Non-reversible tissue fixation retains extracellular vesicles for in situ imaging

Gupta, Mrinali P.; Tandalam, Sangeetha; Ostrager, Shariss; Lever, Alexander S.; Fung, Angus R.; Hurley, David D.; Alegre, Gemstonn; Espinal, Jasmin E.; Remmel, H. Lawrence; Mukherjee, Sushmita; Levine, Benjamin; Robins, Russell P.; Molina, Henrik; Dill, Brian D.; Kenific, Candia M.; Tuschl, Thomas; Lyden, David; D’Amico, Donald J.; Pena, John

doi:10.1038/s41592-019-0623-4

Cited by 19 publications

(17 citation statements)

References 34 publications

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“…The reason for this discrepancy is not known, but renal epithelial cells are, however, highly water permeable 38 , and we speculate that rapid osmotic changes (e.g., 25% sucrose) imposed during tissue preparation are involved. Recently, it was shown that the handling and preparation of tissue samples significantly affect tissue EV abundance through release to, e.g., washing buffers 39 . Our observations agree with this and suggest that careful and specific tissue preparation is essential when analyzing tissue EV levels and intercellular communication.…”

Section: Discussionmentioning

confidence: 99%

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles

Nørgård

Steffensen

Hansen

et al. 2022

Sci Rep

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The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

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Section: Discussionmentioning

confidence: 99%

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles

Nørgård

Steffensen

Hansen

et al. 2022

Sci Rep

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“…That can be accomplished using different methodologies, allowing to determine size, shape, concentration, contents, and surface biochemical-markers. They include: (i) western blotting; (ii) identification and quantification of nucleic-acid contents, using polymerase chain-reaction (PCR), microarray and secondgeneration sequencing (SGS) and third-generation sequencing (TGS), sometimes known with the ambiguous name of "next"generation sequencing (NGS); (iii) lipidomic approaches; (iv) nanoparticle-tracking analyses (NTA) or (v) tunable-resistive pulse sensing (TRPS), both for determination of the size and concentration of particles; (vi) dynamic-light scattering (DLS) or (vii) photon-correlation spectroscopy (PCS), both to measure exosome sizes; (viii) atomic-force microscopy (AFM) or (ix) transmission electron microscopy (TEM), both for visualization and characterization of their structure, morphology, and size; (x) flow cytometry, for the characterization of surface biochemical markers; and (xi) fixation for in situ imaging (Choi et al, 2013;Kreimer et al, 2015;Gupta et al, 2019;Gurunathan et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Extracellular Vesicles Derived From Mesenchymal Stem Cells (MSC) in Regenerative Medicine: Applications in Skin Wound Healing

Casado-Díaz

Quesada‐Gómez

Dorado

2020

Front. Bioeng. Biotechnol.

221

183

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The cells secrete extracellular vesicles (EV) that may have an endosomal origin, or from evaginations of the plasma membrane. The former are usually called exosomes, with sizes ranging from 50 to 100 nm. These EV contain a lipid bilayer associated to membrane proteins. Molecules such as nucleic acids (DNA, mRNA, miRNA, lncRNA, etc.) and proteins may be stored inside. The EV composition depends on the producer cell type and its physiological conditions. Through them, the cells modify their microenvironment and the behavior of neighboring cells. That is accomplished by transferring factors that modulate different metabolic and signaling pathways. Due to their properties, EV can be applied as a diagnostic and therapeutic tool in medicine. The mesenchymal stromal cells (MSC) have immunomodulatory properties and a high regenerative capacity. These features are linked to their paracrine activity and EV secretion. Therefore, research on exosomes produced by MSC has been intensified for use in cell-free regenerative medicine. In this area, the use of EV for the treatment of chronic skin ulcers (CSU) has been proposed. Such sores occur when normal healing does not resolve properly. That is usually due to excessive prolongation of the inflammatory phase. These ulcers are associated with aging and diseases, such as diabetes, so their prevalence is increasing with the one of such latter disease, mainly in developed countries. This has very important socio-economic repercussions. In this review, we show that the application of MSC-derived EV for the treatment of CSU has positive effects, including accelerating healing and decreasing scar formation. This is because the EV have immunosuppressive and immunomodulatory properties. Likewise, they have the ability to activate the angiogenesis, proliferation, migration, and differentiation of the main cell types involved in skin regeneration. They include endothelial cells, fibroblasts, and keratinocytes. Most of the studies carried out so far are preclinical. Therefore, there is a need to advance more in the knowledge about the conditions of production, isolation, and action mechanisms of EV. Interestingly, their potential application in the treatment of CSU opens the door for the design of new highly effective therapeutic strategies.

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“…Recently, it was shown that the handling and preparation of tissue samples significantly affect tissue EV abundance through release to e.g. washing buffers 25 . Our observations agree with this and suggest that careful and specific tissue preparation is important when analyzing tissue EV levels and intercellular communication.…”

Section: Discussionmentioning

confidence: 99%

A new transgene mouse model using an extravesicular EGFP tag to elucidate the in vivo function of extracellular vesicles

Noegaard

Steffensen²,

Hansen³

et al. 2021

Preprint

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The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate. We therefore created an EV reporter using CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9-EGFP expression in cells did not affect EV size and concentration, but allowed for co-precipitation of EV markers TSG101 and ALIX from cell-conditioned medium by anti-GFP immunoprecipitation. We created a transgenic mouse where CD9-EGFP was inserted in the inverse orientation and double-floxed, ensuring Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (AMHC-Cre) and kidney epithelium (Pax8-Cre), respectively. The mice showed tissue-specific EGFP expression, and plasma and urine samples were used to immunoprecipitate EVs. CD9-EGFP EVs was detected in plasma samples from CMV-Cre/CD9-EGFP and AMHC-Cre/CD9-EGFP mice, but not in PAX8-Cre/CD9-EGFP mice. On the other hand, CD9-EGFP EVs were detected in urine samples from CMV-Cre/CD9-EGFP and PAX8-Cre/CD9-EGFP mice, but not AMHC-Cre/CD9-EGFP, indicating that plasma EVs are not filtered to the urine. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to study cell-specific EVs in vivo and gain new insight into their physiological and pathophysiological function.

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Non-reversible tissue fixation retains extracellular vesicles for in situ imaging

Cited by 19 publications

References 34 publications

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles

Extracellular Vesicles Derived From Mesenchymal Stem Cells (MSC) in Regenerative Medicine: Applications in Skin Wound Healing

A new transgene mouse model using an extravesicular EGFP tag to elucidate the in vivo function of extracellular vesicles

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