Bioinspired artificial nanovesicles engineered from 3D spheroid-cultured UC-MSCs enhance angiogenic activity in vitro: functional proof-of-concept for ischemic applications

Abstract

Artificial nanovesicles (aNVs) derived from cells may mimic naturally secreted extracellular vesicles (EVs) and are becoming popular in biomedical research. We isolated aNVs from two-dimensional (2D)- and three-dimensional (3D)-cultured umbilical cord-derived mesenchymal stem cells (UC-MSCs) (aNVs^2D and aNVs^3D, respectively) and characterized them using Western blotting and electron microscopy. The aNVs^3D showed higher expression of IL-6 and SDF-1α than aNVs^2D. In vitro treatment with aNVs^2D and aNVs^3D resulted in their internalization into endothelial cells and the subsequent alteration of endothelial cell proliferation, migration, and tube formation. Both aNVs were positive for EVs and cell markers and were round in shape. Furthermore, aNVs^3D treatment enhanced endothelial cell proliferation, migration, and tube formation more effectively than aNVs^2D treatment. Our study demonstrates that aNVs3D are potent inducers of angiogenesis, indicating their potential in cell-free ischemia treatment.

Keywords: angiogenesis; artificial nanovesicles; ischemia; mesenchymal stem cells; three-dimensional.

Graphical abstract

FIGURE 1

Establishment of 2D and 3D Cultures of Umbilical Cord-Derived Mesenchymal Stem Cells. (A) Schematic representation of 2D and 3D UC-MSC cultures, created with BioRender.com. (B) Images of 2D cultures of MSCs (UC-MSCs^2D) in flat bottom plates and 3D cultures of MSCs (UC-MSCs^3D) in U-shaped 96-well plates, with UC-MSCs^3D cultured up to day 5 (scale bar: 500 pixels).

FIGURE 2

Generation and Characterization of aNVs^2D and aNVs^3D. (A) Graphical illustration of the generation of aNVs^2D and aNVs^3D from UC-MSCs^2D and UC-MSCs^3D, respectively, created with BioRender.com. (B) Western blot analysis of aNVs^2D and aNVs^3D (10 µg protein per lane) using antibodies against ALIX, CD63, flotillin-1, and calnexin. (C) TEM imaging of aNVs^2D and aNVs^3D (scale bar: 200 nm). (D) An array blot incubated with aNVs^2D and aNVs^3D lysate (50 µg per blot), the fold change of highly expressed cytokines was presented in graph.

FIGURE 3

Internalization of aNVs^2D and aNVs^3D into Endothelial Cells. Fluorescence microscopy images demonstrate the internalization of aNVs^2D (DiD) and aNVs^3D (DiD) into endothelial cells (scale bar: 20 µm).

FIGURE 4

aNVs^3D Promoted the Proliferation and Migration of Endothelial Cells. (A) Endothelial cells were treated with aNVs^2D and aNVs^3D (0, 2.5, 5, and 10 μg/mL) for 12 h and 24 h, and cell proliferation (n = 4) was assessed using the CCK-8 assay. (B) Illustration of the experiment to assess endothelial cell migration following treatment with aNVs, created with BioRender.com. (C) Representative image of transwell migration of endothelial cells treated with aNVs^2D and aNVs^3D (5 and 10 μg/mL) at 12 h and 24 h (scale bar: 100 µm). (D) Quantitative analysis of the transwell migration assay presented in (C) (n = 3). Statistical significance is denoted as follows: *p < 0.05; **p < 0.01, ***p < 0.001 and ****p < 0.0001, as determined by Two-way ANOVA.

FIGURE 5

aNVs^3D Promoted Vessel-Like Tube Formation In Vitro. (A,B) Representative microscopic images showing vessel-like tube formation by endothelial cells treated with aNVs^2D and aNVs^3D in Matrigel (scale bar: 100 µm) at 3 h and 6 h (C,D) Quantification of % of vessel percentage area total vessel area, junctions density, and total vessels length analyzed using AngioTool for (A) (n = 3). Statistical significance determined by Student’s t-test is denoted as follows. ns: no significance, *p: <0.05, and **p: <0.01.