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18 pages, 6624 KiB

Open AccessArticle

Lyophilized Small Extracellular Vesicles (sEVs) Derived from Human Adipose Stem Cells Maintain Efficacy to Promote Healing in Neuronal Injuries

by Brianna Jones, Rekha Patel, Bangmei Wang, Theresa Evans-Nguyen and Niketa A. Patel

Biomedicines 2025, 13(2), 275; https://doi.org/10.3390/biomedicines13020275 - 23 Jan 2025

Viewed by 699

Abstract

Background: Traumatic brain injury (TBI) occurs in individuals of all ages, predominantly during sports, accidents, and in active military service members. Chronic consequences of TBI include declined cognitive and motor function, dementia, and emotional distress. Small extracellular vesicles (sEVs), previously referred to as [...] Read more.

Background: Traumatic brain injury (TBI) occurs in individuals of all ages, predominantly during sports, accidents, and in active military service members. Chronic consequences of TBI include declined cognitive and motor function, dementia, and emotional distress. Small extracellular vesicles (sEVs), previously referred to as exosomes, are nano-sized lipid vesicles that play a role in intercellular communication. Our prior research established the efficacy of sEVs derived from human adipose stem cells (hASC sEVs) in accelerating the healing of brain injuries. The hASC sEVs are a biologic therapeutic and need to be stored at −20 °C or −80 °C. This limits their use in translating to everyday use in clinics or their inclusion in first-aid kits for application immediately after injury. To address this, here we demonstrate that hASC sEVs can be stored at room temperature (RT) for two months post lyophilization. Methods: A transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA) were used to validate the morphology of lyophilized RT sEVs. Using in vitro models of neuronal injury mimicking physical injury, inflammation, and oxidative stress, we demonstrate that lyophilized RT hASC sEVs are viable and promote the healing of neuronal injuries. Results: The lyophilized sEVs maintain their purity, size, and morphology upon rehydration. Lyophilized, RT stored sEVs showed better efficacy after two months compared with −80 °C stored sEVs. Conclusions: RT storage of lyophilized hASC sEVs maintains their efficacy to accelerate the healing of injuries in neuronal cells. This will advance the use of hASC sEVs, bringing them closer to use in clinics, home first-aid kits, and on battlefields by active service members. Full article

(This article belongs to the Special Issue Extracellular Vesicles and Exosomes as Therapeutic Agents)

