Adipose-Derived Mesenchymal Stromal Cells Treated with Interleukin 1 Beta Produced Chondro-Protective Vesicles Able to Fast Penetrate in Cartilage

Abstract

The study of the miRNA cargo embedded in extracellular vesicles (EVs) released from adipose-derived mesenchymal stromal cells (ASC) preconditioned with IL-1β, an inflammatory stimulus driving osteoarthritis (OA), along with EVs-cartilage dynamic interaction represent poorly explored fields and are the purpose of the present research. ASCs were isolated from subcutaneous adipose tissue and EVs collected by ultracentrifugation. Shuttled miRNAs were scored by high-throughput screening and analyzed through bioinformatics approach that predicted the potentially modulated OA-related pathways. Fluorescently labeled EVs incorporation into OA cartilage explants was followed in vitro by time-lapse coherent anti-Stokes Raman scattering; second harmonic generation and two-photon excited fluorescence. After IL-1β preconditioning, 7 miRNA were up-regulated, 4 down-regulated, 37 activated and 17 silenced. Bioinformatics allowed to identify miRNAs and target genes mainly involved in Wnt, Notch, TGFβ and Indian hedgehog (IHH) pathways, cartilage homeostasis, immune/inflammatory responses, cell senescence and autophagy. As well, ASC-EVs steadily diffuse in cartilage cells and matrix, reaching a plateau 16 h after administration. Overall, ASCs preconditioned with IL-1β allows secretion of EVs embedded with a chondro-protective miRNA cargo, able to fast penetrate in collagen-rich areas of cartilage with tissue saturation in a day. Further functional studies exploring the EVs dose-effects are needed to achieve clinical relevance.

Keywords: adipose-derived mesenchymal stromal cells; cartilage; extracellular vesicles; interleukin 1 beta; miRNA; nonlinear optical microscopy; osteoarthritis; time-lapse.

Figure 1

Functional enrichment analysis on miRNA up-regulated in ASC-EVs primed with IL-1β. The analysis was conducted by KEGG pathway (a) Reactome (b) and Wikipathway (c). Significant pathways are listed and represented by circles colored according to the significance of the enrichment and their size is proportional to the number of target genes regulating the described signaling pathways.

Figure 2

Functional enrichment analysis on miRNA activated in ASC-EVs primed with IL-1β. The analysis was conducted by KEGG pathway (a) Reactome (b) and Wikipathway (c). Significant pathways are listed and represented by circles colored according to the significance of the enrichment and their size is proportional to the number of target genes regulating the described signaling pathways.

Figure 3

Functional enrichment analysis on miRNA silenced in ASC-EVs primed with IL-1β. The analysis was conducted by KEGG pathway (a) and Wikipathway (b). Significant pathways are listed and represented by circles colored according to the significance of the enrichment and their size is proportional to the number of target genes regulating the described signaling pathways.

Figure 4

ASC-EVs kinetic of penetration in OA cartilage. (a) 3D reconstructions of the cartilage explant during the time-lapse experiment where the cells and the rich lipidic structures are shown in red (CARS signal at 2848 cm⁻¹ from lipid structures), the collagen rich ECM is shown in blue (SHG signal from collagen) end the EVs are shown in green (TPEF signal from EVs fluorescence stain). In the box is shown the scale in μm, while in the upper right corner is depicted the corresponding time of the image during the experiment. The reconstructions are obtained from the acquired images and processed using 3Dviewer with ImageJ. (b) Three graphs of the dynamics of the EVs in the tissue during the time-lapse related to the depth, the area and the volume are showed. For each curve are also plotted the related fitting in blue the double exponential one (Equation (1) in Table S6) and in red the single exponential one (Equation (2) in Table S6).

Figure 5

ASC-EVs diffusion in OA cartilage cells and matrix. (a) 3D surfaces showing the EVs penetration depths extracted from the acquired 3D imaging. In the upper corner are visible the corresponding time in the time-lapse experiment. The 3D surface graphs are obtained from the extracted EVs penetration depths images using the ImageJ3D plugin setting smoothing value at 10 pixels. (b) Dynamics of the co-localization ratios between the cells and the lipidic structure (CARS) and the EVs in red and between the collagen (SHG) in the ECM and the EVs in blue. For each ratio is reported the average (continuous line) and the maximum value (dash-dotted line) in the Z stack for that time step.