During pregnancy, feto-maternal communication can be mediated through extracellular vesicles, specifically exosomes, 30- to 150-nm particles released from each cell. Exosomes carry cellular signals, ...and traffic between fetal and maternal tissues to produce functional changes in recipient cells. Exosomes may function as a biomarker indicative of the physiologic status of their tissue of origin. These properties of exosomes during pregnancy are not well studied.
To test exosome trafficking and function, we used a transgenic mouse model containing membrane-targeted, red fluorescent protein tdTomato and enhanced green fluorescent protein cyclic recombinase−reporter construct expressed only in fetal tissues. This model allows fetal tissues and their exosomes to express tdTomato under normal conditions or green fluorescent protein if fetal tissues are exposed to cyclic recombinase that will excise tdTomato. As maternal tissue remains negative for this construct, tdTomato/green fluorescent protein expression and their switching can be used to determine fetal-specific cell and exosome trafficking.
tdTomato/green fluorescent protein−homozygous male mice were mated with wild-type females to have all fetal tissues express the tdTomato/green fluorescent protein allele. Red fluorescence due to tdTomato expression of the tdTomato/green fluorescent protein allele in fetal tissues (placenta, fetal membranes) was confirmed by confocal microscopy on embryonic day 16. Localization of fetal exosomes in maternal uterine tissues were performed by immunostaining for exosome marker CD81 and tdTomato expression followed by confocal microscopy. Fetal exosomes (tdTomato−positive) in maternal plasma were immunoprecipitated using anti−red fluorescent protein tdTomato, followed by confirmation with flow cytometry. To further illustrate the fidelity of fetal exosomes in maternal samples, exosomes bioengineered to contain cyclic recombinase (1.0 × 1010 exosomes) were injected intraperitoneally on embryonic day 13. On embryonic day 16, fetal (placenta and fetal membranes) tissues were imaged to show tdTomato−to−green fluorescent protein transition. The green fluorescent protein−expressing exomes were localized in maternal tissues (confocal microscopy) and plasma (flow cytometry).
Mating between a male with the tdTomato/green fluorescent protein construct and a null female resulted in fetal tissues and their exosomes expressing tdTomato positivity. Total fetal exosomes in maternal plasma was about 35%. tdTomato−positive exosomes were isolated from maternal plasma and immunostaining localized tdTomato−positive exosomes in maternal uterine tissues. Maternal intraperitoneal injection of cyclic recombinase−enriched exosomes crossed placenta, excised tdTomato from the tdTomato/green fluorescent protein construct in the fetal tissues, and caused green fluorescent protein expression in fetal cells. Furthermore, green fluorescent protein−positive exosomes released from fetal cells were isolated from maternal blood.
In this pilot study, we report feto-maternal and maternal−fetal trafficking of exosomes indicative of paracrine signaling during pregnancy. Exosomes from the maternal side can produce functional changes in fetal tissues. Trafficking of exosomes suggests their potential role in pregnancy as biomarkers of fetal functions and usefulness as a carrier of drugs and other cargo to the fetal side during pregnancy. Isolation and characterization of fetal exosomes can advance fetal research without performing invasive procedures.
Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted ...as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.
Oxidative stresses caused by reactive oxygen species (ROS) can induce rapid depolarization of inner mitochondrial membrane potential and subsequent impairment of oxidative phosphorylation. Damaged ...mitochondria produce more ROS, especially the superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)), which potentiate mitochondria-driven ROS propagation, so-called ROS-induced ROS release (RIRR), via activation of an inter-mitochondria signaling network. Therefore, loss of function in only a fraction of mitochondria might eventually affect cell viability through this positive feedback loop. Since ROS are very short-lived molecules in the biological milieu, mitochondrial network dynamics, such as density, number, and spatial distribution, can affect mitochondria-driven ROS propagation. To address this issue, we developed a mathematical model using an agent-based modeling approach, and tested the effect of mitochondrial network dynamics on RIRR for mitochondria under various conditions. Simulation results show that the intracellular ROS signaling pattern, such as ROS propagation speed and oxidative stress vulnerability, are critically affected by mitochondrial network dynamics. Mitochondrial network dynamics of mitochondrial distribution, density, activity, and size can mediate inter-mitochondrial signaling under certain conditions and determine the identity of the ROS signaling pattern. We further elucidated the potential mechanism of these actions, i.e., conversion of major messenger molecules involved in ROS signaling. If the average distance between neighboring mitochondria is large or mitochondrial distribution becomes randomized, messenger molecule of the ROS signaling network can be switched from O(2)(-) to H(2)O(2). In this case, mitochondria-driven ROS propagation is efficiently blocked by introduction of excess cytosolic glutathione peroxidase 1, while introduction of cytosolic superoxide dismutase has no effect. Together, these results suggest that mitochondrial network dynamics is a major determinant for cellular responses to RIRR through changing the key messenger molecules.