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Schematic of the three processes that occur during lyophilization. The publication license can be found in <a href="#app1-biomedicines-13-00275" class="html-app">Supplementary File S2</a> (Created in BioRender. Jones, B. (2024) <a href="https://BioRender.com/s06h883" target="_blank">https://BioRender.com/s06h883</a>; accessed on 15 December 2024). Full article ">Figure 2
NTA and TEM data showing the sizes and concentrations of sEVs isolated from hASCs. (A) sEV elution from ExoSpin column showing an average size of 80.3 nm and concentration of 9.84 × 108 particles/mL. (B) sEV elution from qEV column showing a peak size of 145 nm and concentration of 9.20 × 107 particles/mL. Measurements in triplicate for each sample. (C) TEM image showing hASC sEVs extracted from a qEV column at 40 k× and 80 k× magnification. Scale bar = 500 nm and 200 nm, respectively. Full article ">Figure 3
TEM images of −80 °C sEVs and lyophilized sEVs at low and high magnifications and NTA data showing the sizes of the −80 °C sEVs and lyophilized sEVs after 2 weeks, 1 month, and 2 months. (A) NTA data showing sizes of hASC sEVs after 2 weeks of storage. (B) NTA data showing sizes of hASC sEVs after 1 month of storage. (C) NTA data showing sizes of hASC sEVs after 2 months of storage. (D) TEM images of sEVs after 2 weeks of storage. (E) TEM images of sEVs after 1 month of storage. (F) TEM images of sEVs after 2 months of storage. (G) NTA data and TEMs of lyophilized sEVs without trehalose after 2 weeks of storage. For 40 k× images, the scale bar = 500 nm. For 50 k×, 80 k×, and 100 k× images, the scale bar = 200 nm. For 150 k× images, the scale bar = 100 nm. Full article ">Figure 4
Cellular uptake and wound closure comparison of −80 °C sEVs and lyophilized RT sEVs at two weeks in HT22 cells. (A) Cellular uptake of DiD and DiD-labeled sEVs over 24 h, imaged at 40× (scale bar = 25 µm). (B) Graphical comparison of DiD intensity. The images for DiD, DAPI, and the overlay are all images of the same section of cells. DiD is being used to stain the sEVs in this study, while DAPI stains the nuclei of the cells. The overlay shows both DiD and DAPI in one image together to visualize how well the DiD-labeled sEVs are taken up by the cells. A high cell colocalization with sEV indicates that a higher number of sEVs are making their way into the cells. Statistical analysis was performed by ANOVA analysis, with no significant differences between −80 °C stored sEVs compared with lyophilized RT sEVs with trehalose. (C) Scratch assay over 24 h, imaged at 4×. (D) Comparison of % wound closure and rate of cell migration between samples. The images in each column shown in the figure demonstrate a scratch in the HT22 cells at different time intervals. The scratch shown at each time interval is the same scratch in the same section of cells to demonstrate how each treatment affects the ability of the cells to close the wound gap. A high % wound closure indicates how well the sEVs are repairing the damage to the cells. Statistical analysis was performed by ANOVA analysis (* p < 0.05, ** p < 0.01, and **** p ≤ 0.0001, no significance (ns)). Full article ">Figure 5
Cellular uptake and wound closure comparison of −80 °C sEVs and lyophilized RT sEVs at one month in HT22 cells. (A) Cellular uptake of DiD-labeled sEVs over 24 h, imaged at 40× (scale bar = 25 µm). (B) Graphical comparison of DiD intensity. The images for DiD, DAPI, and the overlay are all images of the same section of cells. DiD is being used to stain the sEVs in this study, while DAPI stains the nuclei of the cells. The overlay shows both DiD and DAPI in one image together to help visualize how well the DiD-labeled sEVs are taken up by the cells. A high cell colocalization with sEVs indicates that a higher number of sEVs are making their way into the cells. Statistical analysis was performed by two-tailed Student’s t-test, no significance (ns). (C) Scratch assay over 24 h, imaged at 4×. (D) Comparison of % wound closure and rate of cell migration between samples. The images in each column shown in the figure demonstrate a scratch in the HT22 cells at different time intervals. The scratch shown at each time interval is the same scratch in the same section of cells to demonstrate how each treatment affects the ability of the cells to close the wound gap. A high % wound closure indicates how well the sEVs are repairing the damage to the cells. Statistical analysis was performed by ANOVA analysis ** p < 0.01). Full article ">Figure 6
Cellular uptake and wound closure comparison of −80 °C sEVs and lyophilized RT sEVs at two months in HT22 cells. (A) Cellular uptake of DiD-labeled sEVs over 24 h, imaged at 40× (scale bar = 25 µm). (B) Graphical comparison of DiD intensity. The images for DiD, DAPI, and the overlay are all images of the same section of cells. DiD is being used to stain the sEVs in this study, while DAPI stains the nuclei of the cells. The overlay shows both DiD and DAPI in one image together to help visualize how well the DiD-labeled sEVs are taken up by the cells. A high cell colocalization with sEV indicates that a higher number of sEVs are making their way into the cells. Statistical analysis was performed by two-tailed Student’s t-test, no significance (ns). (C) Scratch assay over 24 h, imaged at 4×. (D) Comparison of % wound closure and rate of cell migration between samples after 16 h. Statistical analysis was performed by two-tailed Student’s t-test. (E) Comparison of % wound closure and rate of cell migration between samples after 24 h. The images in each column shown in the figure demonstrate a scratch in the HT22 cells at different time intervals. The scratch shown at each time interval is the same scratch in the same section of cells to demonstrate how each treatment affects the ability of the cells to close the wound gap. A high % wound closure indicates how well the sEVs are repairing the damage to the cells. Statistical analysis was performed by ANOVA analysis *** p < 0.001, and **** p ≤ 0.0001. Full article ">Figure 7
Immunochemistry assay of HT22 cells treated with 5 ng/mL LPS for 6 h, after which the medium was changed, and the cells were treated with 2 µg of sEVs for 18 h. (A) Cells were stained using Ki-67 as a marker for proliferation and DAPI as a nucleus stain and imaged with a Keyence BZx-810 microscope at 20× (scale bar = 50 µm). (B) Determination of colocalization of Ki-67 and DAPI was determined using the Keyence software. The images for Ki67, DAPI, and the overlay are all images of the same section of cells. Ki67 stains proliferating cells, while the DAPI stains the nuclei of the cells. The overlay shows both the Ki67 and DAPI in one image together to visualize cell proliferation occurring from each treatment. Statistical analysis was performed by ANOVA analysis (* p < 0.05, ** p < 0.01, and **** p ≤ 0.0001). Full article ">Figure 8
Immunochemistry assay of HT22 cells treated with 1:1000 hydrogen peroxide for 1 h, after which the medium was changed, and the cells were treated with 2 µg of lyophilized sEVs for 18 h. (A) Cells were stained using Ki-67 as a marker for proliferation and DAPI as a nucleus stain and imaged with a Keyence BZx-810 microscope at 20× (scale bar = 50 µm). (B) Determination of colocalization of Ki-67 and DAPI was determined using the Keyence software. The image for Ki67, DAPI, and the overlay are all images of the same section of cells. Ki67 stains proliferating cells, while the DAPI stains the nuclei of the cells. The overlay shows both the Ki67 and DAPI in one image together to visualize cell proliferation occurring from each treatment. Statistical analysis was performed by ANOVA analysis (** p < 0.01, *** p < 0.001, **** p ≤ 0.0001). Full article ">

