Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: MSC-derived sEV treatment decreases clinical severity scores in murine sepsis (A) Diagram of murine cecal slurry model. Mice were injected with cecal slurry IP, to induce polymicrobial sepsis. At 6 h post-injection, MSC-derived sEVs or sEV-depleted media were administered via tail vein injection. When mice reached a sepsis score of 15 or above or at 24 h post-IP injection, the brain tissue was harvested. (B) MSC-derived sEV treatment ( n = 26) in mice 6 h after the onset of sepsis resulted in improved disease overall severity score as compared with the untreated septic mice ( n = 19) (∗∗∗ p = 0.0005) and lower peak scores (∗∗∗∗ p < 0.0001). (C) MSC-derived sEV treatment improved scores at 24 h in neurological-only parameters (i.e., level of consciousness, activity, response to stimulus) as compared with the untreated septic mice (∗ p = 0.01). Data are represented as mean ± SEM, one-way ANOVA. ANOVA: Analysis of Variance, IP: intraperitoneally, MSC-derived sEV: mesenchymal stem cell-derived small extracellular vesicles, NS: non-significant.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: Derivative Assay, Injection, Activity Assay

Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: Sepsis-induced cerebellar injury is reversed by MSC-derived sEVs (A–D) Representative photomicrographs of H&E and TUNEL staining in the mouse cerebellum show significant histopathological alterations during sepsis. Compared to controls, which exhibited intact cellular architecture with clear, rounded nuclei, the septic mouse cerebellum displayed (A) significant histopathological alterations including shrunken PCs, pyknotic nuclei (black arrows), perineuronal vacuole formation (Materials and Methods: Tissue processing and histological assessment) and (B) increased TUNEL labeled cells (white arrows) indicating DNA fragmentation and cell death. Overall, sepsis resulted in (C) increased neuropathological score and (D) TUNEL+ cells (∗∗∗∗ p < 0.0001 and ∗∗∗∗ p < 0.0001) which both improved with MSC-derived sEV treatment (∗ p = 0.0155 and ∗∗ p = 0.0063). Data are represented as mean ± SEM, one-way ANOVA. Scale bar = 10 μm, Control ( n = 7), Sepsis+media ( n = 8), Sepsis+MSC-derived sEVs ( n = 8). ANOVA: Analysis of Variance, DNA: Deoxyribonucleic Acid, H&E: Hematoxylin and Eosin, NS: non-significant, PC: Purkinje cells, TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling, MSC-derived sEV: mesenchymal stem cell-derived small extracellular vesicles.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: Derivative Assay, TUNEL Assay, Staining, Labeling, Control, End Labeling

Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: RNA-seq reveals MSC-derived sEV-induced changes in cerebellar transcriptome following sepsis (A and B) Predicted activated (green) and inhibited (gray) causal networks and canonical pathways (determined by directional z-scores) in septic mice treated with MSC-derived sEVs compared to untreated septic mice. Ranked based on p value as determined using Fisher’s exact test. (C) Predicted increases (green) and decreases (gray) in cell and molecular functions (determined by directional z-scores) in septic mice treated with MSC-derived sEVs compared to untreated septic mice. Ranked based on p value as determined using Fisher’s exact test. (D) Predicted activated (green) and inhibited (gray) upstream regulators (determined by directional z-scores) in in septic mice treated with MSC-derived sEVs compared to untreated septic mice. Ranked based on p value as determined using Fisher’s exact test. (E) Heatmap of most significantly predicted upstream regulators when septic mice treated with MSC-derived sEVs are compared to untreated septic mice. Boxes are colorized with z-scores (green = activated, gray = inactivated). Source data are provided as a source data file.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: RNA Sequencing Assay, Derivative Assay

Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: MSC-derived sEVs affect cytokine concentration in septic mouse cerebellum TNF-α and IL-17α assessed by immunofluorescence. (A) Representative photomicrograph of TNF-α, PV and IL-17α staining in low (x1.4, left) and high (×60, right) magnification of the area included in the hatched box. TNF-α was expressed and co-localized with the PC and their dendrites. (B and C) TNF-α expression significantly increased in SE compared to controls (5.2 ± 1.1 vs. 1.4 ± 0.2, p = 0.0085), however, treatment with MSC-derived sEVs restored its expression by more than 50% (1.9 ± 0.2, p = 0.03). Notably, TNF-α was not expressed in parvalbumin (PV)+ interneurons (a and c) that surround the PCs. (D) The expression of IL-17α was similar in control and septic mice (5.62E+09 ± 2.7E+08 vs. 7.8E+09 ± 3.7E+08, p = 0.1096), however, treatment with MSC-derived sEVs doubled its expression (1.52E+10 ± 1.3E+09, p =<0.0001). Data are represented as mean ± SEM, one-way ANOVA. Scale bar = 10 μm, Control ( n = 6), Sepsis+media [for TNF-α ( n = 7) and IL-17α ( n = 8)], Sepsis+MSC-derived sEVs [(for TNF-α ( n = 5) and IL-17α ( n = 6)]. MSC-derived sEV: mesenchymal stem cell-derived small extracellular vesicles, NS: non-significant, TNFα: tumor necrosis factor alpha, IL-17α: interleukin 17 alpha, ANOVA: Analysis of Variance, PV: parvalbumin.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: Derivative Assay, Concentration Assay, Immunofluorescence, Staining, Expressing, Control

Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: MSC-derived sEVs restore basal and non-mitochondrial respiration in septic mouse cerebellum Cellular respiration measured with Seahorse technology in mice. (A) Basal respiration decreases in sepsis [236.1 ± 19.1 vs. 311.0 ± 38.2 (∗ p = 0.0226)], but significantly improves with MSC-derived sEV treatment (332.4 ± 40.4, ∗ p = 0.0337 ) to levels similar to controls (311.0 ± 38.2, p = 0.3146 ) . (B) Cerebellar tissue shows lower maximum respiration in sepsis that is trending higher with MSC-derived sEV administration but did not reach significance ( p = 0.07). (C) Non-mitochondrial respiration i.e., OCR attributable to ROS production or pentose phosphate pathway increases under septic conditions [268.2 ± 36.4 vs.127.3 ± 19.3 (∗∗∗ p = 0.0051)], but not with treatment (79.3 ± 8.6, ∗∗∗ p = 0.0048), indicating that MSC-derived sEVs favor OXPHOS-linked ATP production. (D) Although the average ATP-linked respiration showed improvement with sEV treatment, there was no statistically significant difference among the groups, likely due to the short period of time in which observations occurred. Data are represented as mean ± SEM, one-way ANOVA. Control+media ( n = 5), Control+MSC-derived sEVs ( n = 5), Sepsis+media ( n = 5), Sepsis+MSC-derived sEVs ( n = 5). OCR: Oxygen consumption rate, ROS: reactive oxygen species, OXPHOS: oxidative phosphorylation, ATP: adenosine triphosphate, MSC-derived sEV: mesenchymal stem cell-derived small extracellular vesicles, NS: non-significant.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: Derivative Assay, Control

Journal: iScience

Article Title: Mesenchymal stem cell-derived small extracellular vesicles alleviate the immunometabolic dysfunction in murine septic encephalopathy

doi: 10.1016/j.isci.2024.110573

Figure Lengend Snippet: MSC-derived sEVs alter the activation of miRNAs in the septic cerebellum (A) Predicted inhibited (gray) miRNAs (determined by directional z-scores) in septic mice compared to controls. Ranked based on p value as determined using Fisher’s exact test. Most miRNAs of interest are inhibited, indicating that they do not have any predicted inhibitory effects on their target mRNA. (B) Predicted activated (green) and inhibited (gray) miRNAs (determined by directional z-scores) in septic MSC-derived sEV-treated mice compared to untreated septic mice. Ranked based on p value as determined using Fisher’s exact test. Several miRNAs inhibited in septic mice that received sEV-depleted media were predicted to be activated in the septic mice that received MSC-derived sEVs. Source data are provided as a source d file.

Article Snippet: MSC-derived sEVs were isolated from human adipose-derived MSC cell cultures (ADMSC) obtained from Zen-Bio (Durham, NC) for the purpose of this research.

