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1.
Commun Biol ; 6(1): 478, 2023 05 03.
Article in English | MEDLINE | ID: mdl-37137966

ABSTRACT

Extracellular vesicles (EVs) are highly interesting for the design of next-generation therapeutics. However, their preparation methods face challenges in standardization, yield, and reproducibility. Here, we describe a highly efficient and reproducible EV preparation method for monodisperse nano plasma membrane vesicles (nPMVs), which yields 10 to 100 times more particles per cell and hour than conventional EV preparation methods. nPMVs are produced by homogenizing giant plasma membrane vesicles following cell membrane blebbing and apoptotic body secretion induced by chemical stressors. nPMVs showed no significant differences compared to native EVs from the same cell line in cryo-TEM analysis, in vitro cellular interactions, and in vivo biodistribution studies in zebrafish larvae. Proteomics and lipidomics, on the other hand, suggested substantial differences consistent with the divergent origin of these two EV types and indicated that nPMVs primarily derive from apoptotic extracellular vesicles. nPMVs may provide an attractive source for developing EV-based pharmaceutical therapeutics.


Subject(s)
Extracellular Vesicles , Zebrafish , Animals , Reproducibility of Results , Tissue Distribution , Extracellular Vesicles/metabolism , Cell Membrane/metabolism
2.
Front Pharmacol ; 13: 1052091, 2022.
Article in English | MEDLINE | ID: mdl-36506565

ABSTRACT

Cardiac progenitor cell-derived extracellular vesicles (CPC-EVs) have been successfully applied via different delivery routes for treating post-myocardial infarction injury in several preclinical models. Hence, understanding the in vivo fate of CPC-EVs after systemic or local, i.e. myocardial, delivery is of utmost importance for the further therapeutic application of CPC-EVs in cardiac repair. Here, we studied the tissue- and cell distribution and retention of CPC-EVs after intramyocardial and intravenous injection in mice by employing different EV labeling and imaging techniques. In contrast to progenitor cells, CPC-EVs demonstrated no immediate flush-out from the heart upon intramyocardial injection and displayed limited distribution to other organs over time, as determined by near-infrared imaging in living animals. By employing CUBIC tissue clearing and light-sheet fluorescent microscopy, we observed CPC-EV migration in the interstitial space of the myocardium shortly after EV injection. Moreover, we demonstrated co-localization with cTnI and CD31-positive cells, suggesting their interaction with various cell types present in the heart. On the contrary, after intravenous injection, most EVs accumulated in the liver. To potentiate such a potential systemic cardiac delivery route, targeting the cardiac endothelium could provide openings for directed CPC-EV therapy. We therefore evaluated whether decorating EVs with targeting peptides (TPs) RGD-4C or CRPPR connected to Lamp2b could enhance EV delivery to endothelial cells. Expression of both TPs enhanced CPC-EV uptake under in vitro continuous flow, but did not affect uptake under static cell culture conditions. Together, these data demonstrate that the route of administration influences CPC-EV biodistribution pattern and suggest that specific TPs could be used to target CPC-EVs to the cardiac endothelium. These insights might lead to a better application of CPC-EV therapeutics in the heart.

3.
Mol Metab ; 65: 101580, 2022 11.
Article in English | MEDLINE | ID: mdl-36028121

ABSTRACT

OBJECTIVE: Adipose tissue, via sympathetic and possibly sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue are poorly characterized. METHODS AND RESULTS: Taking advantage of recent progress in whole-mount three-dimensional imaging, we identified a network of calcitonin gene-related protein (CGRP)-positive sensory neurons in murine white adipose tissue (WAT). We found that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, is required for arborization of sensory neurons, but not of sympathetic neurons. Time course experiments revealed that adipose mTORC2 is required for maintenance of sensory neurons. Furthermore, loss of sensory innervation in WAT coincided with systemic insulin resistance. Finally, we established that neuronal protein growth-associated protein 43 (GAP43) is a marker for sensory neurons in adipose tissue. CONCLUSION: Our findings indicate that adipose mTORC2 is necessary for sensory innervation in WAT. In addition, our results suggest that WAT may affect whole-body energy homeostasis via sensory neurons.


Subject(s)
Calcitonin Gene-Related Peptide , Insulins , Adipose Tissue/metabolism , Adipose Tissue, White/metabolism , Animals , Calcitonin , Calcitonin Gene-Related Peptide/metabolism , GAP-43 Protein/metabolism , Homeostasis , Insulins/metabolism , Mammals , Mechanistic Target of Rapamycin Complex 2/metabolism , Mice , Sensory Receptor Cells , TOR Serine-Threonine Kinases/metabolism
4.
Cell Rep ; 32(10): 108105, 2020 09 08.
Article in English | MEDLINE | ID: mdl-32905777

ABSTRACT

Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation.