14 pages, 3680 KiB

Open AccessArticle

Grapefruit-Derived Vesicles Loaded with Recombinant HSP70 Activate Antitumor Immunity in Colon Cancer In Vitro and In Vivo

by Luiza Garaeva, Elena Komarova, Svetlana Emelianova, Elena Putevich, Andrey L. Konevega, Boris Margulis, Irina Guzhova and Tatiana Shtam

Biomedicines 2024, 12(12), 2759; https://doi.org/10.3390/biomedicines12122759 - 3 Dec 2024

Viewed by 1069

Abstract

Background/Objectives: Stress protein HSP70 administered exogenously has demonstrated high potential as an efficient adjuvant in antitumor immune response. To enhance the antigen-presenting activity, bioavailability, and stability of exogenous recombinant human HSP70, we propose incorporating it into plant extracellular vesicles. Earlier, we found that [...] Read more.

Background/Objectives: Stress protein HSP70 administered exogenously has demonstrated high potential as an efficient adjuvant in antitumor immune response. To enhance the antigen-presenting activity, bioavailability, and stability of exogenous recombinant human HSP70, we propose incorporating it into plant extracellular vesicles. Earlier, we found that grapefruit-derived extracellular vesicles (GEV) were able to store the protein with no loss of its major function, chaperone activity. Methods: In this study, we tested whether HSP70 loaded into GEV (GEV-HSP70) could elicit an antitumor immune response in cellular and animal models of colorectal cancer. Results: To test the hypothesis in vitro, human and mouse colorectal cancer cell lines were used. We have shown that the addition of HSP70, either in free form or as part of GEVs, increases the sensitivity of human (HCT-116, DLD1) or mouse (CT-26) colon cancer cells to mouse cytotoxic lymphocytes and human NK-92 cells. Moreover, the amount of protein in the form of GEV-HSP70 required to cause the same activation of antitumor immunity was 20 times less than when HSP70 was added in free form. In a colon carcinoma model in vivo, GEV-HSP70 were inoculated subcutaneously into BALB/c mice together with CT-26 cells to form a tumor node. As compared with the control groups, we observed an increase in the lifespan of animals and a decrease in the tumor size, as well as a decrease in the level of TGFB1 IL-10 factors in the blood plasma. In vitro analysis of the immunomodulatory activity of GEV-HSP70 showed that antitumor response in GEV-HSP70-treated mice was associated with the accumulation of CD8+ cells. Conclusions: These results demonstrate the high feasibility and efficacy of the new technique based on HSP70 encapsulated in plant vesicles in activation of the specific response to colon tumors. Full article

(This article belongs to the Special Issue Extracellular Vesicles and Exosomes as Therapeutic Agents)

