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. 2022 Mar;18(3):1113-1126.
doi: 10.1007/s12015-021-10261-4. Epub 2022 Jan 26.

Extracellular Vesicles of Mesenchymal Stromal Cells Can be Taken Up by Microglial Cells and Partially Prevent the Stimulation Induced by β-amyloid

Dorota Kaniowska  1   2 , Kerstin Wenk  3 , Phil Rademacher  3 , Ronald Weiss  3 , Claire Fabian  4 , Isabell Schulz  4 , Max Guthardt  4 , Franziska Lange  4 , Sebastian Greiser  4 , Matthias Schmidt  5 , Ulf-Dietrich Braumann  4   6   7 , Frank Emmrich  4   3 , Ulrike Koehl  4   3   8 , Yarúa Jaimes  4   3   9
Affiliations

Extracellular Vesicles of Mesenchymal Stromal Cells Can be Taken Up by Microglial Cells and Partially Prevent the Stimulation Induced by β-amyloid

Dorota Kaniowska et al. Stem Cell Rev Rep. 2022 Mar.

Abstract

Mesenchymal stromal/stem cells (MSCs) have great capacity for immune regulation. MSCs provide protective paracrine effects, which are partially exerted by extracellular vesicles (EVs). It has been reported that MSCs-derived EVs (MSC-EVs) contain soluble factors, such as cytokines, chemokines, growth factors and even microRNAs, which confer them similar anti-inflammatory and regenerative effects to MSCs. Moreover, MSCs modulate microglia activation through a dual mechanism of action that relies both on cell contact and secreted factors. Microglia cells are the central nervous system immune cells and the main mediators of the inflammation leading to neurodegenerative disorders. Here, we investigated whether MSC-EVs affect the activation of microglia cells by β-amyloid aggregates. We show that the presence of MSC-EVs can prevent the upregulation of pro-inflammatory mediators such as tumor necrosis factor (TNF)-α and nitric oxide (NO). Both are up-regulated in neurodegenerative diseases representing chronic inflammation, as in Alzheimer's disease. We demonstrate that MSC-EVs are internalized by the microglia cells. Further, our study supports the use of MSC-EVs as a promising therapeutic tool to treat neuroinflammatory diseases.Significance StatementIt has been reported that mesenchymal stromal/stem cells and MSC-derived small extracellular vesicles have therapeutic effects in the treatment of various degenerative and inflammatory diseases. Extracellular vesicles are loaded with proteins, lipids and RNA and act as intercellular communication mediators. Here we show that extracellular vesicles can be taken up by murine microglial cells. In addition, they partially reduce the activation of microglial cells against β-amyloid aggregates. This inhibition of microglia activation may present an effective strategy for the control/therapy of neurodegenerative diseases such as Alzheimer's disease.

Keywords: Alzheimer Disease; Amyloid beta; Extracellular vesicles; Mesenchymal stromal/stem cells; Microglia; Neuroinflammation.

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Conflict of interest statement

We have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Isolation and characterization of human adipose derived cells (ASCs). (A) Human subcutaneous adipose tissues (2–10 gr) from different age groups and donors were digested with collagenase IV. After centrifugation, the stromal-vascular fraction was isolated and transferred to flasks completely filled with culture medium. The ASCs were cultured in 20 % FBS DMEM-LG with medium changes every 3–4 days. The cell surface expression of the common ASC markers was evaluated by flow cytometry of 4 immunomodulatory donors (B), showing that the cells did not express hematopoietic lineage markers, such as CD45, and were positive for CD73, CD90 and CD105 n = 4. (C) Cytochemical staining of differentiated and undifferentiated ASCs. Fast Red staining after osteogenic differentiation show a calcium deposition. Alcian blue staining after chondrogenic differentiation show marked deposition of glycosaminoglycans in the matrix. Oil red O stain after induction of adipogenic differentiation show cytoplasmic neutral triglyceride droplets. Scale bars = 100 μm
Fig. 2
Fig. 2
Immunomodulatory screening of extracellular vesicles from adipose stromal/stem cells (ASCs) of different donors. EVs from the ASC supernatant were isolated via differential (ultra)-centrifugation. Resting BV-2 cells were primed for 3 h with 1 µg/ml LPS before addition of 8 EVs/cell (quantification by flow cytometry) for 24 h. The secretion of TNF-α was evaluated by ELISA in the cell-culture supernatant as an activation read-out for the LPS-stimulated microglia cells. (A) The TNF-α levels from all donors (n = 12) were compared. The donors were separated into (B) non-immunomodulatory (n = 6) and (C) immunomodulatory donors (n = 6). Statistics were calculated with the Mann Whitney U-Test. NC = negative control (D) Further characterization and functional analysis of EVs were carried out for the immunomodulatory EVs as shown in the scheme
Fig. 3
Fig. 3
Characterization of EVs isolated from adipose stromal/stem cells (ASCs) culture supernatant. EVs from the ASC supernatant were isolated via differential (ultra)-centrifugation. (A) One representative micrograph of EVs isolated from ASCs taken by scanning helium-ion microscopy. (B) One representative graph of ASC-EV size evaluation using NanoSight nanoparticle tracking (NTA) analysis. ASC-EVs isolated from cell culture supernatant show a mean size of 157 nm. (C) MACSPlex analysis of the ASC-EV surface markers
Fig. 4
Fig. 4
Activation of BV-2 cells with Aβ aggregates. Resting BV-2 cells were primed for 3 h with 1 µg/ml LPS before stimulation with 10 µM of Aβ aggregates for 24 h. The cell culture supernatants were finally analyzed for secretion of (A) TNF-α by ELISA and (B) NO using a Griess reagent. Data represent means ± SEM (n = 3). Statistics were calculated with the Student´s t-test
Fig. 5
Fig. 5
The human ASC-EVs and mouse MSC-EVs treatment partially prevents the upregulation of pro-inflammatory molecules in BV-2 cells after Aβ stimulation. EVs from the human ASC and mouse MSC supernatants were isolated via differential (ultra)-centrifugation. Resting BV-2 cells were primed for 3 h with 1 µg/ml LPS before stimulation with 10 µM of Aβ aggregates in the presence and absence of 8 EVs/cell (flow cytometry quantification). Gene transcription of (A) TNF-α and (B) PTGS2 were evaluated by RT-PCR 6 h after stimulation. After 24 h of stimulation, secretion of the pro-inflammatory molecules (C) TNF-α and (D) NO to the cell culture supernatant were evaluated using ELISA and Griess reagent reactions, respectively. Data represent means ± SEM (n = 3). Statistics were calculated with the Mann Whitney U-Test (A and B) or with the Student´s t-test (C and D)
Fig. 6
Fig. 6
The human ASC-EVs and mouse MSC-EVs affect the expression of CD36 and CD206 of BV-2 cells after Aβ stimulation. EVs from the human ASC and mouse MSC supernatants were isolated via differential (ultra)-centrifugation. Resting BV-2 cells were primed for 3 h with 1 µg/ml LPS before stimulation with 10 µM of Aβ aggregates in the presence and absence of 6.5 × 104 ± 1.5 × 104 EVs/cell (NTA quantification). After 24 h of stimulation surface expressions of (A) CD36 and (B) CD206 on BV-2 cells were evaluated by flow cytometry. Data represent means ± SEM (n = 3). Statistics were calculated with the Mann Whitney U-Test
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