Techniques: Derivative Assay, Activation Assay

Journal: Extracellular vesicles and circulating nucleic acids

Article Title: Milk-borne small extracellular vesicles: kinetics and mechanisms of transport, distribution, and elimination

doi: 10.20517/evcna.2023.25

Figure Lengend Snippet: Tissue distribution of milk sEVs in mice. (A) Distribution of bovine milk sEVs loaded with fluorophore (ATTO)-labeled miR-375 in Balb/c mice. Modified from Manca et al . with permission from the original publisher, Nature Springer . To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ ; (B) Accumulation of enhanced green fluorescence protein (eGFP)-positive milk sEVs in peripheral tissues and the small intestinal mucosa in wild-type (WT) pups fostered to exosome and cargo tracking (ECT) dams and nursed for 17 days. ECT mice secrete sEVs labeled with an eGFP fusion protein in milk. WT pups fostered to WT dams served as controls. From Zhou et al . with permission from the original publisher, Frontiers Media SA . To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ .

Article Snippet: The pharmaceutical industry has recognized the potential for using bovine milk sEVs to deliver anti-cancer drugs, as evidenced by a $1 billion licensing agreement between Roche, Inc. and PureTech Health, Inc. [ ] .

Techniques: Labeling, Modification, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Enhanced therapeutic effect of PEDF-loaded mesenchymal stem cell-derived small extracellular vesicles against oxygen-induced retinopathy through increased stability and penetrability of PEDF

doi: 10.1186/s12951-023-02066-z

Figure Lengend Snippet: Characterisation of MSCs and sEVs. A Representative images of adipogenic (oil red O staining), osteogenic (alizarin red S staining), and chondrogenic (alcian blue staining) differentiation assay. B Analysis of sizes of sEVs from each group using Nanosight. C Representative transmission electron micrograph for each group; scale bar = 200 nm. D Representative western blots for loaded proteins and markers (PEDF, CD63, TSG101, and CD9) in MSC-sEVs. E Flow cytometry for measuring the loading efficiency of PEDF in PEDF-sEVs. FITC-labelled PEDF was used. F The concentrations of PEDF in sEVs and PEDF-sEVs were determined using ELISA ( n = 3/group). The data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: B Analysis of sizes of sEVs from each group using Nanosight.

Techniques: Staining, Differentiation Assay, Transmission Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Brain Tumor Research and Treatment

Article Title: Trends in Developing Extracellular Vesicle-Based Therapeutics

doi: 10.14791/btrt.2024.0027

Figure Lengend Snippet: Development cases of extracellular vesicle-based therapeutics by global biopharmaceutical companies

Article Snippet: Evox Therapeutics (UK) focuses on extracellular vesicle-mediated gene therapy, employing its own platform called DeliverEX ® .

Techniques:

Journal: Journal of Ovarian Research

Article Title: Corpus luteum presence in the bovine ovary increase intrafollicular progesterone concentration: consequences in follicular cells gene expression and follicular fluid small extracellular vesicles miRNA contents

doi: 10.1186/s13048-024-01387-3

Figure Lengend Snippet: Characterization of small extracellular vesicles (sEVs) from follicular fluid (FF). ( A ). Size and ( B ) concentration of FF sEVs analyzed (n=6) by nanotracking analysis equipment. The black line between the error bars represents the group means and the error bars represent the standard error of the means. ( C ) Distribution of size and particles/Ml. ( D ) Western blotting analysis of Alix and Tomm20 in FF sEVs and follicular cells (FCs). The images demonstrated Alix presence, an accessory protein of the ESCRT complex, in sEVs and FCs. Tomm20, a translocase protein of the outer mitochondrial membrane complex, was not detected in sEVs, only in FCs. Western blot images were cropped and the entire membrane image can be found in the supplementary figure S1. ( E ) Transmission electron microscopy of FF sEVs isolated from follicular fluid, demonstrating the cup-shaped phenotype of the nanoparticles (black arrows)

Article Snippet: The FF sEVs analysis by Nanosight was based on five videos of 30 seconds each.