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Size, concentration, and morphology of grapefruit-derived vesicles before (GEV) and after loading procedure (GEV-HSP70). (A,B) Typical examples of nanoparticle tracking analysis (NTA) of the sample of isolated GEV (A) and GEV-HSP70 (B). (C,D) Cryo-EM images of GEV (C) or GEV-HSP70 (D). White arrows indicate vesicles with intact membrane, red arrows indicate vesicles with broken membrane, and black arrows—debris in the sample. The blue arrows depict a lipid bilayer membrane of the vesicle. Scale bars are 50 nm. Inset–size distribution histogram. A total of 100 particles were analyzed. (E,F) Loading efficiency of GEV with HSP70 protein: (E) Example of Western blot (WB) of HSP70 in the initial GEV (line 7) and GEV-HSP70 (lines 5,6) in an amount of 1011 particles per line. Recombinant HSP70 in an amount from 0.2 μg to 2 μg per line (lines 1–4). (F) Cumulative quantification of HSP70 loaded into GEV obtained from WB. Full article ">Figure 2
Effect of free HSP70 and GEV-HSP70 on the proliferative activity of human and murine colon cancer cells when attacked by cytotoxic lymphocytes (CTL) from naïve C3HA mice or human NK-92 cells. (A,B) GEV-HSP70 increases the sensitivity of human colon cancer cells to the NK cells action: HCT-116 (A) and DLD1 (B) cells were seeded to wells of E-plate and incubated or not with GEV-HSP70 for 24 h, and then NK-92 cells were added into wells. Recoding on xCELLigence equipment lasted 45 h. (C–F) Effect of free and GEV-loaded HSP70 on the proliferative activity of CT-26 cells when attacked by CTL from naïve C3HA mice or human NK-92 cells. (C,E) Proliferative activity of CT-26 cells preincubated with HSP70 (10 μg), GEV (0 μg HSP70), and GEV-HSP70 (about 0.5 μg HSP70) when exposed to the CTL. (F,H). Proliferative activity of CT-26 cells preincubated with HSP70 (10 μg or 1 μg), GEV (0 μg HSP70), and GEV-HSP70 (about 0.5 μg HSP70) upon exposure to NK-92 cells. Addition of rHSP70, GEV-HSP70, and naïve GEVs at 24 h of incubation, addition of effector cells at 40 h of incubation. For the (E,F) panels, the last observation time point was chosen for analysis. In panels (A–D), the normalization point for the proliferation curves is chosen to correspond to the time point of effector cells. Pairwise multiple comparisons were performed using ANOVA with Tukey’s posterior test. A statistically significant difference between the is indicated as **** for p < 0.0001, *** for p ≤ 0.0005, * for p < 0.05, ns—not statistically significant. Full article ">Figure 3
Antitumor effect of HSP70 and GEV-HSP70 in a mouse model of colorectal carcinoma. (A) Analysis of the growth rate of the tumor node during the 21st day of observation in 4 groups of animals after inoculation of CT-26 cells (Untreated) or CT-26 cells mixed with GEV, GEV-HSP70 (2 μg HSP70/dose), and with HSP70 (50 μg/dose) (N = 8) (B,C) Twenty-one days after CT-26 cells inoculation tumors were isolated, photographed (B) and weighed (C) (N = 8). (D,E) Analysis of the tumor size by the intravital luminescence imaging system (N = 5). (F) Life expectancy of animals in control and experimental groups (N = 10). Legend *** for p ≤ 0.0005, ** for p ≤ 0.005, * for p < 0.05, ns—not statistically significant. Full article ">Figure 4
GEV-HSP70 and free HSP70 induce a specific antitumor immune response in the CT-26 mouse model of colorectal carcinoma. (A,B) Assessment of the concentration of cytokines IL-10 and TGFB-1 in the blood plasma of mice 21 days after inoculation with CT-26 cells (Untreated), CT-26 cells mixed with GEV, GEV-HSP70 (about 2 μg HSP70/dose) or HSP70 (50 μg/dose) (N = 5). (C) Proliferative activity of CT-26 cells when exposed to the total fraction of lymphocytes isolated from the spleens of mice of experimental and control groups. (D) The influence of the lymphocyte fraction depleted of CD8+ T-lymphocytes on the proliferative activity of CT-26 cells. (E) Cytostatic effect of CD8+ T lymphocytes from mice from experimental and control groups on the proliferation of CT-26 cells (N = 5). Pairwise multiple comparisons were performed using ANOVA with Tukey’s posterior test. p < 0.0001 = ****, p ≤ 0.0005 = ***, p ≤ 0.005 = **, ns—not statistically significant. Full article ">

16 pages, 6155 KiB

Open AccessArticle

Artificial Extracellular Vesicles Generated from T Cells Using Different Induction Techniques

by Ekaterina A. Zmievskaya, Sabir A. Mukhametshin, Irina A. Ganeeva, Elvina M. Gilyazova, Elvira T. Siraeva, Marianna P. Kutyreva, Artur A. Khannanov, Youyong Yuan and Emil R. Bulatov

Biomedicines 2024, 12(4), 919; https://doi.org/10.3390/biomedicines12040919 - 20 Apr 2024

Cited by 4 | Viewed by 2076

Abstract

Cell therapy is at the forefront of biomedicine in oncology and regenerative medicine. However, there are still significant challenges to their wider clinical application such as limited efficacy, side effects, and logistical difficulties. One of the potential approaches that could overcome these problems [...] Read more.

Cell therapy is at the forefront of biomedicine in oncology and regenerative medicine. However, there are still significant challenges to their wider clinical application such as limited efficacy, side effects, and logistical difficulties. One of the potential approaches that could overcome these problems is based on extracellular vesicles (EVs) as a cell-free therapy modality. One of the major obstacles in the translation of EVs into practice is their low yield of production, which is insufficient to achieve therapeutic amounts. Here, we evaluated two primary approaches of artificial vesicle induction in primary T cells and the SupT1 cell line—cytochalasin B as a chemical inducer and ultrasonication as a physical inducer. We found that both methods are capable of producing artificial vesicles, but cytochalasin B induction leads to vesicle yield compared to natural secretion, while ultrasonication leads to a three-fold increase in particle yield. Cytochalasin B induces the formation of vesicles full of cytoplasmic compartments without nuclear fraction, while ultrasonication induces the formation of particles rich in membranes and membrane-related components such as CD3 or HLAII proteins. The most effective approach for T-cell induction in terms of the number of vesicles seems to be the combination of anti-CD3/CD28 antibody activation with ultrasonication, which leads to a seven-fold yield increase in particles with a high content of functionally important proteins (CD3, granzyme B, and HLA II). Full article

(This article belongs to the Special Issue Extracellular Vesicles and Exosomes as Therapeutic Agents)

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28 pages, 6431 KiB

Open AccessArticle

Extracellular Vesicles Derived from Osteogenic-Differentiated Human Bone Marrow-Derived Mesenchymal Cells Rescue Osteogenic Ability of Bone Marrow-Derived Mesenchymal Cells Impaired by Hypoxia

by Chenglong Wang, Sabine Stöckl, Girish Pattappa, Daniela Schulz, Korbinian Hofmann, Jovana Ilic, Yvonne Reinders, Richard J. Bauer, Albert Sickmann and Susanne Grässel

Biomedicines 2023, 11(10), 2804; https://doi.org/10.3390/biomedicines11102804 - 16 Oct 2023

Viewed by 1671

Abstract

In orthopedics, musculoskeletal disorders, i.e., non-union of bone fractures or osteoporosis, can have common histories and symptoms related to pathological hypoxic conditions induced by aging, trauma or metabolic disorders. Here, we observed that hypoxic conditions (2% O₂) suppressed the osteogenic differentiation [...] Read more.