Subject(s)
Cell Hypoxia/immunology , Neoplastic Cells, Circulating/immunology , Proteomics/methods , Animals , Female , Humans , Male , Mice
5.
Dev Biol ; 458(2): 133-140, 2020 02 15.
Article in English | MEDLINE | ID: mdl-31697937

ABSTRACT

The tetrapod limb has long served as a paradigm to study vertebrate pattern formation. During limb morphogenesis, a number of distinct tissue types are patterned and subsequently must be integrated to form coherent functional units. For example, the musculoskeletal apparatus of the limb requires the coordinated development of the skeletal elements, connective tissues, muscles and nerves. Here, using light-sheet microscopy and 3D-reconstructions, we concomitantly follow the developmental emergence of nerve and muscle patterns in chicken wings and legs, two appendages with highly specialized locomotor outputs. Despite a comparable flexor/extensor-arrangement of their embryonic muscles, wings and legs show a rotated innervation pattern for their three main motor nerve branches. To test the functional implications of these distinct neuromuscular topologies, we challenge their ability to adapt and connect to an experimentally altered skeletal pattern in the distal limb, the autopod. Our results show that, unlike autopod muscle groups, motor nerves are unable to fully adjust to a changed peripheral organisation, potentially constrained by their original projection routes. As the autopod has undergone substantial morphological diversifications over the course of tetrapod evolution, our results have implications for the coordinated modification of the distal limb musculoskeletal apparatus, as well as for our understanding of the varying degrees of motor functionality associated with human hand and foot malformations.


Subject(s)
Hindlimb/embryology , Wings, Animal/embryology , Animals , Chick Embryo , Chickens , Extremities/embryology , Muscles/embryology , Nervous System/embryology , Organogenesis/physiology
6.
J Extracell Vesicles ; 8(1): 1663043, 2019.
Article in English | MEDLINE | ID: mdl-31579435

ABSTRACT

Extracellular vesicles (EV) convey biological information by transmitting macromolecules between cells and tissues and are of great promise as pharmaceutical nanocarriers, and as therapeutic per se. Strategies for customizing the EV surface and cargo are being developed to enable their tracking, visualization, loading with pharmaceutical agents and decoration of the surface with tissue targeting ligands. While much progress has been made in the engineering of EVs, an exhaustive comparative analysis of the most commonly exploited EV-associated proteins, as well as a quantification at the molecular level are lacking. Here, we selected 12 EV-related proteins based on MS-proteomics data for comparative quantification of their EV engineering potential. All proteins were expressed with fluorescent protein (FP) tags in EV-producing cells; both parent cells as well as the recovered vesicles were characterized biochemically and biophysically. Using Fluorescence Correlation Spectroscopy (FCS) we quantified the number of FP-tagged molecules per vesicle. We observed different loading efficiencies and specificities for the different proteins into EVs. For the candidates showing the highest loading efficiency in terms of engineering, the molecular levels in the vesicles did not exceed ca 40-60 fluorescent proteins per vesicle upon transient overexpression in the cells. Some of the GFP-tagged EV reporters showed quenched fluorescence and were either non-vesicular, despite co-purification with EVs, or comprised a significant fraction of truncated GFP. The co-expression of each target protein with CD63 was further quantified by widefield and confocal imaging of single vesicles after double transfection of parent cells. In summary, we provide a quantitative comparison for the most commonly used sorting proteins for bioengineering of EVs and introduce a set of biophysical techniques for straightforward quantitative and qualitative characterization of fluorescent EVs to link single vesicle analysis with single molecule quantification.

7.
Sci Rep ; 9(1): 1165, 2019 02 04.
Article in English | MEDLINE | ID: mdl-30718583

ABSTRACT

Confocal microscopy is used today on a daily basis in life science labs. This "routine" technique contributes to the progress of scientific projects across many fields by revealing structural details and molecular localization, but researchers need to be aware that detection efficiency and emission light path performance is of major influence in the confocal image quality. By design, a large portion of the signal is discarded in confocal imaging, leading to a decreased signal-to-noise ratio (SNR) which in turn limits resolution. A well-aligned system and high performance detectors are needed in order to generate an image of best quality. However, a convenient method to address system status and performance on the emission side is still lacking. Here, we present a complete method to assess microscope and emission light path performance in terms of SNR, with a comprehensive protocol alongside NoiSee, an easy-to-use macro for Fiji (available via the corresponding update site). We used this method to compare several confocal systems in our facility on biological samples under typical imaging conditions. Our method reveals differences in microscope performance and highlights the various detector types used (multialkali photomultiplier tube (PMT), gallium arsenide phosphide (GaAsP) PMT, and Hybrid detector). Altogether, our method will provide useful information to research groups and facilities to diagnose their confocal microscopes.