Techniques: Concentration Assay, Western Blot, Membrane, Transmission Assay, Electron Microscopy, Isolation

Journal: Journal of Nanobiotechnology

Article Title: Enhanced therapeutic effect of PEDF-loaded mesenchymal stem cell-derived small extracellular vesicles against oxygen-induced retinopathy through increased stability and penetrability of PEDF

doi: 10.1186/s12951-023-02066-z

Figure Lengend Snippet: Characterisation of MSCs and sEVs. A Representative images of adipogenic (oil red O staining), osteogenic (alizarin red S staining), and chondrogenic (alcian blue staining) differentiation assay. B Analysis of sizes of sEVs from each group using Nanosight. C Representative transmission electron micrograph for each group; scale bar = 200 nm. D Representative western blots for loaded proteins and markers (PEDF, CD63, TSG101, and CD9) in MSC-sEVs. E Flow cytometry for measuring the loading efficiency of PEDF in PEDF-sEVs. FITC-labelled PEDF was used. F The concentrations of PEDF in sEVs and PEDF-sEVs were determined using ELISA ( n = 3/group). The data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: PEDF FITC -sEVs prepared using sonication were used to measure the loading efficiency on a FACSCalibur flow cytometer (BD Biosciences, USA), and the data were analysed using flow cytometry software (FlowJo, USA).

Techniques: Staining, Differentiation Assay, Transmission Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Nanobiotechnology

Article Title: Enhanced therapeutic effect of PEDF-loaded mesenchymal stem cell-derived small extracellular vesicles against oxygen-induced retinopathy through increased stability and penetrability of PEDF

doi: 10.1186/s12951-023-02066-z

Figure Lengend Snippet: PEDF-sEVs suppress VEGF-induced angiogenic effects and VEGF-downstream signalling in HRECs. A Starved HRECs were treated with PEDF under VEGF stimulation for 24 h. Cell proliferation was measured using a CCK assay (OD = 450 nm, n = 8). B Starved HRECs were treated with sEVs under VEGF stimulation for 24 h. Cell proliferation was measured using a CCK assay (OD = 450 nm, n = 7). C Starved HRECs were treated with PEDF (400 ng/mL), sEVs (10 µg/mL), a mixture of PEDF (400 ng/mL) and sEVs (10 µg/mL) without sonication, or PEDF-sEVs (10 µg/mL) under VEGF (10 ng/mL) stimulation for 24 h. Cell proliferation was measured using a CCK assay (OD = 450 nm, n = 8). D Representative images and E Quantification of the Transwell assay of HRECs treated as in C after 24 h ( n = 5); scale bar = 100 μm. F Representative images and G Quantification of the scratch migration assay in HRECs treated as in C after 12 h ( n = 5), scale bar = 200 μm. H Representative images and I Quantification of the tube formation assay in HRECs treated as in C after 2–4 h ( n = 6), scale bar = 200 μm. J Starved HRECs were pre-treated with PEDF, sEVs, or PEDF-sEVs for 24 h and then stimulated with 10 ng/mL VEGF for 20 min. Representative western blots for pAKT, AKT, pERK1/2, and ERK1/2 showing VEGF-downstream signalling. K Quantitative analysis for qERK/ERK and qAKT/AKT in HRECs ( n = 6). The data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: PEDF FITC -sEVs prepared using sonication were used to measure the loading efficiency on a FACSCalibur flow cytometer (BD Biosciences, USA), and the data were analysed using flow cytometry software (FlowJo, USA).

Techniques: Sonication, Transwell Assay, Migration, Tube Formation Assay, Western Blot

Journal: Journal of Nanobiotechnology

Article Title: Enhanced therapeutic effect of PEDF-loaded mesenchymal stem cell-derived small extracellular vesicles against oxygen-induced retinopathy through increased stability and penetrability of PEDF

doi: 10.1186/s12951-023-02066-z

Figure Lengend Snippet: PEDF-sEVs suppress the expression of inflammatory cytokines in HRECs. A Starved HRECs were pre-treated with PEDF, sEVs, or PEDF-sEVs under stimulation with 10 ng/mL VEGF for 24 h. Representative western blot images showing the expression of ICAM-1 in HRECs. B Relative expression of ICAM-1 compared with that of GAPDH ( n = 3). C , D , E . PCR analysis of the expression of TNF-α, VCAM-1, and IL-1β in HRECs ( n = 5). The data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: PEDF FITC -sEVs prepared using sonication were used to measure the loading efficiency on a FACSCalibur flow cytometer (BD Biosciences, USA), and the data were analysed using flow cytometry software (FlowJo, USA).