In orthopedics, musculoskeletal disorders, i.e., non-union of bone fractures or osteoporosis, can have common histories and symptoms related to pathological hypoxic conditions induced by aging, trauma or metabolic disorders. Here, we observed that hypoxic conditions (2% O₂) suppressed the osteogenic differentiation of human bone marrow-derived mesenchymal cells (hBMSC) in vitro and simultaneously increased reactive oxygen species (ROS) production. We assumed that cellular origin and cargo of extracellular vesicles (EVs) affect the osteogenic differentiation capacity of hBMSCs cultured under different oxygen pressures. Proteomic analysis revealed that EVs isolated from osteogenic differentiated hBMSC cultured under hypoxia (hypo-osteo EVs) or under normoxia (norm-osteo EVs) contained distinct protein profiles. Extracellular matrix (ECM) components, antioxidants and pro-osteogenic proteins were decreased in hypo-osteo EVs. The proteomic analysis in our previous study revealed that under normoxic culture conditions, pro-osteogenic proteins and ECM components have higher concentrations in norm-osteo EVs than in EVs derived from naïve hBMSCs (norm-naïve EVs). When selected for further analysis, five anti-hypoxic proteins were significantly upregulated (response to hypoxia) in norm-osteo EVs. Three of them are characterized as antioxidant proteins. We performed qRT-PCR to verify the corresponding gene expression levels in the norm-osteo EVs’ and norm-naïve EVs’ parent cells cultured under normoxia. Moreover, we observed that norm-osteo EVs rescued the osteogenic ability of naïve hBMSCs cultured under hypoxia and reduced hypoxia-induced elevation of ROS production in osteogenic differentiated hBMSCs, presumably by inducing expression of anti-hypoxic/ antioxidant and pro-osteogenic genes. Full article

(This article belongs to the Special Issue Extracellular Vesicles and Exosomes as Therapeutic Agents)

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14 pages, 3348 KiB

Open AccessArticle

The Influence of Exercise-Associated Small Extracellular Vesicles on Trophoblasts In Vitro

by Shuhiba Mohammad, Jayonta Bhattacharjee, Velislava Tzaneva, Kelly Ann Hutchinson, Madeeha Shaikh, Danilo Fernandes da Silva, Dylan Burger and Kristi B. Adamo

Biomedicines 2023, 11(3), 857; https://doi.org/10.3390/biomedicines11030857 - 11 Mar 2023

Cited by 1 | Viewed by 2238

Abstract

Exercise induces the release of small extracellular vesicles (sEVs) into circulation that are postulated to mediate tissue cross-talk during exercise. We previously reported that pregnant individuals released greater levels of sEVs into circulation after exercise compared to matched non-pregnant controls, but their biological [...] Read more.

Exercise induces the release of small extracellular vesicles (sEVs) into circulation that are postulated to mediate tissue cross-talk during exercise. We previously reported that pregnant individuals released greater levels of sEVs into circulation after exercise compared to matched non-pregnant controls, but their biological functions remain unknown. In this study, sEVs isolated from the plasma of healthy pregnant and non-pregnant participants after a single bout of moderate-intensity exercise were evaluated for their impact on trophoblasts in vitro. Exercise-associated sEVs were found localized within the cytoplasm of BeWo choriocarcinoma cells, used to model trophoblasts in vitro. Exposure to exercise-associated sEVs did not significantly alter BeWo cell proliferation, gene expression of angiogenic growth factors VEGF and PLGF, or the release of the hormone human chorionic gonadotropin. The results from this pilot study support that exercise-associated sEVs could interact with trophoblasts in vitro, and warrant further investigation to reveal their potential role in communicating the effects of exercise to the maternal–fetal interface. Full article

(This article belongs to the Special Issue Extracellular Vesicles and Exosomes as Therapeutic Agents)

