Interaction with hyaluronan matrix and miRNA cargo as contributors for in vitro potential of mesenchymal stem cell-derived extracellular vesicles in a model of human osteoarthritic synoviocytes

Abstract

Background: Osteoarthritis (OA) is the most prevalent joint disease, and to date, no options for effective tissue repair and restoration are available. With the aim of developing new therapies, the impact of mesenchymal stem cells (MSCs) has been explored, and the efficacy of MSCs started to be deciphered. A strong paracrine capacity relying on both secreted and vesicle-embedded (EVs) protein or nucleic acid-based factors has been proposed as the principal mechanism that contributes to tissue repair. This work investigated the mechanism of internalization of extracellular vesicles (EVs) released by adipose-derived MSCs (ASCs) and the role of shuttled miRNAs in the restoration of homeostasis in an in vitro model of human fibroblast-like synoviocytes (FLSs) from OA patients.

Methods: ASC-EVs were isolated by differential centrifugation and validated by flow cytometry and nanoparticle tracking analysis. ASC-EVs with increased hyaluronan (HA) receptor CD44 levels were obtained culturing ASCs on HA-coated plastic surfaces. OA FLSs with intact or digested HA matrix were co-cultured with fluorescent ASC-EVs, and incorporation scored by flow cytometry and ELISA. ASC-EV complete miRNome was deciphered by high-throughput screening. In inflamed OA FLSs, genes and pathways potentially regulated by ASC-EV miRNA were predicted by bioinformatics. OA FLSs stimulated with IL-1β at physiological levels (25 pg/mL) were treated with ASC-EVs, and expression of inflammation and OA-related genes was measured by qRT-PCR over a 10-day time frame with modulated candidates verified by ELISA.

Results: The data showed that HA is involved in ASC-EV internalization in FLSs. Indeed, both removal of HA matrix presence on FLSs and modulation of CD44 levels on EVs affected their recruitment. Bioinformatics analysis of EV-embedded miRNAs showed their ability to potentially regulate the main pathways strictly associated with synovial inflammation in OA. In this frame, ASC-EVs reduced the expression of pro-inflammatory cytokines and chemokines in a chronic model of FLS inflammation.

Conclusions: Given their ability to affect FLS behavior in a model of chronic inflammation through direct interaction with HA matrix and miRNA release, ASC-EVs confirm their role as a novel therapeutic option for osteoarthritic joints.

Keywords: Adipose-derived mesenchymal stem cells; Extracellular vesicles; Hyaluronan coat; Osteoarthritis; Synoviocytes; miRNA.

Fig. 1

Characterization of ASCs and ASC-EVs. a Representative dot plots of MSC (CD44-73-90-105) and hemato-endothelial (CD34-45) markers in ASCs. One representative cell isolate is shown. b NTA analysis of representative cell culture supernatant showing the presence of particles in the range between 50 and 500 nm. c Electron micrographs of ASC-derived small size (asterisk: possible exosomes) and large size (number sign: microvesicles) vesicles. d Setting up the EV-dedicated flow cytometer. The resolution of the reference bead mix indicates the flow cytometer performance in light scattering at default settings. Left cytogram shows side scatter (SSC) versus forward scatter (FSC). Right cytogram depicts FSC versus 535/35 (green fluorescence triggering) channel. Four fluorescent populations (100, 300, 500, and 900 nm) were resolved from the instrument noise. e CFSE-labeled ASC-EVs stained with stem cell (CD44) and EV (CD63) markers. ASC-EVs were first gated in the FITC channel (CFSE positive)

Fig. 2

Characterization FLSs. a, b Representative FLS population at passage 0 (P0) and 1 (P1) shows the initial presence and steady reduction of CD14+ monocyte with enrichment of CD73+ events. c Synovial cells (CTRL) with added red blood cells to demonstrate clear pericellular zones. As control of HA coat involvement in erythrocyte exclusion, synovial cells pre-treated for 24 h with 4-MU to block HA coat synthesis are shown (4-MU). Bright field. d FLSs stained for CD73 surface marker shows thin lines representing cell protrusions

Fig. 3

ASC-EVs are taken up by synoviocytes. a CFSE-EVs incorporated by FLSs after 24-h co-culture at different EV:FLS ratios. Number of incorporated EVs has been calculated comparing FLS fluorescence with signal given by a pre-determined number of isolated CFSE-labeled EVs (Quantification of data from three independent FLSs, each incubated with three independent ASC-EVs, is shown as mean ± SD. ****p < 0.0001; ***p < 0.001; **p < 0.01). b Flow cytometry of CFSE-EV-treated FLSs at 100,000:1 ratio showing that all cells incorporate fluorescent vesicles. c Confocal microscopy images of FLSs after 24-h co-culture with labeled ASC-EVs. Uptaken EVs localize in perinuclear areas. d Kinetics of EV incorporation (100,000 EVs per FLS, n = 3 independent experiments, each FLS incubated with a (1:1:1) mix of ASC-EVs from three independent ASCs. Data are presented as mean ± SD. *p ≤ 0.05). e FLSs, after 24-h incubation with CFSE-EVs, are able to release fluorescent vesicles (box). Representative plot of three independent analyses