Techniques: Expressing, Western Blot

Journal: Journal of Nanobiotechnology

Article Title: Enhanced therapeutic effect of PEDF-loaded mesenchymal stem cell-derived small extracellular vesicles against oxygen-induced retinopathy through increased stability and penetrability of PEDF

doi: 10.1186/s12951-023-02066-z

Figure Lengend Snippet: PEDF-sEVs suppress retinal neovascularisation and vaso-obliteration in OIR mice. A A schematic diagram showing the treatment of OIR mice with PBS, PEDF, sEVs, a mixture of PEDF and sEVs without sonication, anti-VEGF drugs, or PEDF-sEVs through intravitreal injection on P12. Pups were sacrificed and eyes were dissected on P17, P25, or P42. B The expression of PEDF and VEGF in the retinas of NOR and OIR mice on P12, P14, and P17. NOR refers to the normal C57BL/6J mice kept in room air that have not undergone hyperoxia. C Relative expression of PEDF and VEGF compared with that of GAPDH ( n = 6 mice/group). D PCR analysis of PEDF and VEGF gene expression in the retinas of NOR and OIR mice on P12, P14, and P17 ( n = 6 mice/group). E Representative confocal images of retinal vasculature stained with IsoB4 of OIR mice treated with PBS, PEDF, sEVs, a mixture, anti-VEGF drugs, or PEDF-sEVs on P17; scale bar = 500 μm. F Quantitative analysis of avascular area/total retinal area ( n = 5 mice/group). G Quantitative analysis of neovascularisation/total retinal area ( n = 5 mice/group). H VEGF expression in the retinas of OIR mice treated with PBS, PEDF, sEVs, and PEDF-sEVs on P17. I Relative expression of VEGF compared with that of GAPDH ( n = 9 mice/group). J PCR analysis of VEGF gene expression in the retinas of OIR mice treated with PBS, PEDF, sEVs, and PEDF-sEVs on P17 ( n = 6 mice/group). K VEGF expression in the retinas of OIR mice treated with PBS, anti-VEGF drugs, and PEDF-sEVs on P17. L Relative expression of VEGF compared with that of GAPDH ( n = 6 mice/group). The data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: PEDF FITC -sEVs prepared using sonication were used to measure the loading efficiency on a FACSCalibur flow cytometer (BD Biosciences, USA), and the data were analysed using flow cytometry software (FlowJo, USA).

Techniques: Sonication, Injection, Expressing, Staining

Journal: Journal of Nanobiotechnology

Article Title: Enhanced therapeutic effect of PEDF-loaded mesenchymal stem cell-derived small extracellular vesicles against oxygen-induced retinopathy through increased stability and penetrability of PEDF

doi: 10.1186/s12951-023-02066-z

Figure Lengend Snippet: PEDF-sEVs suppress retinal inflammation in OIR mice. A GFAP expression in the retinas of NOR and OIR mice on P12, P14, and P17. B Relative expression of GFAP compared with that of GAPDH ( n = 6 mice/group). C PCR analysis of GFAP gene expression in the retinas of NOR and OIR mice on P12, P14, and P17 ( n = 6 mice/group). D GFAP expression in the retinas of OIR mice treated with PBS, PEDF, sEVs, and PEDF-sEVs treated on P17. E Relative expression of GFAP compared with that of GAPDH ( n = 9 mice/group). F PCR analysis of GFAP gene expression in the retinas of OIR mice treated with PBS, PEDF, sEVs, and PEDF-sEVs on P17 ( n = 6 mice/group). G GFAP expression in the retinas of OIR mice treated with PBS, anti-VEGF drugs, and PEDF-sEVs on P17. H Relative expression of GFAP compared with that of GAPDH ( n = 6 mice/group). I Representative immunofluorescence staining images of retinas showing GFAP expression in NOR and OIR mice treated with PBS, PEDF, sEVs, anti-VEGF drugs, and PEDF-sEVs on P17; scale bar = 50 μm. J The fluorescence intensity was measured by integrated density using the ImageJ software ( n = 3 mice/group, 3 sections per mouse, at least 3 images per section were analysed and the values were averaged). K , L PCR analysis of TNF-α and IL-1β gene expression in the retinas of OIR mice treated with PBS, PEDF, sEVs, and PEDF-sEVs on P17 ( n = 6 mice/group). The data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. RGC: Retinal ganglion cell; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium

Article Snippet: PEDF FITC -sEVs prepared using sonication were used to measure the loading efficiency on a FACSCalibur flow cytometer (BD Biosciences, USA), and the data were analysed using flow cytometry software (FlowJo, USA).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Software