Summary
Mesenchymal stem cells (MSCs) are one of the most easily accessible stem cells that can be obtained from various human tissues. They have raised considerable interests for their potential ...applications in tissue repair, anti‐cancer therapy, and inflammation suppression. Stem cell‐based therapy was first used to treat muscular dystrophies and has been studied intensively for its efficacy in various disease models, including myocardial infarction, kidney injuries, liver injuries, and cancers. In this review, we summarized the potential mechanisms underlying MSC‐derived EVs therapy as a drug delivery platform. Additionally, based on currently published data, we predicted a potential therapeutic role of cargo proteins shuttled by EVs from MSCs. These data may support the therapeutic strategy of using the MSC‐derived EVs to accelerate this strategy from bench to bedside. Stem Cells Translational Medicine 2019;8:880&886
Exosomes are cell-secreted nano-sized vesicles which deliver diverse biological molecules for intercellular communication. Due to their therapeutic potential, exosomes have been engineered in ...numerous ways for efficient delivery of active pharmaceutical ingredients to various target organs, tissues, and cells. In vivo administered exosomes are normally delivered to the liver, spleen, kidney, lung, and gastrointestinal tract and show rapid clearance from the blood circulation after systemic injection. The biodistribution and pharmacokinetics (PK) of exosomes can be modulated by engineering various factors such as cellular origin and membrane protein composition of exosomes. Recent advances accentuate the potential of targeted delivery of engineered exosomes even to the most challenging organs including the central nervous system. Major breakthroughs have been made related to various imaging techniques for monitoring in vivo biodistribution and PK of exosomes, as well as exosomal surface engineering technologies for inducing targetability. For inducing targeted delivery, therapeutic exosomes can be engineered to express various targeting moieties via direct modification methods such as chemically modifying exosomal surfaces with covalent/non-covalent bonds, or via indirect modification methods by genetically engineering exosome-producing cells. In this review, we describe the current knowledge of biodistribution and PK of exosomes, factors determining the targetability and organotropism of exosomes, and imaging technologies to monitor in vivo administered exosomes. In addition, we highlight recent advances in strategies for inducing targeted delivery of exosomes to specific organs and cells.
Process of manufacturing therapeutics exosome development for commercialization. The development of exosome treatment starts at the bench, and in order to be commercialized, it goes through the ...manufacturing, characterization, and formulation stages, production under Good Manufacturing Practice (GMP) conditions for clinical use, and close consultation with regulatory authorities. Exosome, a type of nanoparticles also known as small extracellular vesicles are gaining attention as novel therapeutics for various diseases because of their ability to deliver genetic or bioactive molecules to recipient cells. Although many pharmaceutical companies are gradually developing exosome therapeutics, numerous hurdles remain regarding manufacture of clinical-grade exosomes for therapeutic use. In this mini-review, we will discuss the manufacturing challenges of therapeutic exosomes, including cell line development, upstream cell culture, and downstream purification process. In addition, developing proper formulations for exosome storage and, establishing good manufacturing practice facility for producing therapeutic exosomes remains as challenges for developing clinicalgrade exosomes. However, owing to the lack of consensus regarding the guidelines for manufacturing therapeutic exosomes, close communication between regulators and companies is required for the successful development of exosome therapeutics. This review shares the challenges and perspectives regarding the manufacture and quality control of clinical grade exosomes.
Senescent astrocytes in aging brain express senescence-associated secretory phenotype (SASP) and link with increased brain aging and its related diseases. In order to determine whether ginsenosides ...ameliorate the astrocytic senescence in vitro, human astrocytic CRT cells and primary rat astrocytes were used in the present study. Ginsenosides Rg1, Re, Rb1 and Rg3 (5 μg/mL) could effectively prevent the astrocytic senescence induced by H₂O₂ exposure. However, these ginsenosides did not reverse the astrocytic senescence. Importantly, senescent astrocytes herein produce SASP. The expression of major components of SASP, IL-6 and IL-8, are greatly increased in senescent astrocytes. Ginsenoside Rg3 (10 μg/mL) effectively suppressed the expressions of IL-6 and IL-8, which is associated with regulations of NF-κB and p38MAPK activation. In addition, after incubation with Rg3, conditioned medium from senescent astrocytic CRT cells significantly decreased the ability to promote the proliferation of astrocytoma U373-MG, U87-MG and U251-MG cells compared with non-treated senescent samples. Similar patterns were confirmed in chemotherapy-induced glioblastoma senescent cells. The present study explored a potential candidate for amelioration of astrocytic senescence and SASP in brain aging, which provided a basis for developing strategies to reduce the dark side of senescence in normal or pathological aging process.
Delivering therapeutics to the central nervous system (CNS) is difficult because of the blood-brain barrier (BBB). Therapeutic delivery across the tight junctions of the BBB can be achieved through ...various endogenous transportation mechanisms. Receptor-mediated transcytosis (RMT) is one of the most widely investigated and used methods. Drugs can hijack RMT by expressing specific ligands that bind to receptors mediating transcytosis, such as the transferrin receptor (TfR), low-density lipoprotein receptor (LDLR), and insulin receptor (INSR). Cell-penetrating peptides and viral components originating from neurotropic viruses can also be utilized for the efficient BBB crossing of therapeutics. Exosomes, or small extracellular vesicles, have gained attention as natural nanoparticles for treating CNS diseases, owing to their potential for natural BBB crossing and broad surface engineering capability. RMT-mediated transport of exosomes expressing ligands such as LDLR-targeting apolipoprotein B has shown promising results. Although surface-modified exosomes possessing brain targetability have shown enhanced CNS delivery in preclinical studies, the successful development of clinically approved exosome therapeutics for CNS diseases requires the establishment of quantitative and qualitative methods for monitoring exosomal delivery to the brain parenchyma in vivo as well as elucidation of the mechanisms underlying the BBB crossing of surface-modified exosomes.
The characterization of white blood cells (WBCs) is crucial for blood analyses and disease diagnoses. However, current standard techniques rely on cell labeling, a process which imposes significant ...limitations. Here we present three-dimensional (3D) optical measurements and the label-free characterization of mouse WBCs using optical diffraction tomography. 3D refractive index (RI) tomograms of individual WBCs are constructed from multiple two-dimensional quantitative phase images of samples illuminated at various angles of incidence. Measurements of the 3D RI tomogram of WBCs enable the separation of heterogeneous populations of WBCs using quantitative morphological and biochemical information. Time-lapse tomographic measurements also provide the 3D trajectory of micrometer-sized beads ingested by WBCs. These results demonstrate that optical diffraction tomography can be a useful and versatile tool for the study of WBCs.
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