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Exercise-associated sEVs were found within BeWo cells in vitro. Representative confocal microscopy images of BeWo cells after overnight (16 h) incubation with 2.5 µg/mL PKH26-labeled sEVs from pregnant (A) and non-pregnant (B) plasma post-exercise. Panel (C) shows a representative vehicle control image (PBS). Blue represents DAPI staining for nuclei, magenta depicts phalloidin staining, and green shows PKH26-labeled sEVs. “Magenta” and “Green” lookup tables were used to display phalloidin and sEV labeling, respectively. For each panel, orthogonal projections show the XY (main image), YZ (right of main image), and XZ (top image) planes. All images were taken using a 63× objective lens with oil immersion. Scale bar = 10 µm for all images. Full article ">Figure 2
Exercise-associated sEVs did not affect BeWo cell proliferation. There were no differences in proliferation when no treatment (PBS Ctrl) was compared to treatment with exercise-associated sEVs from pregnant and non-pregnant individuals (F = 0.469, p = 0.642) (A). Representative merged fluorescence images of each condition (B) and a negative control (Neg Ctrl) where primary antibody was omitted are shown, where blue depicts DAPI staining for nuclei and green shows Ki67 positive signal. All experiments were conducted in triplicate, with sEVs obtained from n = 3 pregnant and n = 3 non-pregnant participants, with corresponding PBS controls (n = 5). Scale bar = 200 µm. Neg ctrl, negative control; PBS Ctrl, phosphate-buffered saline control. Full article ">Figure 3
The effect of exercise-associated sEVs on relative gene expression of angiogenic growth factors and b-hCG production in BeWo cells. Gene expression of VEGF (A) and PLGF (B) was not altered upon exposure to circulating exercise-associated sEVs (10 µg/mL for 24 h) obtained from pregnant and non-pregnant individuals compared to PBS control (PBS Ctrl) treatment (F = 1.98, p = 0.200, and F = 0.726, p = 0.513, respectively). (C) There were no differences in b-hCG cell media levels when no treatment was compared to treatment with exercise-associated sEVs from pregnant and non-pregnant participants (F = 0.885, p = 0.450). All experiments were conducted in triplicate, with sEVs obtained from n = 3 pregnant and n = 3 non-pregnant participants, with corresponding PBS controls (n = 5). Full article ">

14 pages, 1082 KiB

Open AccessArticle

Antimicrobial and Immunomodulatory Potential of Cow Colostrum Extracellular Vesicles (ColosEVs) in an Intestinal In Vitro Model

by Samanta Mecocci, Livia De Paolis, Roberto Zoccola, Floriana Fruscione, Chiara Grazia De Ciucis, Elisabetta Chiaradia, Valentina Moccia, Alessia Tognoloni, Luisa Pascucci, Simona Zoppi, Valentina Zappulli, Giovanni Chillemi, Maria Goria, Katia Cappelli and Elisabetta Razzuoli

Biomedicines 2022, 10(12), 3264; https://doi.org/10.3390/biomedicines10123264 - 15 Dec 2022

Cited by 4 | Viewed by 2479

Abstract

Extracellular Vesicles (EVs) are nano-sized double-lipid-membrane-bound structures, acting mainly as signalling mediators between distant cells and, in particular, modulating the immune response and inflammation of targeted cells. Milk and colostrum contain high amounts of EVs that could be exploited as alternative natural systems [...] Read more.

Extracellular Vesicles (EVs) are nano-sized double-lipid-membrane-bound structures, acting mainly as signalling mediators between distant cells and, in particular, modulating the immune response and inflammation of targeted cells. Milk and colostrum contain high amounts of EVs that could be exploited as alternative natural systems in antimicrobial fighting. The aim of this study is to evaluate cow colostrum-derived EVs (colosEVs) for their antimicrobial, anti-inflammatory and immunomodulating effects in vitro to assess their suitability as natural antimicrobial agents as a strategy to cope with the drug resistance problem. ColosEVs were evaluated on a model of neonatal calf diarrhoea caused by Escherichia coli infection, a livestock disease where antibiotic therapy often has poor results. Colostrum from Piedmontese cows was collected within 24 h of calving and colosEVs were immediately isolated. IPEC-J2 cell line was pre-treated with colosEVs for 48 h and then infected with EPEC/NTEC field strains for 2 h. Bacterial adherence and IPEC-J2 gene expression analysis (RT-qPCR) of CXCL8, DEFB1, DEFB4A, TLR4, TLR5, NFKB1, MYD88, CGAS, RIGI and STING were evaluated. The colosEVs pre-treatment significantly reduced the ability of EPEC/NTEC strains to adhere to cell surfaces (p = 0.006), suggesting a role of ColosEVs in modulating host–pathogen interactions. Moreover, our results showed a significant decrease in TLR5 (p < 0.05), CGAS (p < 0.05) and STING (p < 0.01) gene expression in cells that were pre-treated with ColosEVs and then infected, thus highlighting a potential antimicrobial activity of ColosEVs. This is the first preliminarily study investigating ColosEV immunomodulatory and anti-inflammatory effects on an in vitro model of neonatal calf diarrhoea, showing its potential as a therapeutic and prophylactic tool. Full article

(This article belongs to the Special Issue Extracellular Vesicles and Exosomes as Therapeutic Agents)

