Figure 2. iPSC-derived EVs are internalized by HSCs and reduce HSC profibrogenic phenotype.

Representative confocal microphotographs of human HSCs exposed to (A) PKH26-labeled EVs (red) or (B) EV-free supernatant for 6 hours. Original magnification, ×40 (A), ×10 (B) (by confocal microscopy). (C) Quantitative PCR expression graphs of profibrogenic genes α–smooth muscle actin (αSMA), CollagenIα1, and tissue inhibitor of metalloproteinases–1 (TIMP-1) in HSCs primed with the profibrogenic agent transforming growth factor–β (TGF-β) and exposed to iPSC-EVs or EV-free supernatant for 24 hours. β₂m, β₂-microglobulin. (D) Gene expression levels of proliferation marker cyclin D determined by quantitative PCR. β₂m was used as a housekeeping gene for quantitative PCR. (E) Representative Western blot gels and corresponding densitometry analyses of fibrosis markers αSMA and fibronectin in HSCs primed with TGF-β and exposed to iPSC-EVs or EV-free supernatant for 24 hours. Actin was used as a loading control. (F) Quantification graph of proliferation assay assessed in human HSCs primed with TGF-β and exposed to iPSC-EVs or EV-free supernatant for 48 hours. (G) Representative microphotographs and (H) quantitation histogram of chemotaxis assay performed on human HSCs primed with TGF-β and exposed to iPSC-EVs or EV-free supernatant for 6 hours. Original magnification, ×10. Values represent mean ± SD from at least 3 independent experiments. *P < 0.05; Kruskal-Wallis test with post hoc Mann-Whitney test and Bonferroni’s correction were used for statistical analysis.