Recently, extracellular vesicles (EVs)—including exosomes, microvesicles, and others—have attracted interest as cell-derived biotherapeutics and drug delivery vehicles for a variety of applications. ...This interest stems from favorable properties of EVs, including their status as mediators of cell-cell communication via transfer of biological cargo and their reported ability to cross biological barriers that impede many delivery systems. However, there are many challenges to translation and widespread application of EV-based therapeutics. One such challenge that has yet to be extensively studied involves EV preservation and storage, which must be addressed to enable use of therapeutic EVs beyond resource-intensive settings. Studies to date suggest that the most promising mode of storage is − 80°C; however, understanding of storage-mediated effects is still limited. Additionally, the effects of storage appear to vary with sample source. The lack of knowledge about and standardization of EV storage may ultimately hinder widespread clinical translation. This mini-review reports current knowledge in the field of EV preservation and storage stability and highlights future directions in the area that could be critical to eventual development of EV therapies.
Extracellular vesicles (EVs) are membrane-delimited particles that are secreted by nearly all cell types. EVs mediate crucial physiological functions and pathophysiological processes in the CNS. As ...carriers of diverse bioactive cargoes (e.g., proteins, lipids, and nucleic acids) that can be modified in response to external stimuli, EVs have emerged as pathological mediators following neurotrauma such as spinal cord injury (SCI). We discuss the roles of endogenous EVs in the CNS as well as crosstalk with peripheral EVs in relation to neurotrauma, with a particular focus on SCI. We then summarize the status of EV-based therapeutic advances in preclinical animal models for these conditions. Finally, we discuss new bioengineering strategies that are poised to enhance CNS-specific therapeutic capabilities of EVs.
Extracellular vesicles (EVs) have functional roles in neuron–glia and CNS–periphery communication following neurotrauma by regulating neuronal function, axonal regeneration and remyelination, metabolic activity, and the inflammatory milieu.Understanding EV signaling following spinal cord injury (SCI) is limited, but recent studies have identified differences in circulating EV counts and miRNA cargoes that may contribute to remote inflammatory changes.The therapeutic potential of EVs for SCI treatment depends on the phenotype of the source cell and is linked to the repertoire of the EV-associated cargo.New bioengineering approaches such as 3D printing demonstrate improved control over the production microenvironment of EVs and the cellular phenotype that can accelerate the development of high-potency therapeutic EVs for applications in neurotrauma, including SCI, in a reproducible and scalable fashion.
We have previously reported that a group of host cellular microRNAs (miRNAs; miR-34a-5p, miR-122-5p, miR-145-5p, miR-146a-5p, miR-210-3p) are released into the blood during sepsis, some of which are ...capable of inducing complement activation, cytokine production, and leukocyte migration. Extracellular vesicles (EVs) have been proposed as vehicles for extracellular miRNA-mediated intercellular communication. However, the biological function of plasma EVs and the associated miRNAs in sepsis are largely unknown. In this study, we tested the hypothesis that plasma EVs in sepsis are proinflammatory and EV-associated miRNAs are responsible for EV-induced cytokine production. Compared with those of sham mice, the plasma EVs from septic mice were slightly smaller (157 ± 2 versus 191 ± 6 nm,
< 0.0001), but more abundant (1.6 ± 0.14) × 10
versus (0.93 ± 0.14) × 10
/ml plasma,
< 0.003. miRNA array revealed that among 65 miRNAs, 8 miRNAs exhibited >1.5-fold increase in septic EVs compared with sham EVs, including miR-126-3p, miR-122-5p, miR-146a-5p, miR-145-5p, miR-26a-5p, miR-150-5p, miR-222-3p, and miR-181a-5p. Septic but not sham EVs were proinflammatory, promoting IL-6, TNF-α, IL-1β, and MIP-2 production. The effects of EVs were resistant to polymyxin B (an endotoxin inhibitor) but significantly inhibited by anti-miR inhibitors against miR-34a, miR-122, and miR-146a. Moreover, the septic EV-induced cytokine production was attenuated in TLR7
or MyD88
cells but remained the same in TLR3
or Trif
cells. In vivo, mice i.p. injected with septic EVs had marked peritoneal neutrophil migration, which was significantly attenuated in MyD88
mice. Taken together, these data demonstrate that plasma EVs of septic animals play an important role in inflammation, and EV-associated miRNAs likely mediate the cytokine production via TLR7-MyD88 signaling.
Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular ...transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.
Biogeochemical reactions associated with stream nitrogen cycling, such as nitrification and denitrification, can be strongly controlled by water and solute residence times in the hyporheic zone (HZ). ...We used a whole‐stream steady state 15N‐labeled nitrate (15NO3−) and conservative tracer (Cl−) addition to investigate the spatial and temporal physiochemical conditions controlling the denitrification dynamics in the HZ of an upland agricultural stream. We measured solute concentrations (15NO3−, 15N2 (g), as well as NO3−, NH3, DOC, DO, Cl−), and hydraulic transport parameters (head, flow rates, flow paths, and residence time distributions) of the reach and along HZ flow paths of an instrumented gravel bar. HZ exchange was observed across the entire gravel bar (i.e., in all wells) with flow path lengths up to 4.2 m and corresponding median residence times greater than 28.5 h. The HZ transitioned from a net nitrification environment at its head (short residence times) to a net denitrification environment at its tail (long residence times). NO3− increased at short residence times from 0.32 to 0.54 mg‐N L−1 until a threshold of 6.9 h and then consistently decreased from 0.54 to 0.03 mg‐N L−1. Along these same flow paths, declines were seen in DO (from 8.31 to 0.59 mg‐O2 L−1) and DOC (from 3.0 to 1.7 mg‐C L−1). The rates of the DO and DOC removal and net nitrification were greatest during short residence times, while the rate of denitrification was greatest at long residence times. 15NO3− tracing confirmed that a fraction of the NO3− removal was via denitrification as 15N2 was produced across the entire gravel bar HZ. Production of 15N2 across all observed flow paths and residence times indicated that denitrification microsites are present even where nitrification was the net outcome. These findings demonstrate that the HZ is an active nitrogen sink in this system and that the distinction between net nitrification and denitrification in the HZ is a function of residence time and exhibits threshold behavior. Consequently, incorporation of HZ exchange and water residence time characterizations will improve mechanistic predictions of nitrogen cycling in streams.