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Morphological characterization of isolated colosEVs: Transmission electron microscopy low (A) and high (B) magnification micrographs showing single and clustered colosEVs indicated by red arrows. Scale bar: A. 100 nm; B. 200 nm; (C) Western blot images obtained using Ab against Tsg101 (Tumor Susceptibility Gene 101 protein) and CD81 (Cluster of Differentiation 81) that are both mEV antigens and calnexin as negative cellular debris control of two colosEV samples (1 and 2); (D) nanoparticle tracking analysis graph indicating colosEV size distribution. Full article ">Figure 2
Viability of IPEC-J2 after colosEVs exposure at 48 h. The different concentrations of colosEVs did not determine a significant difference in terms of cell viability after 24 h (see <a href="#app1-biomedicines-10-03264" class="html-app">Figure S1</a>), whereas 150 µg colosEVs determined a significant reduction in IPECJ2 vitality at 48 h (p < 0.0001, indicated by *** in the graph). Data are expressed as optical density (OD) ± SD. Differences were evaluated through the Kruskal–Wallis test and applying the post hoc Dunn’s multiple comparison test. Full article ">Figure 3
Effect of 48 h +1.5 µg colosEVs on E. Coli-inflamed IPEC-J2 cells. IPEC-J2-tested conditions were: inflamed (E. coli, pink), inflamed 1.5 µg colosEVs (E. coli + 1.5 µg colosEVs, light yellow), untreated (control, dark yellow). Significant differences are reported with respect to infected cells with E. coli. Differences were evaluated through the Kruskal–Wallis test and applying the post hoc Dunn’s multiple comparison test. The asterisks indicate the statistical significance: * p < 0.05, ** p < 0.01 and *** p < 0.001. Full article ">Figure 4
Adhesion of E. coli strains on 1.5 µg colosEVs pre-treated cells compared with untreated ones. Data are expressed as log10 CFU of adherent, E. coli/5 × 105 cells and mean value of 3 experiments ± 1 standard deviation. Differences were evaluated through the Student’s t-test. The asterisks indicate the statistical significance: ** p < 0.01. Full article ">

Review

Jump to: Research

37 pages, 2081 KiB

Open AccessReview

Effects of Physical Cues on Stem Cell-Derived Extracellular Vesicles toward Neuropathy Applications

by Danyale Berry, Justice Ene, Aakash Nathani, Mandip Singh, Yan Li and Changchun Zeng

Biomedicines 2024, 12(3), 489; https://doi.org/10.3390/biomedicines12030489 - 22 Feb 2024

Cited by 2 | Viewed by 2110

Abstract

The peripheral nervous system undergoes sufficient stress when affected by diabetic conditions, chemotherapeutic drugs, and personal injury. Consequently, peripheral neuropathy arises as the most common complication, leading to debilitating symptoms that significantly alter the quality and way of life. The resulting chronic pain [...] Read more.

The peripheral nervous system undergoes sufficient stress when affected by diabetic conditions, chemotherapeutic drugs, and personal injury. Consequently, peripheral neuropathy arises as the most common complication, leading to debilitating symptoms that significantly alter the quality and way of life. The resulting chronic pain requires a treatment approach that does not simply mask the accompanying symptoms but provides the necessary external environment and neurotrophic factors that will effectively facilitate nerve regeneration. Under normal conditions, the peripheral nervous system self-regenerates very slowly. The rate of progression is further hindered by the development of fibrosis and scar tissue formation, which does not allow sufficient neurite outgrowth to the target site. By incorporating scaffolding supplemented with secretome derived from human mesenchymal stem cells, it is hypothesized that neurotrophic factors and cellular signaling can facilitate the optimal microenvironment for nerve reinnervation. However, conventional methods of secretory vesicle production are low yield, thus requiring improved methods to enhance paracrine secretions. This report highlights the state-of-the-art methods of neuropathy treatment as well as methods to optimize the clinical application of stem cells and derived secretory vesicles for nerve regeneration. Full article

(This article belongs to the Special Issue Extracellular Vesicles and Exosomes as Therapeutic Agents)

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19 pages, 1275 KiB

Open AccessReview

Potential for Therapeutic-Loaded Exosomes to Ameliorate the Pathogenic Effects of α-Synuclein in Parkinson’s Disease

by David J. Rademacher

Biomedicines 2023, 11(4), 1187; https://doi.org/10.3390/biomedicines11041187 - 17 Apr 2023

Cited by 8 | Viewed by 2838

Abstract

Pathogenic forms of α-synuclein (α-syn) are transferred to and from neurons, astrocytes, and microglia, which spread α-syn pathology in the olfactory bulb and the gut and then throughout the Parkinson’s disease (PD) brain and exacerbate neurodegenerative processes. Here, we review attempts to minimize [...] Read more.