8.
ACS Comb Sci ; 18(5): 209-19, 2016 05 09.
Article in English | MEDLINE | ID: mdl-27057765

ABSTRACT

The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community.


Subject(s)
Combinatorial Chemistry Techniques , High-Throughput Screening Assays/methods , Microscopy, Fluorescence/methods , Drug Discovery/methods , Microspheres , Peptide Library , Protein Array Analysis , Protein Binding
9.
J Cell Biol ; 213(2): 173-84, 2016 04 25.
Article in English | MEDLINE | ID: mdl-27114500

ABSTRACT

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.


Subject(s)
Endoplasmic Reticulum/metabolism , Endosomes/metabolism , Exosomes/metabolism , Pseudopodia/physiology , Biological Transport , Endoplasmic Reticulum/ultrastructure , Endosomes/ultrastructure , Exosomes/physiology , Exosomes/ultrastructure , HEK293 Cells , Humans , Microscopy, Electron, Scanning , Pseudopodia/ultrastructure
10.
Nanomedicine ; 11(4): 879-83, 2015 May.
Article in English | MEDLINE | ID: mdl-25659648

ABSTRACT

Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media types such as stem cell media, which is of huge significance for future clinical applications involving EVs. FROM THE CLINICAL EDITOR: Recent evidence suggests extracellular vesicles (EVs) as another route of cellular communication. These EVs may be utilized for future therapeutics. In this article, the authors compared ultrafiltration with size-exclusion liquid chromatography (UF-LC) and ultra-centrifugation (UC) for EV recovery.


Subject(s)
Cell-Derived Microparticles/chemistry , Cell-Derived Microparticles/ultrastructure , Chromatography, Gel , HEK293 Cells , Humans , Ultrafiltration
11.
EMBO J ; 32(8): 1115-27, 2013 Apr 17.
Article in English | MEDLINE | ID: mdl-23511973

ABSTRACT

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing.


Subject(s)
Endoplasmic Reticulum/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Argonaute Proteins/analysis , DEAD-box RNA Helicases/analysis , Endoplasmic Reticulum/chemistry , HeLa Cells , Humans , Immunoprecipitation , RNA-Binding Proteins/analysis , Ribonuclease III/analysis
12.
J Control Release ; 157(2): 297-304, 2012 Jan 30.
Article in English | MEDLINE | ID: mdl-21884740

ABSTRACT

Chitosans are naturally occurring polymers widely used in life science to mediate intracellular uptake of nucleic acids such as siRNA. Four chitosans of fungal origin (Agaricus bisporus; molecular weights MW=44, 63, 93 and 143 kDa) were used in this study and profiled for size, viscosity and hydrodynamic radius using gel permeation chromatography (GPC). Polyplexes made of these chitosans and siRNA were developed and optimized for transfection efficacy in vitro. The characteristics of these polyplexes were low chitosan:siRNA ratios (4-8; N:P) similar positive zeta potential (20-30 mV) and comparable particle sizes (about 150 nm). Endogenous luciferase reporter gene down-regulation in human epithelial H1299 cells at nanomolar concentrations (37.5-150 nM) was significantly stronger for the lower molecular weight chitosans. The impact of these low N:P polyplexes on the cellular viability was minimal also at 150 nM. To help develop an understanding of these differences, an energetic profile of the molecular interactions and polyplex formation was established by isothermal titration calorimetry (ITC). The four polyplexes exhibited strong binding enthalpies delta H(bind)(-84 to -102 kcal/mol) resulting in nanomolar dissociation constants. Intracellular trafficking studies using rhodamine labeled siRNA revealed that polyplexes made from smaller MW chitosans exhibited faster cellular uptake kinetics than their higher MW counterpart. Transmission electron microscopy and small angle X-ray scattering studies (SAXS) revealed that the 44 kDa derived polyplexes exhibited regular spherical structure, whereas the 143 kDa chitosan polyplex was rather irregularly shaped. With regards to adverse effects these low N:P chitosan/siRNA formulations represent an interesting alternative to so far reported chitosan polyplexes that used vast N:P excess to achieve similar bioactivity.


Subject(s)
Chitosan/chemistry , Nanoparticles/chemistry , RNA, Small Interfering/chemistry , Cell Line , Chitosan/administration & dosage , Gene Silencing , Genes, Reporter/genetics , Humans , Luciferases/genetics , Microscopy, Confocal , Microscopy, Electron, Transmission , Nanoparticles/administration & dosage , Nanoparticles/ultrastructure , Particle Size , RNA, Small Interfering/administration & dosage , Transfection
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