Fig. 4

HA coat has a crucial role in EV incorporation. a Ratio of ASC-EV incorporation with (+) or without (−) HA coat, after 24 h [100,000 EVs per FLS, n = 3 independent experiments, each FLS incubated with a (1:1:1) mix of ASC-EVs from three independent ASCs]. Data are presented as mean ± SD. **p < 0.01, *p ≤ 0.05, ns > 0.05. b ASC-EVs release from FLSs after coat digestion with hyaluronidase (+HYAL) for 10 m at 37 °C. Digestion of FLSs with (+EV) or without (−EV) 24-h pre-incubation with ASC-EVs is presented. Released vesicles in the green box. c Ratio of ASC-EV incorporation with (+) or without (−) 2 mg/ml soluble high molecular weight HA in growth medium, after 24 h [100,000 EVs per FLS, n = 3 independent experiments, each FLS incubated with a mix (1:1:1) of ASC-EVs from three independent ASCs]. Data are presented as mean ± SD. **p < 0.01, *p ≤ 0.05, and ns > 0.05

Fig. 5

CD44 is involved in ASC-EVs uptake in FLSs. a Representative cytogram of CD44 expression in ASCs grown with (ASC01HA) or without (ASC01) HA coating of culture flask. b Representative CD44 cytogram of ASC-EVs obtained from “non-primed” ASCs (ASC-EV) or “HA-primed” ASCs (ASCHA-EV) for 24 h. P2 represents CD44-positive particles, after gating on CFSE-positive events as explained in Fig. 1e. c Ratio of EV incorporation in FLS between “CD44-boosted” EVs (ASCHA-EV) and “normal” EVs (ASC-EV), after 24 h (100,000 EVs per FLS, n = 3 independent experiments, each FLS incubated with a mix of ASC-EVs or ASCHA-EVs from three independent ASCs). Data are presented as mean ± SD. **p < 0.01, *p ≤ 0.05, ns > 0.05). d Representative cytogram of FLSs incubated with ASC-EVs (FLS + EV) or with ASC-EVs pre-treated with aCD44 Ab (FLS + EVCD44), and FLSs co-cultured with ASCHA-EVs (FLS + EVHA) or ASCHA-EVs blocked with aCD44 Ab (FLS + EVHACD44)

Fig. 6

Comparison of genome-wide miRNA expression profiles between ASC-EVs under study. a Correlation of miRNA expression levels (normalized C_RT) between the three ASC-EVs under study. b Box plot of normalized C_RT values for shared and averaged 267 miRNAs embedded in ASC-EVs. The percentage of total expression for each quartile is shown. First quartile miRNAs have been used for target mining

Fig. 7

IL-1β and EV effect on FLSs. a Experimental plan for IL-1β inflammation and EVs supplementation in FLSs. IL-1β or IL-β + EVs (100,000 EVs:FLS) were freshly added with medium change each 48 h. b IL-1β at low concentration activates inflammation markers. Synoviocytes were treated with 25 pg/mL IL-1β and, after 6 days, at time points 0, 2, and 10 days, 11 genes related to inflammation were scored by qRT-PCR. The data are presented as ^−ΔΔCt relative to the untreated control for each time point. *p ≤ 0.05, **p < 0.01, ***p < 0.001. c EVs are able to reduce secretion of chemokines and cytokines under inflammation stimuli. FLSs treated with 25 pg/mL of IL-1β for 6 days were supplemented with EVs pooled from three ASC supernatants and OA-related genes scored after 2 and 10 days. Quantification of data is shown as mean ± SD and normalized for TBP. The data are presented as ^−ΔΔCt relative to IL-1β only treated FLSs. *p ≤ 0.05, **p < 0.01, ***p < 0.001. d ELISA assays confirm reduction of inflammation-related CCL2/CCL5 chemokines and IL-6 after 10-day EV exposure. Quantification of data is shown as mean ± SD. *p ≤ 0.05, ***p < 0.001, ****p < 0.0001

Fig. 8

ASC-EVs interact with inflamed synoviocyte hyaluronan matrix enhancing their uptake and eventually release therapeutic miRNAs