Pathogenic forms of α-synuclein (α-syn) are transferred to and from neurons, astrocytes, and microglia, which spread α-syn pathology in the olfactory bulb and the gut and then throughout the Parkinson’s disease (PD) brain and exacerbate neurodegenerative processes. Here, we review attempts to minimize or ameliorate the pathogenic effects of α-syn or deliver therapeutic cargo into the brain. Exosomes (EXs) have several important advantages as carriers of therapeutic agents including an ability to readily cross the blood–brain barrier, the potential for targeted delivery of therapeutic agents, and immune resistance. Diverse cargo can be loaded via various methods, which are reviewed herein, into EXs and delivered into the brain. Genetic modification of EX-producing cells or EXs and chemical modification of EX have emerged as powerful approaches for the targeted delivery of therapeutic agents to treat PD. Thus, EXs hold great promise for the development of next-generation therapeutics for the treatment of PD. Full article

(This article belongs to the Special Issue Extracellular Vesicles and Exosomes as Therapeutic Agents)

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24 pages, 1822 KiB

Open AccessReview

Regenerative Effects of Exosomes-Derived MSCs: An Overview on Spinal Cord Injury Experimental Studies

by Giovanni Schepici, Serena Silvestro and Emanuela Mazzon

Biomedicines 2023, 11(1), 201; https://doi.org/10.3390/biomedicines11010201 - 13 Jan 2023

Cited by 13 | Viewed by 3072

Abstract

Spinal cord injury (SCI) is a devastating condition usually induced by the initial mechanical insult that can lead to permanent motor and sensory deficits. At present, researchers are investigating potential therapeutic strategies to ameliorate the neuro-inflammatory cascade that occurs post-injury. Although the use [...] Read more.

Spinal cord injury (SCI) is a devastating condition usually induced by the initial mechanical insult that can lead to permanent motor and sensory deficits. At present, researchers are investigating potential therapeutic strategies to ameliorate the neuro-inflammatory cascade that occurs post-injury. Although the use of mesenchymal stromal/stem (MSCs) as a potential therapy in application to regenerative medicine promoted anti-inflammatory and neuroprotective effects, several disadvantages limit their use. Therefore, recent studies have reported the effects of exosomes-derived MSCs (MSC-EXOs) as an innovative therapeutic option for SCI patients. It is noteworthy that MSC-EXOs can maintain the integrity of the blood-spinal cord barrier (BSCB), promoting angiogenic, proliferative, and anti-oxidant effects, as well as immunomodulatory, anti-inflammatory, and antiapoptotic properties. Therefore, in this study, we summarized the preclinical studies reported in the literature that have shown the effects of MSC-EXOs as a new molecular target to counteract the devastating effects of SCI. Full article

(This article belongs to the Special Issue Extracellular Vesicles and Exosomes as Therapeutic Agents)

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Figure 1
The Prisma flow diagram illustrates the methodology that was used to select the in vivo and in vitro studies used for the writing of the review. Duplicate articles were excluded from the total of the studies that were found. Conversely, articles that highlight the role of MSCs derived exosomes in promoting injury repair and restoring functional deficits are described (The PRISMA Statement is published in [<a href="#B9-biomedicines-11-00201" class="html-bibr">9</a>]. Full article ">Figure 2
Pathophysiology of spinal cord injury (SCI). This schematic diagram illustrates the phase of SCI and its pathophysiology. Immediately after primary injury, the activation of resident astrocytes and microglia, and the subsequent infiltration of blood immune cells, induce an important neuroinflammatory response. This acute neuroinflammatory response plays a key role in the secondary injury mechanisms in the sub-acute and chronic phases that lead to cell death and tissue degeneration, as well as the formation of the glial scar, axonal degeneration and demyelination. During the acute phase, monocyte-derived macrophages occur in the central area of the injury to scavenge tissue damage. In these phases, the loss of oligodendrocytes leads to axonal demyelination, followed by spontaneous remyelination. Instead, astrocytes and pericytes, normally present in the spinal cord parenchyma, after damage proliferate and migrate to the site of injury and contribute to the formation of the glial scar. The image was created using the image bank of Servier Medical Art (Available online: <a href="http://smart.servier.com/" target="_blank">http://smart.servier.com/</a>, accessed on 1 December 2022), licensed under a Creative Commons Attribution 3.0 Unported License (Available online: <a href="https://creativecommons.org/licenses/by/3.0/" target="_blank">https://creativecommons.org/licenses/by/3.0/</a>, accessed on 1 December 2022). Full article ">Figure 3
Repair of the nervous system following SCI post exosomes transplantation. Exosomes secreted by donor cells can cross to the blood-brain barrier, reducing both neuroinflammation and neuronal apoptosis, thus promoting vascular remodeling, neurogenesis, microglia activation and axonal remodeling in the nervous system. The image was created using the image bank of Servier Medical Art (Available online: <a href="http://smart.servier.com/" target="_blank">http://smart.servier.com/</a>, accessed on 1 December 2022), licensed under a Creative Commons Attribution 3.0 Unported License (Available online: <a href="https://creativecommons.org/licenses/by/3.0/" target="_blank">https://creativecommons.org/licenses/by/3.0/</a>, accessed on 1 December 2022). Full article ">

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