Vascularization is a crucial process during the growth and development of bone 1, yet it remains one of the main challenges in the reconstruction of large bone defects. The use of in vitro coculture ...of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) has been one of the most explored options. Both cell types secrete specific growth factors that are mutually beneficial, and studies suggested that cell-cell communication and paracrine secretion could be affected by a number of factors. However, little is known about the effect of cell patterning and the distance between cell populations on their crosstalk. In the present study, we showed that the separation and distance between ECs and MSCs populations affects angiogenesis by modulating cell-cell communication. HUVECs grown farther apart from MSCs (˃400 μm) presented characteristics of an early stage of angiogenesis (migration/proliferation). Results showed an increase in the up-regulation of VEGF, FGF-2, and ITGA3 (integrins) but a smaller fold change in the expression of VE-Cadherin and Ang-1. HUVECs were also still highly proliferative. On the contrary, HUVECs incubated closer (≤200 μm) to MSCs, showed signs of stabilization, mainly an increase in Ang-1 and VE-cadherin expression, as well as tighter monolayers. Conditioned media collected from HUVECs and MSCs grown ≤200 μm apart preferentially promoted tube formation, a later stage of angiogenesis, due in part to a significant increase in Ang-1 paracrine secretion. In addition, in groups in which fibers were printed farther apart (400 μm), cells produced EVs with a significantly increase cargo. Finally, in vivo experiment results showed an increase in blood vessels density and new bone thickness after 12 weeks of implantation in rat cranial defect, further suggesting the higher efficiency of indirect ECs/MSCs contact in prompting the release of paracrine signals that stimulate the angiogenesis of local tissues, and enhanced subsequent bone regeneration.
Lake Superior summer (July–September) surface water temperatures have increased approximately 2.5°C over the interval 1979–2006, equivalent to a rate of (11 ± 6) × 10−2°C yr−1, significantly in ...excess of regional atmospheric warming. This discrepancy is caused by declining winter ice cover, which is causing the onset of the positively stratified season to occur earlier at a rate of roughly a half day per year. An earlier start of the stratified season significantly increases the period over which the lake warms during the summer months, leading to a stronger trend in mean summer temperatures than would be expected from changes in summer air temperature alone.
The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy ...in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-β superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.
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•Heterochronic parabiosis reverses age-related cardiac hypertrophy•The antihypertrophic effect is not due to hemodynamic or behavioral factors•GDF11 is a circulating TGF-β family member that declines with age•Restoration of youthful GDF11 levels reverses age-related cardiac hypertrophy
Parabiosis experiments in mice unravel a role for the TGF-β family member GDF-11 as a negative regulator of age-associated cardiac hypertrophy.
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Extracellular vesicles (EVs) have garnered significant interest in the biotechnology field due to their intrinsic therapeutic properties as well as their ability to serve as vehicles ...for bioactive cargo. However, the lack of an established biomanufacturing platform and limited potency of EVs in vivo remain critical bottlenecks for clinical translation. In this study, we utilized a 3D-printed scaffold-perfusion bioreactor system to assess the response of dynamic culture on extracellular vesicle production from endothelial cells (ECs). We also investigated whether ethanol conditioning, which was previously shown to enhance vascularization bioactivity of EC-derived EVs produced in standard 2D culture conditions, could be employed successfully for the same purpose in a 3D production system. Our results indicate that dynamic culture in a perfusion bioreactor significantly enhances EV production from human ECs. Moreover, the use of ethanol conditioning in conjunction with dynamic culture induces pro-vascularization bioactivity of EC-derived EVs that is correlated with increased EV levels of pro-angiogenic lncRNAs HOTAIR and MALAT1. Thus, this study represents one of the first reports of rationally-designed EV potency enhancement that is conserved between static 2D and dynamic 3D EV production systems, increasing the potential for scalable biomanufacturing of therapeutic EC-derived EVs for a variety of applications.
Extracellular vesicles (EVs) have substantial therapeutic potential in a variety of applications. However, translation of EV-based therapies may be hindered by biomanufacturing challenges. EV production to date has predominantly involved the use of tissue culture flasks. Here, we report, for the first time, the use of a tubular perfusion bioreactor system with an integrated 3D-printed biomaterial scaffold for EV production from human endothelial cells. This system increases EV yield by over 100-fold compared to conventional tissue culture systems. Further, we show that an ethanol-conditioning approach that our group previously developed in 2D culture for enhancing EV potency is compatible with this new system. Thus, potency enhancement of EVs for vascularization applications is possible even with significantly increased production rate.
Cancers often evade immune surveillance by adopting peripheral tissue–tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent ...antitumor immunity. Here, we show that cyclindependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)–induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4⁺ T cell–mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells.
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