-function studies, we demonstrate that miR-126, one of the most enriched miRNAs in CD34exo, plays a critical role in regulating the mRNA modifications. We conclude that miRNAs enriched in CD34exo mediate their cardioprotective effect at least in part, by regulating the mRNA epitranscriptome of the target cell. Our new data suggests hypoxia as a novel regulator of the mRNA epitranscriptome and provides novel insights to post-transcriptional gene regulation in the heart.

Introduction: Clinical application of human CD34+ stem cells is associated with improved exercise tolerance and therapeutic angiogenesis in patients with myocardial ischemia. We reported the first description of independent therapeutic potential of CD34+ stem cell-derived exosomes (CD34Exo) to that of parent cell by mechanisms that still remain poorly understood. Hypothesis: Herein, we tested the hypothesis that CD34Exo may selectively carry non-coding RNA (ncRNA) cargo targeted for pro-angiogenic signaling and ischemic tissue repair. Methods and Results: Murine models of myocardial ischemia employed throughout the study. Cell-free CD34Exo replicated the therapeutic activity of their parent cells by significantly improving myocardial ischemia (ejection fraction, 42±4 v 22±6%; capillary density, 113±7 v 66±6/HPF; fibrosis, 27±2 v 48±7%; p Conclusion: Our results reveal specific CD34Exo-shuttled microRNAs and pathways regulated in the ischemic myocardium. Our work presents a molecular framework for CD34Exo mechanism and function in therapeutic angiogenesis. Precise understanding of CD34Exo mechanisms could significantly amplify the therapeutic benefits of CD34Exo in ischemic tissue regeneration and repair.

Previous studies in our lab have revealed a novel mechanism that therapeutically important human CD34+ stem cells secrete exosomes (Exo) to extracellular milieu that induce angiogenic activity independent of the cells. Further studies demonstrated that CD34+ exosomes (CD34Exo) carry and transfer of proangiogenic miRNAs, such as miR-126, which affect the therapeutic function of the exosomes. Herein we hypothesized that hypoxic treatment of CD34+ stem cells can modulate the miRNA content and regenerative efficacy of CD34Exo. Methods and Results: Exosomes from human CD34+ cells cultured under hypoxia (H-Exo) were more proliferative, anti-apoptotic and angiogenic in vitro, as compared to exosomes from cells under normoxia (N-Exo). In a mouse model of hind limb ischemia, H-Exo treatment significantly enhanced perfusion (ratio: 0.93±0.05 v 0.77±0.02), increased capillary density (1.6±0.2 v 1.1±0.1/HPF) and prevented ischemic limb amputation (0% v 37.5%) as compared to N-Exo (p Conclusion: Hypoxia induced miR-126 expression in CD34 cell-derived exosomes stimulating exosomes-mediated angiogenesis and therapeutic recovery via ETS-transcriptional pathway. Our work has important clinical implications for therapeutic angiogenesis, especially in diabetic and cardiovascular patients who have stem cells with diminished angiogenic potential.

Exosomes, also known as microvesicles (EMVs), are nano-sized membrane particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation and suppression, and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein cargo, which can dictate pathogenicity. While some mechanisms have been proposed for the regulation of EMV cargo selection, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from the overall cellular membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we explore further the role of glycosylation in EMV cargo selection. We identify specific glycosylated targets and associated proteins and demonstrate alteration of their recruitment as a functio...