Journal: Journal of Nanobiotechnology

Article Title: Enhanced therapeutic effect of PEDF-loaded mesenchymal stem cell-derived small extracellular vesicles against oxygen-induced retinopathy through increased stability and penetrability of PEDF

doi: 10.1186/s12951-023-02066-z

Figure Lengend Snippet: PEDF-sEVs are superior to anti-VEGF drugs in protecting retinal structure and in ameliorating retinal function in OIR mice. A Representative OCT images of NOR and OIR mice treated with PBS, PEDF, sEVs, anti-VEGF drugs, and PEDF-sEVs on P25 and P42. Red arrows indicate the total retina and green arrows indicate photoreceptor layer. B Quantitative analysis of total retina and photoreceptor layer thickness on P25 ( n = 8–10 mice/group). C Quantitative analysis of total retina and photoreceptor layer thickness on P42 ( n = 6–7 mice/group). D H&E staining of retinal tissues in NOR and OIR mice treated with PBS, PEDF, sEVs, anti-VEGF drugs, and PEDF-sEVs on P25 and P42 ( n = 3 mice/group); scale bar = 50 μm. E Representative TUNEL assay images of eye sections of retinas from OIR mice treated with PBS, PEDF, sEVs, anti-VEGF drugs, and PEDF-sEVs on P17; scale bar = 50 μm. F Quantitative analysis of TUNEL-positive cells in the retinas ( n = 5 mice/group, 5 sections per mouse, at least 5 images per section were analysed and the values were averaged). G Representative images of ERG response at a flash strength of 1.0 log (cd•s/ m 2 ) in NOR and OIR mice treated with PBS, PEDF, sEVs, anti-VEGF drugs, and PEDF-sEVs on P25 and P42. H ERG analysis of a and b wave amplitudes on P25 ( n = 7–11 mice/group). I ERG analysis of a and b wave amplitudes on P42 ( n = 7–9 mice/group). The data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. RGC: Retinal ganglion cell; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium

Article Snippet: PEDF FITC -sEVs prepared using sonication were used to measure the loading efficiency on a FACSCalibur flow cytometer (BD Biosciences, USA), and the data were analysed using flow cytometry software (FlowJo, USA).

Techniques: Staining, TUNEL Assay

Journal: Journal of Nanobiotechnology

Article Title: Enhanced therapeutic effect of PEDF-loaded mesenchymal stem cell-derived small extracellular vesicles against oxygen-induced retinopathy through increased stability and penetrability of PEDF

doi: 10.1186/s12951-023-02066-z

Figure Lengend Snippet: PEDF-sEVs effectively reduced PEDF degradation both in vitro and in vivo. A DiD-labelled sEVs and FITC-labelled PEDF were used to detect the distribution of PEDF and sEVs, respectively. PEDF-sEVs and a mixture of PEDF and sEVs without sonication were added to HRECs in culture. Representative confocal images show the cellular uptake of PEDF and sEVs in HRECs after 24 and 48 h; scale bar = 50 μm. B FITC fluorescence intensity in HRECs was measured by integrated density using the ImageJ software ( n = 6/group). C PEDF-sEVs and a mixture were added to HRECs in culture. After 24 and 48 h, cells were collected for flow cytometry to analyse the cellular uptake efficiency of PEDF. D PEDF and PEDF-sEVs were added to HRECs in culture. After 6, 24, 48, and 72 h, the concentrations of PEDF in supernatants were measured using ELISA ( n = 3/group). E Ocular distribution of PEDF and PEDF-sEVs in cryosections of OIR retinas on P13 and P17 after intravitreal injection on P12; scale bar = 50 μm. F FITC fluorescence intensity in cryosections of retinas was measured by integrated density using the ImageJ software ( n = 6 mice/group; 3 sections per mouse, and at least 3 images per section were analysed and the values were averaged). The data are represented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. RGC: Retinal ganglion cell; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium

Article Snippet: PEDF FITC -sEVs prepared using sonication were used to measure the loading efficiency on a FACSCalibur flow cytometer (BD Biosciences, USA), and the data were analysed using flow cytometry software (FlowJo, USA).

Techniques: In Vitro, In Vivo, Sonication, Fluorescence, Software, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Injection